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The properties and functions of bacterial aminopeptidases

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Acta Microbiologica Polonica
2003, Vol. 52, No 3

CONTENTS

MINIREVIEW

The properties and functions of bacterial aminopeptidases

JANKIEWICZ U., BIELAWSKI W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Horizontal DNA transfer between bacteria in the environment
WOLSKA K.I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

ORIGINAL PAPERS

Rearrangements between differently replicating DNA strands in asymmetric bacterial genomes

MACKIEWICZ D., MACKIEWICZ P., KOWALCZUK M., DUDKIEWICZ M., DUDEK M.R.,
CEBRAT S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Synthesis of siderophores by strains of Staphylococcus cohnii isolated from various
environments
SZARAPIÑSKA-KWASZEWSKA J., FARKAS £.I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Differences in inhibition of apoptosis depending on the virulence of used hHerpes Simplex Virus
type 1 strains. Function of interferon alpha in apoptotic death of virus infected cells
RECHNIO M., LITWIÑSKA B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Enteropathogenic activity and invasion of Hep-2 cells by Aeromonas caviae clinical isolates
KRZYMIÑSKA S., KAZNOWSKI A., LINDNER K., MNICHOWSKA M. . . . . . . . . . . . . . . . . . 277
Amlodipine: a cardiovascular drug with powerful antimicrobial property
KUMAR K.A., GANGULY K., MAZUMDAR K., DUTTA N.K., DASTIDAR S.G.,
CHAKRABARTY A.N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Comparative delta-endotoxins of Bacillus thuringiensis against mosquito vectors
(Aedes aegypti and Culex pipiens)
LONC E., KUCIÑSKA J., RYDZANICZ K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Improvement of rifemycins production by Amycolatopsis mediterranei in batch and fed-batch
cultures
EL-ENSHASY H.S., BESHAY U.I., EL-DIWANY A.I., OMAR H.M., EL-KHOLY A.G.E.,
EL-NAJAR R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

BOOK REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

INSTRUCTIONS TO AUTHORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

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Acta Microbiologica Polonica
2003, Vol. 52, No 3, 217–231

The Properties and Functions of Bacterial Aminopeptidases

URSZULA JANKIEWICZ* and WIES£AW BIELAWSKI

Department of Biochemistry, Warsaw Agricultural University,

ul. Rakowiecka 26/30, 02-528 Warsaw, Poland

Received 14 August 2003

Abstract

Aminopeptidases are enzymes that release N-terminal amino residues from oligopeptides,
polypeptides and proteins. The classification of aminopeptidases has often been based on mecha-
nism of catalysis, structure of active site, substrate specificity kinetic and molecular properties. In
terms of catalytic mechanism bacterial aminopeptidases can be divided into three main catalytic
groups: metallo-, cysteine- and serine aminopeptidases. According to their substrate specificity
the enzymes can be ordered into two sub-groups: having broad or narrow specificity. Almost half
of the characterized aminopeptidases show a subunit structure. Enzymes having a quaternary
structure are most often built of a combination of 2, 4, 6 subunits. Bacterial aminopeptidases may
be localised in the cytoplasm, on membranes, associated with the envelope or secreted into the
extracellular media. Regulation of the synthesis of aminopeptidases is assumed to take place
mainly at the level of transcription. Most genes encoding the enzymes are monocistronic and
contain a promotor characteristic for the genes transcribed by RNA polymerase associated with
the factor F70. Aminopeptidases play an important role in the initial and final steps of protein
turnover and they are involved in several specific regulatory functions.

K e y w o r d s: aminopeptidases, functions, localisation, synthesis

Introduction

Aminopeptidases are exopeptidases that catalyse the cleavage of N-terminal amino

acids from proteins or peptides. The enzymes are present in every prokaryotic and
eukaryotic cell. Their activity is very high, which may indicate their importance in
the metabolism of all living organisms. Aminopeptidases embrace an exceptionally
numerous and differentiated group of peptidases. According to the classification of
the International Union of Biochemistry and Molecular Biology the enzymes are given
the number EC 3.4.11. Peptidases, including aminopeptidases, are further divided
into families according to the homology of the primary structure and the sequence
of functional groups participating in catalysis and into clans according to the similar-
ity of the quarternary structure and the sequence of amino acids surrounding the
active site (R a w l i n g s and B a r r e t t, 1993). Moreover, substrate specificity,

* Correspondence to: [email protected]

218 Jankiewicz U., Bielawski W. 3

mechanism of catalysis, sensitivity to bestatin, subcellular location and optimum pH

are often used classification parameters of aminopeptidases. The division of bac-
terial aminopeptidases into several types marked with letters, e.g. N, C, A, P based
on catalytic and molecular properties is common in the literature. The names of
the remaining aminopeptidases are formed depending on their substrate specificity,
e.g. methionine aminopeptidase or arginine aminopeptidase (T a y l o r, 1993a;
G o n z a l e s and R o b e r t - B a u d o u y, 1996).

The mechanism of enzymatic catalysis

Because of the mechanism of catalysis and the structure of their active site, bacte-
rial aminopeptidases may be sub-divided into three main catalytic groups: metallo-
aminopeptidases, cysteine aminopeptidases and serine aminopeptidases.
Metallo-aminopeptidases are the most numerous group comprising 60% of these
enzymes. These are enzymes whose activity is inhibited by metal chelating compounds
e.g. EDTA, hydroxyquinoline, and 1, 10 phenanthroline and also by 3-amino-2-hy-
droxy-4-phenylbutanoyl-L-leucine (bestatin) and by 3-amino-2-hydroxy-5-methyl-
hexanoyl-L-valil- L-valil-L-aspartic acid (amastatin). The metal ion is most often
combined through coordination bonds with two residues of histidine and with one
residue of glutamic acid. The catalysis requires, apart from metal ligands, also at least
one residue of an amino acid, often glutamic acid, arginine or lysine. Mechanism of
reactions catalysed by these enzymes is not yet fully explained. It is assumed that the
cation of a metal markedly increases the reactivity of combined water molecule mak-
ing easier the nucleophilic attack on carbonyl carbon of the hydrolysed peptide bond
and in addition it stabilises the transitional stage of the reaction (R a w l i n g s, 1998;
H o l z, 2002). In spite of the fact that the identification of the metal ion participating
in catalysis is not always possible, it was found that the most numerous group are zinc
dependent aminopeptidases (G o n z a l e s and R o b e r t - B a u d o u y, 1996). Two
sub-groups have been distinguished within this group. The first comprises aminopep-
tidases with two zinc ions in the catalytic site of the enzyme. Here belong bacterial
intracellular leucine aminopeptidases called Pep A showing a homology of amino
acid sequences with the animal leucine aminopeptidase (S t i r l i n g et al., 1989;
B u r l e y et al., 1992; T a y l o r, 1993a) and extracellular bacterial leucine amino-
peptidases (P r e s c o t t and W i l k e s, 1966; S p u n g i n and B l u m b e r g,
1989; C a h a n et al., 2001; H o l z, 2002). Aminopeptidases of the second group
contain at least one zinc ion in the catalytic site. Aminopeptidases of the N and A type
belong to this subgroup (G e i s s et al., 1985; N i v e n et al., 1995; K l e i n, 1998;
B u t l e r, 1998). A group of enzymes containing in the active site of each subunit two
Co+2 ions was also distinguished from among metal dependent aminopeptidases. Such
mechanism of catalysis was described for methionine aminopeptidase from Escherichia
coli (R o d e r i c k and M a t t h e w s, 1993, L o w t h e r and M a t t h e w s, 2000),
from Lactobacillus plantarum (Macedo et al., 2003) and for extracellular aminopep-
tidase from Bacillus sp. N2 (L e e et al., 1998). Type T aminopeptidases synthesised
3 Minireview 219

only by thermophilic and extremophilic strains of bacteria were also included among
metallo-aminopeptidases containing zinc or cobalt in their active sites (M o t o s h i m a
and K a m i n a g a w a, 1998, F e r n a n d e z - E s p l a and R u l, 1999).
Additional types of catalysis were also found in aminopeptidases dependent on
other than the mentioned metal ions. Mn+2 and Mg+2 focused particular interest since
they are able to activate enzymes or to remove the activity of some metal dependent
aminopeptidases. In the active site of aminopeptidase P isolated from E. coli there
are two ions of Mn+2 (Y o s h i m o t o et al., 1989; Z h a n g, 1998) while aminopep-
tidase isolated from the strain of Thermatoga maritime was classified as magnesium
dependent (R a t n a y a k e et al., 2003).
Cysteine aminopeptidases contain SH groups of cysteine and most often histidine,
asparagine and glutamic acid residues in the active site. In contrast to metallo-ami-
nopeptidases, they do not contain an ionic co-factor in their structure. The reaction
starts with the nucleophilic attack of the sulfur of the sulphydril group on the carbo-
nyl carbon of the peptide bond. The enzymes are specifically inhibited by
iodoacetamide, iodoacetate, N-ethyl-maleimide, p-chloromercuribenzoate (pCMB),
trans-epoxysuccinyl-L-leucylamido-4-guanidino-butane (E-64). The activity of this
group of enzymes is decreased by some serine inhibitors: TLCK, TPCK, leupeptin,
antipain. Activators of this group are: cysteine, dithiothreitol (DTT) and some chelat-
ing agents like EDTA. The group comprises enzymes, whose mechanism of catalysis
is much better understood than that of metal-dependent aminopeptidases. Type C ami-
nopeptidases and other cysteine aminopeptidases belong to this group. The former
have been purified only from lactic acid fermentation bacteria. This is a family of
cytoplasmic aminopeptidases, closely related in structure and activity to bleomycin
hydrolase (N e v i a n i et al., 1989; C h a p o t - C h a r t i e r et al., 1993; C h a p o t -
C h a r t i e r et al., 1994; M i s t o u and G r i p o n, 1998; d e P a l e n c i a et al.,
2000). Thiol aminopeptidases that do not show homology of amino acid sequences
with aminopeptidases type C are distinguished in the literature as other cysteine ami-
nopeptidases. Here belong acrylamidase from the strain of Neisseria catarrhalis
(B e h a l and C o x, 1968) and pyrrolidone carboxyl peptidases (A w a d é et al.,
1994; P a t t i et al., 1995; L e S a u x and R o b e r t - B a u d o u y, 1997). Ami-
nopeptidase specifically inhibited by cysteine enzyme inhibitors was also isolated
from the Pseudomonas sp. (J a n k i e w i c z and B i e l a w s k i, 2002a).
Bacterial serine aminopeptidases are the least numerous group of aminopeptidases
that do not belong to any of the known families of serine proteolytic enzymes repre-
sented by trypsin and subtilisin (R a w l i n g s and B a r r e t t, 1994). Diagnostic
inhibitors for serine aminopeptidases are diisopropyl fluorophosphate (DFP) and
phenylmethylsulfonyl fluoride (PSMF). Serine aminopeptidases are also inhibited by
ketones: N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-p-Tosyl-L-
lysine-chloromethyl ketone (TLCK) but these are not specific inhibitors since they
act also on other cysteine aminopeptidases.
Among the serine aminopeptidases are proline aminopeptidases which have also
been called prolineiminopeptidases (PIP), containing a triad of serine, histidine and
aspartic acid in the active site (K i t a z a n o et al., 1994; M o r e l et al., 1999) and
220 Jankiewicz U., Bielawski W. 3

D-aminopeptidase, in which four amino acid residues (3 of Ser and 1 of Lys) are found
in the active site. Lysine plays a role of proton acceptor during the nucleophilic attack of
the serine hydroxyl group on carbonyl carbon of the peptide bond (A s a n o, 1998;
F a n u e l et al., 1999, A s a n o and L u b b e h u s e n 2000, K o m e d a et al., 2003).

Substrate specificity

According to their substrate specificity bacterial aminopeptidases can be divided

into two sub-groups: of broad and narrow specificity. Aminopeptidases of broad speci-
ficity are able to release many different N-terminal amino acids whereas aminopepti-
dases of narrow specificity cleave only a single type of amino acid residues. All ami-
nopeptidases are stereospecific enzymes hydrolysing as a rule L-forms of amino acids
from the NH2 terminus of substrates. The exception is D-aminopeptidase from
O. anthropi which hydrolyses N-end residues of glycine, D-alanine and D-serine
(A s a n o et al., 1989, F a n u e l et al., 1999). Aminopeptidases of broad substrate
specificity are usually not able to hydrolyse peptide bonds formed by acidic amino
acids (Asp, Glu, pGlu) in the P1 position or of proline in the P1 or P’1 position.
Common in microbial cells leucine aminopeptidases, aminopeptidases N and C belong
to this group of enzymes. The leucine aminopeptidases show broad substrate specificity
with preference for N-end leucine or Met and Phe. Well characterised are leucine ami-
nopeptidases (Pep A) present in E. coli and Salmonella typhimurium (M i l l e r and
M a c k i n n o n,  1974; M i l l e r and G r e e n, 1983) and those synthesised by the
strains of Vibrio proteolyticus – formerly Aeromonas proteolytica (P r e s c o t t and
W i l k e s, 1966; W o o d, 1998; Z h a n g et al., 2000), Streptomyces griseus
(Vo s b e c k et al., 1975, M a r a s et al., 1996) and Pseudomonas aeruginosa
(C a h a n et al., 2001). Aminopeptidases of the pep A type, in contrast to other leu-
cine aminopeptidases, are not able to detach N-end proline from substrates (M i l l e r
and G r e e n, 1983). Very interesting results were demonstrated by B a y l i s s and
P r e s c o t t (1986) on the change of substrate specificity of leucine aminopeptidase
isolated from Vibrio proteolyticus. After intensive dialysis of the enzyme in EDTA
containing solution its activity was reactivated with ions of Zn+2, Co+2, Cu+2 and Ni+2.
The kind of bound ion decided of substrate specificity of the enzyme. Similar results
were obtained in experiments carried out on other aminopeptidases of similar struc-
ture of the catalytic site (A j a b n o o r and W a g n e r, 1979; B e n - M e i r et al.,
1993). This is a specific type of activity regulation described exclusively for metallo-
aminopeptidases. Another enzyme of broad specificity is aminopeptidase N showing
specificity for the N-end alkaline and aliphatic amino acids. Aminopeptidases N iso-
lated from strains of Lactobacillus delbrueckii, Lactobacillus curvatus, Lactcoccus
lactis and Streptococcus thermophilis show broad substrate preference to peptides
with lysine or leucine at their N-end and, to a lesser extent, to those having alanine,
phenylalanine, arginine and methionine (T a n and K o n i n g s, 1990; B a a n k r e i s
and E x t e r k a t e, 1991; N i v e n et al., 1995; M c D o n n e l l et al., 1999;
C h a w a g n a t et al., 1999; M a g b o u l and M c S w e e n e y, 1999). Intracellular
3 Minireview 221

aminopeptidases specific to N-end alanine, arginine, leucine and lysine were also pu-
rified from strains of and Pseudomonas fluorescens ATCC 948 and Pseudomonas sp.
(G o b b e t t i et al., 1995; J a n k i e w i c z and B i e l a w s k i, 2001; J a n k i e w i c z
and B i e l a w s k i, 2002b).
Type C aminopeptidases belongs also to those of broad substrate specificity. They
release N-terminal residues of Ala, Lys, Arg, Met and Phe most easily. Enzymes of
that group show a lack or very low activity with substrates containing proline in the
P1 and P1’ position (Ve s a n t o et al., 1994; W o l h r a b and B o c k e l m a n n,
1993, d e P a l e n c i a et al., 2000). From among enzymes of broad substrate speci-
ficity there are also cysteine aminopeptidases specifically inhibited by p-CMB and
showing no homology in amino acid sequences with aminopeptidases C.
Aminopeptidases of a narrow substrate specificity are divided into 6 sub-groups,
with the kind of cleaved N-end amino acid being the criterion for distinction.
1. Methionine aminopeptidases called aminopeptidases M. These are enzymes pre-
ferring substrates that contain methionine in the N-end position of peptide chain.
Aminopeptidase M was characterised from e.g. strains of E. coli and Bacillus
subtilis (N a k a m u r a et al., 1990; R o d e r i c k and M a t t h e w s, 1993).
2. Aspartate aminopeptidases A called also glutamyl aminopeptidases. Enzymes of
this group show substrate specificity for peptides containing residues of aspartic
of glutamic acid (E x t e r k a t e and d e Ve e r, 1987; B a c o n et al., 1994).
3. Pyrrolidone carboxyl peptidase (pyrase) prefers substrates containing pyroglutamic
acid which forms as a result of spontaneous, intracellular cyclization of glutamic
acid (D o o l i t t l e, 1970).
4. Arginine aminopeptidases – enzymes of this group demonstrate specificity to sub-
strates with the N-end arginine. Arginine aminopeptidases were isolated from only
several strains of Streptococcus and E. coli (I s h i n o et al., 1987; F l o d e r u s,
1990, G o l d s t e i n et al., 2002; J o b i n and G r e n i e r, 2003)
5. Aminopeptidases P – detach N-end amino acid from substrates containing proline
in the P1’ position. Aminopeptidase P was obtained from Streptomyces lividans
and Salmonella typhimurium (M c H u g h and M i l l e r, 1974; B u t l e r et al.,
1993; B u t l e r et al., 1994, M c D o n n e l et al., 1997).
6. Proline aminopeptidases are specific for substrates that contain proline at the
N-end. Such aminopeptidases were isolated e.g. from E. coli, bacteria of the
genus Lactobacillus, and from Arthrobacter nicotianae (K u n j i et al., 1996;
S m a c c h i et al., 1999).
The activity of aminopeptidases of narrow specificity is often determined not only
by amino acid situated in the last position at the N-end but also by that in the last
but one or further positions. Proline iminopeptidase hydrolyses peptide bonds in
substrates containing N-end proline on condition that neither Lys nor Phe is the next
in polypeptide chain (Y o s h i m o t o et al., 1983). Similarly, methionyl aminopepti-
dase shows activity against substrates with N-terminal methionine only if P1’ posi-
tion is occupied by alanine, glycine, proline, serine or threonine. The presence of
arginine, leucine, lysine or phenylalanine totally inhibits the activity of this aminopep-
tidase (B e n - B a s a d et al., 1987). Amino acids in positions P1, P1’, P2’, P3’ and
222 Jankiewicz U., Bielawski W. 3

P4’ affect the kinetic constant of the reaction of aminopeptidase P from E. coli
(Y o s h i m o t o et al., 1994). Aminopeptidases of a narrow specificity e.g. proline
or glutamine aminopeptidase can hydrolyse substrates with N-end amino acids unaf-
fected by aminopeptidases of broad specificity.

Localization of the aminopeptidases

Finding the subcellular location of some aminopeptidases appears difficult at the

present stage of genetic studies. It is known that aminopeptidases, which do not possess
the signal sequence at their N-end are cytoplasmic enzymes. Most aminopeptidases,
however, are enzymes located in soluble cell fractions, most often in the cytoplasm
and the periplasm of gram-negative bacteria (G o n z a l e s and R o b e r t -
B a u d o u y, 1996). All found Pep C are described as cytoplasmic enzymes (T a n
et al., 1992). Aminopeptidases may be also located in cell walls of gram-positive
bacteria (F l o d e r u s and L i n d e r, 1990; B l a n c et al., 1993, L i n d e r et al.,
1996). Only several aminopeptidases associated with membranes have been found
yet (I s h i n o et al., 1987; F l o d e r u s et al., 1990). Some aminopeptidases are
associated with the internal side of cytoplasmic membrane. Therefore, the activity of
these enzymes is found partly in the cytoplasm and partly in the cell membrane frac-
tion. It is assumed that the location of the aminopeptidase activity in the close
neighbourhood of cell membranes results from their function in the transport and
degradation of extracellular peptides. Such subcellular location was described for
aminopeptidase N in Pseudomonas aeruginosa (B e r t h o d et al., 1984) and for
aminopeptidase A in Lactococcus (B a a n k r e i s, 1992; B a c o n et al., 1994).
Extracellular enzymes are the minority among bacterial aminopeptidases. Enzymes
synthesised by Pseudomonas aeruginosa (C a h a n et al., 2001) and some strains of
Vibrio, Alteromonas and Streptococcus are described (T o m a and H o n m a, 1996,
M e r k e l et al., 1981, G o l d s t e i n et al., 2002).

Subunit structure

Almost half of the already known aminopeptidases show a subunit structure. En-
zymes having a quaternary structure are most often built of a combination of 2, 4, 6
subunits. Most frequent is the homomeric subunit system. Only several yet described
aminopeptidases have the heteromultimeric structure. Subunit structure possess intra-
cellular bacterial leucine aminopeptidases i.a. enzymes isolated from Pseudomonas
putida of a mass of 400 kDa built of 8 identical subunits (H e r m e s et al., 1993),
those isolated from Brevibacterium linens SR 3 built of 12 subunits 18 kDa each
(F e r n a n d e z et al., 2000) or aminopeptidase A from E. coli combined of
6 homomeric subunits 55 kDa each (Vo g t, 1970). Aminopeptidase isolated from
Mycoplasma salivarium has a heteromeric subunit structure. It consists of subunits
with a mass of 50 and 47 kDa (S h i b a t a et al., 1987). Aspartate aminopeptidases
3 Minireview 223

or aminopeptidases type C from bacteria of lactic acid fermentation also show subunit
structure (N i v e n, 1991; B a a n k r e i s, 1992; d e P a l e n c i a et al., 2000).
Interesting results were presented by S t o l l et al. (1973) from their studies on
metallo-aminopeptidase from Bacillus stearothermophilus. The enzyme was built
of 12 identical subunits, 36 kDa each. Analysis of the amino acid sequence of this
enzymatic protein proved the existence of two distinct subunits types. The enzymes
of monomeric structure involve also aminopeptidases type N of a molecular mass
between 78 and 99 kDa (T a n et al., 1990; R u l et al., 1994; C h a v a g n a t et al.,
1999). The so far recognised extracellular aminopeptidases belong also to monomeric
enzymes with the exception of dimeric (2×33 kDa) aminopeptidase excreted by
Alteromomonas B-207 (M e r k e l et al., 1981). The mass of extracellular leucine
aminopeptidases is small and usually varies between 20 and 30 kDa (S p u n g i n
and B l u m b e r g, 1989; V i t a l e et al., 1986; T o m a and H o n m a, 1996) while
those of arginine aminopeptidase and prolineiminopeptidase are 70 and 53 kDa,
respectively (G o l d s t e i n et al., 2002, S m a c c h i et al., 1999).

Regulation of enzyme synthesis

Though the amino acid sequence is known and the homology of encoding genes is
established e.g. for leucine, C and N aminopeptidases from various bacterial strains,
information on the regulation of these genes expression is still incomplete. Regula-
tion of the synthesis of enzymatic proteins in Procaryotes is assumed to take place
mainly at the level of transcription. Most genes encoding bacterial aminopeptidases
are monocistronic and contain a promotor characteristic for the genes transcribed
by RNA polymerase associated with the factor F70 (G o n z a l e s and R o b e r t -
B a u d o u y, 1996). Regulation of aminopeptidase encoding genes is most often stud-
ied in Enterobacteria and particularly in E. coli and Salmonella typhimurium. The
relatively best characterised enzyme in E. coli is aminopeptidase N. The enzyme
is synthesised during the whole life cycle of the bacteria but the expression of the
gene encoding this enzyme increases in the case of phosphorus or oxygen deficit
even four-fold while carbon and nitrogen deficiencies do not affect the activity
(L a z d u ñ s k i et al., 1975, G h a r b i et al., 1985). The molecular base of this
regulation is not known. The synthesis of pyroglutamic aminopeptidase in Pseudo-
monas fluorescens is induced under iron deficiency and in the presence of the product
– pyroglutamic acid (L e S a u x and R o b e r t - B a u d o u y, 1997). In spite of the
fact that catabolic repression is the common model of regulation of the proteolytic
enzymes biosynthesis, only one gene regulated in that way was characterised in bac-
teria. The expression of a gene encoding aminopeptidase E (dipeptide aminopepti-
dase) with the specificity for N-end Asp in S. typhimurium is controlled by catabolic
repression (C o n l i n et al., 1994). The physiological basis of such type of regula-
tion is not fully explained. The authors suggest that this might be a way of adaptation
of bacteria to limited carbon resources. Released aspartic acid can serve as a source of
carbon or energy for the bacteria end may enable the synthesis of other amino acids
224 Jankiewicz U., Bielawski W. 3

like Asn, Lys, Met. Protein molecules of extracellular aminopeptidases undergo post-
translational modifications. Extracellular aminopeptidases are synthesised in the form
of inactive precursors (preproenzymes) built of four or three domains: typical signal
sequence, proteolytic domain, N-end propeptide and often of C-end propeptide. N-end
propeptide is given a function of an intracellular chaperon responsible for proper fold-
ing of the particle of enzymatic protein. Moreover, it was found that N-end propeptides
are often inhibitors of the activity of the proteolytic domain. C-end propeptide partici-
pates in the secretion of this protein. After cleavage off the signal sequence and usu-
ally the double maturation at the N- and C-terminus they appear as active enzymes
(N i r a s a w a et al., 1999; Z h a n g et al., 2000; T a n g et al., 2002).
Another mechanism of regulation of the enzyme activity was described for E. coli,
where an endogenous competitive inhibitor of N-aminopeptidase was found in the
cell (Y a n g and S o m e r w i l l e, 1976).
Many studies on the effect of environmental factors on the production of amino-
peptidases have been carried out. However, the obtained results concerned enzyme
activities only and therefore they could not be used to determine of the level of regu-
lation, i.e. transcriptional, translational or post-translational.
Both constitutive and induced enzyme synthesis take place in various growth
phases of bacteria and are affected by various factors. Induced expression of the gene
encoding aminopeptidase synthesised by Bacillus stearothermophilus takes place at
a high temperature (M o s e r et al., 1970; S t o l l et al., 1972). It is known that the
presence of peptides in culture medium induces the expression of dipeptidase in bac-
teria of the lactic acid fermentation (A t l a n et al., 1989), which might suggest that
the enzyme plays a role in supplying the cell with free amino acids. In a strain of
Lactococcus lactis, however, the activity of aminopeptidase N decreased in the pres-
ence of dipeptides at high concentrations in medium (M e i j e r et al., 1996). Results
of experiments on the effect of medium composition on the biosynthesis of several
aminopeptidases of different substrate specificity in Pseudomonas fluorescens proved
the presence of both induced and constitutive aminopeptidases in this strain
(G o b e t t i and R o s s i, 1992). C h o i et al., (1996) presented results on the regu-
lation mechanism of the synthesis of extracellular aminopeptidase. The experiment
was carried out on two strains of L. casei cultured on various media. Casein contain-
ing medium induced the synthesis of extracellular aminopeptidases while medium
with glucose did not bring such effect. Most experiments carried out so far did not
give a clear picture of the regulation of aminopeptidase synthesis in bacteria and there-
fore, the problem still focuses much attention.

The functions of bacterial aminopeptidases

Bacterial aminopeptidases have often been proposed to be involved in limited pro-

teolysis. The process is assumed to be responsible for e.g. post translation modifica-
tion and maturing of proteins, for transformation of inactive proenzymes into biologi-
cally active forms and for removing signal peptides from proteins transported across
3 Minireview 225

membranes (L a z d u ñ s k i, 1989; T a y l o r, 1993a, b). Many studies were devoted

to methionine aminopeptidase responsible for removing N-terminal methionine of
newly synthesised polypeptide chains in both Prokaryota and Eukaryota (B e n -
B a s s a t et al., 1987; N a k a m u r a et al., 1990; R o d e r i c k and M a t t h e w s,
1993). This was confirmed in studies on strains of E. coli and S. typhimurium, in which
the gene encoding methionyl aminopeptidase was mutated (C h a n g et al., 1989;
M i l l e r et al., 1989). Mutation of the gene appeared lethal. It was assumed that due
to the lack of methionyl aminopeptidase in the cell, main cell proteins were inacti-
vated in the stage of methionyl precursors. Bacteria do not probably have any alterna-
tive ways of post translation maturing of proteins in the cell. Similar experiments
carried out on yeasts did not bring lethal effects, which might suggest the existence of
an alternative mechanism of removing N-end methionine (C h a n g et al., 1992).
Apart from limited proteolysis, a constant process of degradation and synthesis of
intracellular proteins takes place in the cell. The rate of protein degradation depends
on the growth phase of bacteria. A bacterial cell in the logarithmic growth phase
degrades 1–2% of its protein per hour while in the stationary phase – 5–10%
(G o t t e s m a n and M a u r i z i, 1992). The cell is able to adapt to extreme condi-
tions and in deficiency of C, N and amino acids it can degrade normally stable pep-
tides and use them as a source for the synthesis of new peptides (L a z d u ñ s k i,
1989; G o n z a l e s and R o b e r t - B a u d o u y, 1996). Moreover, protein catabo-
lism is necessary for removing defective proteins and for regulating the level of par-
ticular protein particles. Aminopeptidases are thought to be engaged in the stage initi-
ating protein catabolism in the cell and in removing N-end amino acids responsible
for the stability of a protein molecule (Y e n et al., 1980). According to the N-end
principle, the protein has different half-life depending on the type of amino acid at
N-terminus. If the theory is true then aminopeptidases are engaged in both the initial
stage of protein degradation and in the final stage leading to free amino acids
(M i l l e r and G r e e n, 1981). Controlled protein degradation is, however, still not
fully understood. It is known also that the process may be based on mechanism other
than the N-end principle (T a y l o r, 1993b). Genetic studies with mutated gene
encoding aminopeptidases showed that at a lack of these enzymes, the final stage of
intracellular protein degradation and hydrolysis of proteins to free amino acids did
not occur in the cell. This resulted in the reduction of new synthesised proteins and,
consequently, in the reduction of cell viability (Y e n et al., 1980).
Aminopeptidases play important functions in the uptake of nutrients from envi-
ronment by the bacterial cell (G o b b e t t i and R o s s i, 1992; J a n k i e w i c z and
B i e l a w s k i, 2002c). Many studies were devoted to lactic acid fermentation bacte-
ria with particularly high demand for nitrogen compounds (A t l a n et al., 1989,
C o g a n et al., 1993). The significant role of aminopeptidases may be probved
by the lack of carboxypeptidase activity in these bacteria. The results suggest
that casein degradation takes place from the amine end of the polypeptide chain.
Aminopeptidases synthesised by these bacteria show a particularly high preference
for substrates containing N-end proline. It is important due to a high content of this
amino acid in casein.
226 Jankiewicz U., Bielawski W. 3

Apart from the mentioned functions, bacterial aminopeptidases play some other
roles in the cell. S t i r l i n g et al. (1989) reported on the particular structural role of
aminopeptidase A in E. coli in stabilising plasmid COL E. An additional role is played
also by D aminopeptidase, which participates in the synthesis and degradation of pep-
tidoglycan (A s a n o et al., 1992). It is expected that some aminopeptidases take
place in the activation and transport of antibiotics into the cell. Aminopeptidase N in
E. coli and aminopeptidases A and N in S. typhimurium are responsible for the activa-
tion of the antibiotic albomycin in the cytoplasm. Mutation of genes encoding amino-
peptidase N in E. coli and aminopeptidases A and N in S. typhimurium resulted in
the insensitivity of the bacteria to this antibiotic (B r a u n et al., 1983). The partici-
pation of aminopeptidases in the degradation of toxic peptides and inactivation of
physiologically important proteins has also been suggested (M i l l e r et al., 1975).

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Acta Microbiologica Polonica
2003, Vol. 52, No 3, 233–243

Horizontal DNA Transfer between Bacteria

in the Environment
KRYSTYNA I. WOLSKA*

Department of Bacterial Genetics, Institute of Microbiology, University of Warsaw,

Miecznikowa 1, 02-096 Warsaw, Poland

Received 27 June 2003

Abstract

In the environment horizontal DNA transfer between various bacterial species and genera takes
place by transformation, transduction, but mainly by conjugation. Conjugation is responsible for
the spread of genes coding for antibiotic resistance and xenobiotic degradation. Transfer events
are reported in animal, rhizosphere and phylloplane ecosystems and in non polluted and polluted
water and soil. Genetic exchange between Bacteria and Archaea is also observed. Evaluation of
the extent of interspecies gene transfer is crucial in view of the deliberate release of a variety
of unmodified and genetically modified microorganisms into the natural environments.

K e y w o r d s: conjugation, transformation, transduction, natural environment

Introduction

Horizontal DNA transfer between various bacterial species and genera is quite
common in the environment. Of the three mechanisms of genes transfer – transfor-
mation, transduction and conjugation, the last one facilitated by conjugative plas-
mids and transposons is the most often observed (D a v i d s o n, 1999). Conjugation
is responsible for the spread of the antibiotic resistance genes (D a v i e s, 1996;
M a n z e l and D a v i e s, 1999) and xenobiotic degradation genes (V a n d e r
M e e r et al., 1992). The presence of efficient donors in heterologous bacterial
populations can accelerate plasmid transfer and spread by several orders of magni-
tude (D i o n i s i o et al., 2002).
The extent of horizontal transfer can be estimated by an analysis of microbial
genome sequences and codon usage patterns (B u s h m a n, 2002a). It was shown
that even different strains of the same species display remarkable diversity. 17.6%
of Escherichia coli genes have been received by horizontal transfer and the ge-
nomes of the laboratory K-12 strain and pathogenic O157:H7 strain are remarkably
* phone (48 22) 554 1302; e-mail: [email protected]
234 Wolska K.I. 3

different. The two genomes differ by more than a megabase, the pathogenic strain
being larger. Differences are also observed in various Helicobacter pylori and
Chlamydia trachomatis strains (after B u s h m a n, 2002a). The discovery of exten-
sive lateral transfer in bacteria causes that their tree of life appears to be highly
reticulated rather than branched in an orderly way (B u s h m a n, 2002b).
The detection of environmental gene transfer creates severe problems. Only when
easily selectable phenotypes are available, genetic transfer experiments may be per-
formed under natural conditions and the relevant phenotype selected. The use of
green fluorescent protein, GFP (D a h l b e r g et al., 1998) removes the need to
select the phenotype and to cultivate transconjugants. It should be mentioned here
that less than 1% of bacteria are cultivable using the available techniques (A m a n n
et al., 1995). The ability to detect gene transfer may be interfered with restriction
systems present in the recipient cells, the inability of a plasmid to replicate in a new
recipient, the inability of chromosomal DNA to recombine with the recipient chro-
mosome and the lack of incoming transposon integration (D a v i d s o n, 1999). Also
mismatch repair systems (M a t i c et al., 1996) negatively influence recombination
and therefore also the detection of gene transfer. Most studies on environmental
gene transfer have been performed in microcosms designed to represent natural
environmental conditions but permitting their manipulation (D a v i d s o n, 1999).
The knowledge about the extent of horizontal gene transfer is extremely important
in view of the deliberate release of a variety of nonrecombinant and recombinant
microorganisms into the environment for such purposes as nitrogen fixation, phos-
phate solubilization, control of phytopathogenic fungi and bacteria, plant growth stimu-
lation, insect and weed control and bioremediation (W i l s o n and L i n d o w, 1993).
In this review gene transfer events between various bacteria reported in different
environments are described with special emphases on conjugation. Transfer between
Bacteria and Archaea is also noticed. Transfer between Bacteria and Eukarya e.g.
from Agrobacterium tumefaciens to plants (Z u p a n and Z a m b r y s k i, 1995),
to yeast (B u n d o c k and H o o y h a a s, 1996), from E. coli to Saccharomyces
cerevisiae (H e i n e m a n and S p r a g u e, 1989), from transgenic plants to
phytosphere (D r o g e et al., 1998) and terrestial bacteria (N i e l s e n et al., 1998),
DNA transfer to animal cells by intracellular bacteria (G r i l l o t - C o u r v a l i n
et al., 1998) and from Bacteria to Eukarya via endosymbiosis (D o o l i t t l e, 1998)
are beyond the scope of this minireview.

Conjugation

The vast majority of bacterial gene transfer in the environment involves conjuga-
tion. Important, however arbitrarily chosen, examples of conjugational events in the
environment are summarized in Table I. Only in some cases DNA is transferred
from a known bacterial donor to known recipient, more often the donor or the recipi-
ent may be unknown indigenous bacteria. Conjugative events were reported in ani-
mal ecosystems, rhizosphere, plant leaves, nonpolluted and polluted water and soil.
3 Minireview 235

Table I
Bacterial conjugation in various ecosystems

Donor Recipient Environment Marker Reference

Animal ecosystem
E. coli S. flexneri Urinary tract? AR-P T a u x e et al., 1989
E. coli S. enteritidis Human intestine A -P
R
B a l i s et al., 1996
Prevotella sp. B. fragilis Human intestine? AR-P N i k o l i c h et al., 1994
Klebsiella sp. Klebsiella sp. Human intestine? AR-P P r o d i n g e r et al., 1996
B. thuringiensis B. thuringiensis Leptidopterous larvae Bt-P J a r r e t et al., 1990
E. cloacae E. cloacae Cutworm insect gut A -P
R
A r m s t r o n g et al., 1990
Rhizosphere and plant leaves
M. loti Mesorhizobium sp. Rhizosphere or soil sym-I S u l l i v a n et al., 1995
S. freedi R. leguminosarum Non-sterile soil sym-P K i n k l e et al., 1991
Rhizosphere P. fluorescens Beet rhizosphere A -P
R
L i l l e y et al., 1997
bacteria
P. syringae P. syringae Pear leaves AR, MR-P S u n d i n et al., 1994
P. putida P. putida Bush bean leaves cat, gfp-P N o r m a n d e r et al, 1998
Nonpolluted water and soil; polluted water and soil and sludges
P. putida P. fluorescens Oligotrophic river AR, cat-P B a l e et al., 1988
epiliton
P. putida Indigenous Phenol cat-P P e t e r s et al., 1997
Pseudomonas sp. contaminated site
Starved Vibrio Vibrio and E. coli Oligotrophic marine AR-P G o o d m a n et al., 1993
and E. coli microcosms
Streptomyces S. lividans TK24 Sterile soil MR-P R a v e l et al., 2000
Acaligenes sp. Indigenous Xenobiotic -polluted cat-Tn F u l t h o r p e and
bacteria freshwater W y n d h a m, 1991; 1992
Pseudomonas sp. P. putida F1 Activated sludge cat-Tn? R a v a t n et al., 1998
B13 microcosms

MR-P, heavy metal resistant plasmid; AR-P, antibiotic resistant plasmid; cat-P, catabolic plasmid; cat-Tn, catabolic
transposon; sym-P, nitrogen fixing symbiotic plasmid ; sym-I, chromosomal, nitrogen fixing symbiotic island;
Bt-P, B. thuringiensis insect toxin plasmid; gfp-P, green fluorescent protein gene; ?, possible or unknown
From D a v i d s o n, 1999, modified

Conjugative transfer of genes coding for antibiotic resistance among the bacteria
in the human and animal intestine tracts has taken place quite often as was proved
by the analysis of plasmid profiles in different gram-positive and gram-negative
bacteria (B a l i s et al., 1996; B r a t o e v a and J o h n, 1994; P r o d i n g e r
et al., 1996; T a u x e et al., 1989). It was shown that, in spite of peristaltic move-
ment in the gut, the intestinal environment displays transfer kinetics different from
those expected of the mixed, liquid culture, but quite similar to those of a biofilm
236 Wolska K.I. 3

(L i c h t et al., 1994). Direct examination of the nucleotide sequence of resistance

genes tetM and tetQ isolated from various bacterial species showed that it is almost the
same, suggesting recent horizontal transfer (N i k o l i c h et al., 1994; S l a y e r s
and S h o e m a k e r, 1996). Conjugative transfer of multiple drug resistance plas-
mids between bacterial pathogens of human, animal and fish origins and strains
from different ecological niche was demonstrated in food-processing environments
(K r u s e and S ø u r m, 1994). Horizontal transfer of multi-drug resistance plas-
mid between coliform bacteria of human and bovine origin in a farm environment
was also reported (O p p r e g a a r d et al., 2001).
Transfer of plasmids was observed in insects, e.g. transfer of plasmid
R388::Tn127 between Enterobacter cloacae strains in digestive tract of cutworm
Peridroma saucia (A r m s t r o n g et al., 1990) and plasmids coding for delta-
endotoxins between different Bacillus thuringiensis strains in lepidopterous larvae
Galleria mellonella and Spodoptera littoralis (J a r r e t and S t e p h e n s o n, 1990).
Transfer of large plasmid (Sym) containing genes for symbiosis, nitrogen fixa-
tion and host specificity from Sinorhizobium freedi to Rhizobium leguminosarum
in the soil was proved (K i n k l e et al., 1991). Later S u l l i v a n et al. (1995)
demonstrated the conjugative transfer of sym-I – chromosomal, nitrogen fixing
symbiosis island from Mesorhizobium loti to other nonsymbiotic Mezorhizobium
species. L i l l e y and B a i l e y (1997) observed the transfer of different large Tra+,
mercury resistance plasmids present in indigenous bacteria in sugar beet phytosphere
to newly introduced Pseudomonas fluorescens labeled with LacZ and KmR-xylE
cassettes. At the same time T r o x l e r et al. (1997) proved chromosome gene trans-
fer after mobilization by RP1 and R64.53 conjugative plasmids in P. fluorescens
grown in wheat rhizosphere ecosystem.
Plasmid transfer was also reported on the leaf surface. The donor strains were
Pseudomonas syringae, Erwinia herbicola and Pseudomonas putida, the recipient
were P. syringae and leaf surface bacteria (L a c y et al., 1984) or various strains of
P. syringae (S u n d i n et al., 1994). Transfer of the TOL plasmid labeled with gfp
gene from P. putida to leaf surface bacteria was followed directly by green fluores-
cence expressed only in the recipient cells after derepression (N o r m a n d e r et al.,
1998). Gene spread on the leaf surface was also efficient when conditions did not
allow the bacterial population to grow, as was shown for intraspecies conjugation of
plasmid RP1 among P. syringae inoculated onto the leaves of common bean
(B j ö r k l o f et al. 2000).
In many cases the transfer of genetic material in uncontaminated soil and water
has been observed. Water and soil ecosystems are oligotrophic so bacteria are in
a state of starvation (v a n Ve e n et al., 1997). Addition of a carbon source greatly
increases the in situ conjugation frequencies (D a v i d s o n, 1999). Because only
a low portion of naturally occurring bacteria can be cultivated, the use of GFP marker
allowing precise monitoring DNA transfer in situ is recommended.
The early works of B a l e et al. (1987; 1988) proved the transfer of HgR plas-
mid between Pseudomonas strains colonizing river stones. Later a vast amount of
data demonstrated the transfer of plasmids (even of Tra– Mob– phenotype) to the
3 Minireview 237

indigenous water and soil bacterial population. Plasmid transfer to indigenous soil
bacteria is highly stimulated by the activity of earth worms which act as a vector for
dispersal of bacteria (D a a n e et al., 1996). Efficient dissemination of catabolic
plasmids among desiccation-tolerant bacteria in soil microcosms was reported
(W e e k e r s et al., 2001). Recently it was shown that gene transfer occurs with
enhanced efficiency in biofilms and induces enhanced stabilization of the biofilm
structure (M o l i n and T o l k e r - N i e l s e n, 2003).
Bacteria have been isolated that are able to degrade most man-made pollutants
and most of the degradative genes are part of the operons localized at wild host-
range, conjugative or mobilizable plasmids (V a n d e M e e r et al., 1992). Bacte-
rial genes for degradation of pollutants have been studied for their potential use in
bioremediation of the contaminated sites. For example, an operon comprising genes
for degradation of chlorobenzoate (cbaAB) was found in a conjugative plasmid of
Alcaligenes isolated in contaminated soil. The cba genes were subsequently found
to be associated with transposon Tn5271, a part of this plasmid. In an environment
simulating a chlorobenzoate-contaminated river the initial host bacteria did not
survive well but the plasmid was found to transfer to a wide range of indigenous
species (F u l t h o r p e and W y d h a m, 1989; 1991; 1992). PCR analysis, using
primers located within the catabolic genes showed that transposon Tn5271-like
sequences were present in diverse bacterial species from a bioremediation system
treating contaminated water from a chemical landfill site (W y n d h a m et al.,
1994). The transfer of degradative plasmids was subsequently demonstrated between
various bacterial species in polluted soil and activated sludge (D i G i o v a n n i
et al., 1999; R a v a t n et al., 1998; H a l l i e r - S o u l i e r et al., 1999).
Studies of a river in Estonia contaminated from a subterranean oil-shale fire demon-
strated the horizontal spread of pheBA, genes encoding catehol 1,2-dioxygenase and
a single-component phenol monooxygenase, a part of plasmid PaW85 originally
present in phenol-degrading P. putida to indigenous bacteria – P. corrugata, P. fragi,
P. stuzeri and P. fluorescent (P e t e r s et al., 1997).

Transformation

DNA transformation, the process whereby naked DNA is transferred to bacteria

plays an important role in DNA exchange between bacteria in natural settings,
however transformation events are not as common as conjugation. Many species of
bacteria are naturally transformable (L o r e n z and W a c k e r n a g e l, 1994), e.g.
Streptococcus pneumoniae becomes competent in the natural course of its life cycle
(L u n s f o r d, 1998), Neisseria gonorrhoeae is always competent (L o r e n z and
W a c k e r n a g e l, 1994) and E. coli can develop natural competence at low tempera-
tures in mineral water containing low concentrations of CaCl2 (B a u r et al., 1996).
DNA is relatively common in all environments and can be protected from degra-
dation up to 1000-fold by adsorption to sand and clay particles (R o m a n o w s k i
et al., 1993). Futhermore, both bacteria and DNA may cluster in biofilms or small
238 Wolska K.I. 3

Table II
Bacterial transformation in the environment

Bacterial host Environment Marker Reference

P. stutzeri Marine water microcosms Chromosomal rif R
S t e w a r t and S i n i g a l l i o,
1991
Pseudomonas sp. Marine water Plasmid multimers P a u l et al., 1991
and sediment microcosm
A. calcoaceticus River epilithon Chromosomal his W i l l i a m s et al., 1996
A. calcoaceticus Soil micrcosms Chromosomal DNA N i e l s e n et al., 1997
+ KmR, GmR
E. coli River and spring water plasmid B a u r et al., 1996

From B u s h m a n, 2002a, modified

particles, thereby greatly increasing the local concentration of both, and therefore
the frequency of transformation (B a u r et al., 1996).
DNA transfer by transformation has been demonstrated in several natural set-
tings (Table II). S t e w a r t and S i n i g a l l i a n o (1991) demonstrated transfor-
mation of Pseudomonas stutzeri to rifampicin resistance by chromosomal DNA in
sterile and nonsterile marine sediments and P a u l et al. (1991) detected the trans-
formation by a broad host-range plasmid in a marine Pseudomonas sp. Studying the
efficiency of P. stutzeri transformation in soil, it was shown that highly different
levels of natural transformation are associated with the genomic subgroups within
a local population (S i k o r s k i et al., 2002). W i l l i a m s and coworkers (1996)
assayed biofilms of Acinetobacter calcoaceticus growing on river stones for incor-
poration of a DNA marked with a his gene and documented the transfer of this
marker. Environmental conditions, such as soil type, soil moisture and nutrient
accessibility affect the efficiency of A. calcoaceticus transformation (N i e l s e n
et al., 1997a; b). Recently genetic transformation of clinical isolates of E. coli under
naturally occurring conditions in oligotrophic, aquatic environments containing
physiologic concentrations of calcium was described. In contrast, transformation
was suppressed in a nitrogen-rich body fluid like urine, a common habitat of
uropathogenic strains (W o e g e r b u n e r et al., 2002).

Transduction

In the process of transduction bacterial genes are transferred to another bacte-

rium by bacteriophage. Bacteriophages, including temperate ones, are very common
in the environment. Examples of gene transfer by phage transduction in the environ-
ment are listed in Table III.
A marine phage T-phiHSIC was shown to facilitate the transduction of a wide
host-range plasmid to marine bacteria from Tampa Bay (J i a n g and P a u l, 1998).
3 Minireview 239

Table III
Transfer of genes by phage transduction under environmental conditions

Bacterial
Bacterial donor Phage Environment Reference
recipient
Marine bacteria Marine bacteria T-phiHSIC Seawater-nutrient mix J i a n g and P a u l, 1998
P. aeruginosa P. aeruginosa F116 Fresh water M o r r i s o n et al., 1978
P. aeruginosa P. aeruginosa F116 Leaf surface S a y e et al., 1987
E. coli E. coli P1 Soil Z e p h and S t o t z k y, 1989

From B u s h m a n, 2002a, modified

Using a mathematical model, the rate of transduction in Tampa Bay Estuary was
estimated at about 1.3×1014 events per year. Moreover, it was shown that 43% of
bacterial population isolated from this bay lysed after treatment with mitomycin C,
yielding phage particles or particle-like structures (B u s h m a n, 2002a).
Transduction of both chromosomal and plasmid markers by P. aeruginosa phage
F116 was seen in fresh-water reservoir (M o r r i s o n et al., 1978) and on the leaf
surface (K i d a m b i et al., 1994). Another phage, UT1, able to promote trans-
duction in P. aeruginosa and as well as in members of the indigenous population of
natural lake-water environments was also isolated (R i p p et al., 1994).
It should be mentioned here that bacteriophages encoding virulence factors
expressed upon lysogenization can also be transferred to a new hosts. This phenom-
enon explain the distribution of pyrogenic exotoxin C determinants among Strepto-
coccus pyogenes (K a p u r et al., 1992), cholera toxin determinants among Vibrio
cholerae (W a l d o r and M e k a l a n o s, 1996) and shiga toxin determinants in
pathogenic E. coli O157::H7 (M u n i e s a and J o f r e, 1998).

Gene transfer between Bacteria and Archaea

The recent focus on the potential for life in extreme environments has generated
a great deal of interest in the Archaea because of their adaptation to extremes of
temperature, salinity and anaerobicity. The discovery of genetic transfer systems for
the Archaea allows the understanding of their genetic mechanisms allowing to study
their unique adaptive strategies (S o w e r s and S c h r e i e r, 1999). In such stud-
ies special focus is on hyperthermophilic Archaea (N o l l and Va r g a s, 1997).
Among the best characterized archaeal representatives all mechanisms of transfer
such as transduction, conjugation and transformation have been discovered (L u o
and W a s s e r f a l l e n, 2001). Conjugative transfer of ING family of conjugative
plasmids from the extremely thermophilic archaeon Sulfolobus islandicus was
proved (S t e d m a n et al., 2000) and transfer of pyr chromosomal marker was
described between the various strains of Sulfolobus acidocaldarius, another
hyperthermophilic archaeon (R e i l l y and G o r g a n, 2001).
240 Wolska K.I. 3

The comparison of genome sequences proved the presence in archaeal and bacte-
rial genomes of a comparable amount of mobile DNA (L a w r e n c e and
O c h m a n, 1998). Moreover there are many bacterial and archaeal genes that have
the strongest similarities to genes from another domain, suggesting lateral transfer.
Aquifex and Thermatoga, both extremeophile bacteria contain 16% and 24% archaeal
genes, respectively; an archaeon Archaeoglobus fulgidis contains a large set of
bacterial genes for fatty acids metabolism (B u s h m a n, 2002b).

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Acta Microbiologica Polonica
2003, Vol. 52, No 3, 245–261

Rearrangements between Differently Replicating DNA Strands

in Asymmetric Bacterial Genomes
DOROTA MACKIEWICZ1, PAWE£ MACKIEWICZ1, MARIA KOWALCZUK1,
MA£GORZATA DUDKIEWICZ1, MIROS£AW R. DUDEK2 and STANIS£AW CEBRAT1*

1 Institute of Genetics and Microbiology, Wroc³aw University,

ul. Przybyszewskiego 63/77, 51-148 Wroc³aw, Poland;
2 Institute of Physics, University of Zielona Góra,

ul. Wojska Polskiego 69, 65-246 Zielona Góra, Poland,

Received 21 May 2003

Abstract

Many bacterial genomes are under asymmetric mutational pressure which introduces composi-
tional asymmetry into DNA molecule resulting in many biases in coding structure of chromo-
somes. One of the processes affected by the asymmetry is translocation changing the position of
the coding sequence on chromosome in respect to the orientation on the leading and lagging
DNA strand. When analysing sets of paralogs in 50 genomes, we found that the number of
observed genes which switched their positions on DNA strand is lowest for genomes with the
highest DNA asymmetry. However, the number of orthologs which changed DNA strand
increases with the phylogenetic distance between the compared genomes. Nevertheless, there is
a fraction of coding sequences that stay on the leading strand in all analysed genomes, whereas
there are no sequences that stay always on the lagging strand. Since sequences diverge very fast
after switching the DNA strand, this bias in mobility of sequences is responsible, in part, for
higher divergence rates among some of coding sequences located on the lagging DNA strand.

K e y w o r d s: DNA asymmetry, divergence, leading, lagging strand, mutation pressure,

rearrangements

Introduction

Rearrangements are common in bacterial genomes (M u s h e g i a n and K o o n i n,

1996; Ta t u s o v et al., 1996; K o l s t o, 1997; Wa t a n a b e et al., 1997; B e l l g a r d
et al., 1999; I t o h et al., 1999; H u g h e s, 2001) but this phenomenon has not been
analysed with respect to leading/lagging strand asymmetry of bacterial chromosomes
which seems to be a characteristic (if not universal) feature of these genomes (e.g.

* Address for correspondence: [email protected]; tel. + 48-71-3756-303; fax: + 48-

71-3252-151
246 Mackiewicz D. et al. 3

L o b r y, 1996; F r e e m a n et al., 1998; G r i g o r i e v, 1998; M c L e a n et al.,

1998; M a c k i e w i c z et al., 1999a; R o c h a et al., 1999; T i l l i e r and C o l l i n s,
2000a; see for review: F r a n c i n o and O c h m a n, 1997; M r a z e k and K a r l i n,
1998; F r a n k and L o b r y, 1999; K o w a l c z u k et al., 2001a). Rearrangements
of genes in bacterial chromosomes follow very specific rules. In very closely related
genomes, many observed rearrangements are symmetric with respect to the origin or
terminus of replication (E i s e n et al., 2000; R e a d et al., 2000; T i l l i e r and
C o l l i n s, 2000b; S u y a m a and B o r k, 2001). T i l l i e r and C o l l i n s (2000b)
claim that such rearrangements are a result of higher frequency of recombination
events at the replication forks which might be recombination hot spots. Another ex-
planation involves the role of selection, and is supported by many genetic and experi-
mental analyses (S c h m i d and R o t h, 1983; M a h a n and R o t h, 1988; 1991;
R e b o l l o et al., 1988; S e g a l l et al., 1988; S e g a l and R o t h, 1989;
F r a n c o i s et al., 1990; L i u and S a n d e r s o n, 1995; 1996; S a n d e r s o n and
L i u, 1998; A l o k a m et al., 2002). The distance from the origin of replication
determines copy number of a gene (dosage effect). Thus, genes should be located in
optimal distances from the origin, according to their required expression level. There
is also a trend to keep the same size of both replichores which ensures the shortest
time of chromosome replication. Furthermore, since inversions of sequences resulting
in switching the position of the coding sequence with respect to leading/lagging role
of DNA strand is connected with a higher mutational pressure (T i l l i e r and
C o l l i n s, 2000c; R o c h a and D a n c h i n, 2001; S z c z e p a n i k et al., 2001),
there could be a higher probability that such a sequence will be eliminated by selec-
tion (M a c k i e w i c z et al., 2001a). (In the terminology, a coding sequence is sup-
posed to be positioned on the leading strand if its sense strand is on the leading DNA
strand, respectively the same for the lagging DNA strand). An inversion of a chromo-
some fragment which encompasses the origin or the terminus of replication does not
change the positions of sequences in respect to the leading/lagging role of the DNA
strand (M a c k i e w i c z et al., 2001b). This could lead to a bias in the observed
rearrangements. Actually, experimental analyses have shown that permissive (viable)
chromosome rearrangements include the origin or terminus of replication (S c h m i d
and R o t h, 1983; M a h a n and R o t h, 1991; A l o k a m et al., 2002). Neverthe-
less, this feature of keeping the same distance from the origin of replication disap-
pears very fast with phylogenetic distance between analysed genomes which leaves
an impression that there is no structural correlation between chromosomes of distant
genomes (E i s e n et al., 2000; T i l l i e r and C o l l i n s, 2000b). On the other
hand, there are some other phenomena, which could introduce some correlation or
structural bias across genomes even at higher phylogenetic distances. Such a phe-
nomenon is a differentiated mutational pressure for coding sequences located on the
leading and the lagging strands (T i l l i e r and C o l l i n s, 2000c; R o c h a and
D a n c h i n, 2001; S z c z e p a n i k et al., 2001). There appears to be some prefer-
ence in the accumulation of translocated coding sequences from the lagging to the
leading strand rather than in the opposite direction (M c I n e r n e y, 1998;
M a c k i e w i c z et al., 2001a). Again, mechanisms of selection are blamed for this
3 Rearrangements in asymmetric bacterial genomes 247

bias rather than bias in frequency of translocations themselves. A significant surplus

of genes on the leading strand has been observed in many genomes (B r e w e r, 1988;
F r a s e r et al., 1995; K u n s t et al., 1997; F r e e m a n et al., 1998; M c L e a n
et al., 1998). Knowing that the divergence rate of coding sequences depends on their
location on the leading/lagging DNA strand (S z c z e p a n i k et al., 2001), we should
expect also a correlation between the function of genes and their position on chromo-
some as well as differentiated frequency of switching the position of genes lying on
the two DNA strands.
One of the main mechanisms of genome evolution is gene duplication, which en-
ables further independent evolution of the structure and function of the two copies
(O h n o, 1970). These copies can be seen in genomes as paralogs – homologous
sequences occurring in the same genome (F i t c h, 1970). It was found that both,
duplication and elimination of paralogs should be ruled by some strict mechanisms,
since the number of paralogs follows a very specific numerical law (H u y n e n and
N i m w e g e n, 1998; S l o n i m s k i et al., 1998; Q i a n et al., 2001). What we
observe is a final result of duplication itself and the paralogs elimination. Duplication
of sequences could be connected with a transfer of a new copy into the other DNA
strand (inversion) or the copy could stay at the same strand. The mutation rate in
sequences after inversion is higher, thus there should be a higher elimination rate of
inverted copies. We have already shown that it is true (M a c k i e w i c z et al., 1999a).
Genes which have switched DNA strand accommodate very quickly to a new muta-
tional pressure and, in respect to their nucleotide composition, become similar to genes
of the new strand (L a f a y et al., 1999; T i l l i e r and C o l l i n s, 2000c; R o c h a
and D a n c h i n, 2001).
In this paper we present the results of analysis of fully sequenced bacterial genomes
which revealed asymmetry in frequency of translocations (viable inversions) of genes
lying on the leading and the lagging DNA strands and we have shown how this affects
the divergence rate of genes classified according to the criteria of their mobility.

Experimental

Materials and Methods

Data for analysis. Prokaryotic genomic sequences and gene annotations have been downloaded
from the Genbank (ftp://www.ncbi.nlm.nih.gov). Boundaries between leading and lagging strands (posi-
tions of origins and termini of replication) and decisions concerning the location of genes on one of
these strands were set on the basis of experimental results or on the basis of the results of DNA walks
describing nucleotide compositional bias of differently replicating DNA strands (M a c k i e w i c z et al.,
1999b, see also: http://smorfland.microb.uni.wroc.pl). The asymmetry of the genomes was measured
by the absolute value of the difference between the GC3 skews of the genes in the leading strand and the
ones in the lagging strand:
)GC3 skew = |(Gd–Cd)/(Gd+Cd) – (Gg–Cg)/(Gg+Cg)|
where: Gd and Cd – numbers of guanine and cytosine in the third codon positions of the leading strand
genes; Gg and Cg – numbers of guanine and cytosine in the third codon positions of the lagging strand
genes. The AT skew and GC skew values proved to be good parameters describing asymmetry of DNA
strands (L o b r y , 1996).
248 Mackiewicz D. et al. 3

Paralogs for 50 genomes (listed in Table I) showing leading/lagging strand asymmetry were ex-
tracted from the TIGR database (http://www.tigr.org). In the analysis only paralogs with minimum 50%
identity were chosen.
Classification of genes to orthologous groups and their amino acid sequences were extracted from
Clusters of Orthologous Groups (COGs) downloaded from ftp://www.ncbi.nlm.nih.gov/pub/COG in Sep-
tember 2001. COGs contain protein sequences which are supposed to have evolved from one ancestral
protein (K o o n i n et al., 1998; T a t u s o v et al., 2001). In the analyses only the best matches for
each ortholog (the closest orthologs) have been chosen.
Analyses of all orthologous sequences have been done on the two sets of bacterial genomes showing
evident compositional asymmetry between leading and lagging strands.
– 7 genomes belonging to (-subdivision of Proteobacteria group compared with each other: E. coli
K12-MG1655 (EcK), E. coli O157:H7 EDL933 (EcE), H. influenzae (Hi), P. multocida (Pm),
P. aeruginosa (Pa), V. cholerae (Vc), X. fastidiosa (Xf);
– 14 genomes compared with E. coli O157:H7 EDL933 (EcE): E. coli K12-MG1655 (EcK),
V. cholerae (Vc), P. multocida (Pm), P. aeruginosa (Pa), X. fastidiosa (Xf), N. meningitidis MC58
(Nm), B. subtilis (Bs), R. prowazekii (Rp), M. tuberculosis H37Rv (Mt), C. jejuni (Cj), T. pallidum
(Tp), H. pylori 26695 (Hp), C. pneumoniae CWL029 (Cp), P. horikoshii (Ph).
Moreover, from the 7 genomes of the (-Proteobacteria group, the 7 sets of 1521 orthologs present in
all the genomes, being the “best hits” for E. coli EDL933 sequences (the closest orthologs), were with-
drawn. Similarly, from the set of 14 genomes compared with E. coli EDL933, the 14 sets of 233 orthologs
present in all the genomes, being the “best hits” for E. coli EDL933 sequences, were extracted.
For each pair of genomes, orthologs and paralogs were classified into three groups according to their
strand location: pairs of sequences lying on the leading strands, pairs of sequences lying on lagging
strands, and pairs of sequences of which one is lying on the leading and the other on the lagging strand.
For each case fractions of the three groups of sequences have been counted.
Phylogenetic analysis. The amino acid sequences of each COG were aligned by the CLUSTAL
W 1.8 v. program (T h o m p s o n et al., 1994). Pairwise evolutionary distances (expressed by the mean
number of amino acid substitutions per site) between sequences of each COG were calculated using the
WAG model of amino acid substitution (W h e l a n and G o l d m a n, 2001) as implemented in the
TREE-PUZZLE program version 5.0 (S c h m i d t et al., 2002). The analyses of divergence of the three
groups of orthologs were shown for the sets of 1521 orthologs present in all 7 (-Proteobacteria genomes.
For each of the three groups of orthologs a mean value of the evolutionary distances was calculated.
Nonparametric analyses by Mann-Whitney U, Kolmogorov-Smirnov and ANOVA Kruskal-Wallis tests
(S o k a l and R o h l f, 1995) were carried out to assess statistical significance of differences between
these groups.
Evolutionary distances between 16S rRNA sequences (measured by the number of substitutions per
site) were calculated by the MEGA 2.1 program (K u m a r et al., 1993) assuming Tamura-Nei model of
nucleotide substitutions (T a m u r a and N e i, 1993).

Results and Discussion

In highly asymmetric genomes, the mutational pressure after inversion should be

relatively higher than for genomes with low asymmetry – there are stronger differ-
ences in substitution rates for the leading and lagging DNA strands in the asymmetric
genomes (K o w a l c z u k et al., 2001b; R o c h a and D a n c h i n, 2001). Thus,
we have anticipated and found a negative correlation between the chromosome asym-
metry and the frequency of occurring paralogs in the trans-positions in the genome
(one paralog on the leading strand, the other one on the lagging strand – we call these
sequences “trans-paralogs”). In Table I we show data for each analysed genome and
3 Rearrangements in asymmetric bacterial genomes 249

in Fig. 1 we show the relation between the fraction of trans-paralogs in the genome
and the asymmetry of chromosomes measured by )GC3 skew. The observed negative
correlation (Spearman correlation coefficient, r = –0.715) is statistically significant
with high confidence (p = 5.6×10–9). There are two possible explanations for the ob-
served negative correlation. One, assuming a higher mutation rate and in consequence
higher elimination rate of gene copies translocated to the other DNA strand in highly
asymmetric genomes. The second, to us less plausible, refers to the influence of fre-
quency of rearrangements on the maintenance of chromosomal asymmetry. If a global
frequency of rearrangements in a genome is low, it does not disturb chromosomal
asymmetry established by the mutational pressure. On the contrary, high frequency of
rearrangements should diminish this asymmetry.
We have performed a pairwise analysis of orthologs found in compared genomes
belonging to (-Proteobacteria. For each pair of genomes, the orthologs were divided
into three groups: i/ pairs of orthologs which are in both compared genomes on the
leading strand, ii/ pairs of orthologs which are in both genomes on the lagging strand
and iii/ pairs of orthologs of which one is located on the leading and the second on the

60
y = -73.61x + 47.34
r = -0.715
50 p = 5.6E-09
fraction of trans-paralogs

40

30

20

10

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
∆ GC3 skew

Fig. 1. Relation between the fraction of trans-paralogs (one paralog on the

leading strand, the other one on the lagging strand) in 50 analysed genomes
and the asymmetry of chromosomes measured by )GC3 skew.
Spearman correlation coefficient (r) and its statistical significance (p) are shown.
250 Mackiewicz D. et al. 3

Table I
Number of all paralogs, the fraction of trans-paralogs and DGC3 skew for 50 analysed genomes
number fraction
genome )GC3 skew
of all paralogs of trans-paralogs
Agrobacterium tumefaciens C58 Cereon 1096 46.8 0.08
Agrobacterium tumefaciens C58 Uwash 1117 46.6 0.08
Bacillus halodurans C-125 2421 39.2 0.17
Bacillus subtilis 168 558 31.4 0.15
Borrelia burgdorferi B31 11 9.1 0.62
Brucella melitensis 16M 314 38.2 0.13
Campylobacter jejuni NCTC 11168 79 30.4 0.41
Caulobacter crescentus CB15 511 44.2 0.05
Chlamydia muridarum Nigg 19 0.0 0.49
Chlamydia pneumoniae AR39 111 6.3 0.29
Chlamydia pneumoniae CWL029 112 6.3 0.30
Chlamydia pneumoniae J138 100 7.0 0.29
Chlamydia trachomatis serovar D 6 16.7 0.45
Clostridium perfringens 13 217 29.0 0.41
Deinococcus radiodurans R1 282 46.5 0.01
Escherichia coli O157:H7 EDL933 3604 26.9 0.10
Escherichia coli K12-MG1655 919 47.0 0.09
Escherichia coli VT2-Sakai 4020 24.1 0.10
Haemophilus influenzae KW20 73 15.1 0.16
Helicobacter pylori 26695 198 51.5 0.12
Helicobacter pylori J99 109 41.3 0.12
Lactococcus lactis IL1403 811 42.9 0.22
Listeria innocua CLIP 11262 349 18.9 0.18
Listeria monocytogenes EGD-e 255 31.4 0.20
Mesorhizobium loti MAFF303099 1414 42.9 0.04
Mycobacterium leprae TN 121 47.1 0.13
Mycobacterium tuberculosis CDC1551 2417 43.7 0.09
Mycobacterium tuberculosis H37Rv 2279 45.1 0.08
Neisseria meningitidis MC58 595 35.0 0.20
Neisseria meningitidis Z2491 874 42.3 0.22
Pasteurella multocida PM70 86 23.3 0.23
Pseudomonas aeruginosa PAO1 786 44.8 0.11
Pyrococcus abyssi GE5 118 48.3 0.04
Pyrococcus horikoshii shinkaj OT3 147 42.2 0.07
Ralstonia solanacearum GMI1000 784 50.8 0.08
Rickettsia conorii Malish 7 478 40.4 0.21
3 Rearrangements in asymmetric bacterial genomes 251

Table I continued

number fraction
genome )GC3 skew
of all paralogs of trans-paralogs
Salmonella enterica Typhi CT18 679 35.3 0.13
Salmonella typhimurium LT2 SGSC1412 1280 41.0 0.12
Sinorhizobium meliloti 1021 460 48.0 0.05
Staphylococcus aureus Mu50 228 16.2 0.28
Staphylococcus aureus N315 482 15.1 0.29
Streptococcus pneumoniae R6 759 28.5 0.30
Streptococcus pneumoniae TIGR4 479 38.2 0.31
Streptococcus pyogenes SF370 M1 130 26.2 0.26
Thermoplasma acidophilum DSM 1728 41 39.0 0.03
Thermotoga maritima MSB8 216 49.5 0.06
Treponema pallidum Nichols 72 43.1 0.34
Vibrio cholerae El Tor N16961 868 16.0 0.17
Xylella fastidiosa 9a5c 779 23.7 0.33
Yersinia pestis CO92 8551 49.1 0.09

The set of paralogs (with minimum 50 % identity) was extracted from TIGR database.

lagging strand. If we assume that there is no bias in the frequency of inversions of

genes located on the leading and on the lagging DNA strands, we should expect that
the fractions of orthologs staying at the same strand in both genomes of the compared
pair would decrease with the phylogenetic distance between genomes but the decrease
should be proportional to the initial values on the two strands. The results of analyses
do not follow these expected rules.
For each pair of compared genomes we have plotted (Fig. 2) the fractions of the
three groups of orthologs against the evolutionary distance measured by divergence
of 16S rRNA genes between the two compared genomes. The fraction of orthologs
lying on the same strand decreases with evolutionary distance while fraction of
orthologs which have switched their strands increases rapidly with divergence and
become saturated for long evolutionary distances. The same results we have obtained
for similar analysis when we compared the E. coli EDL933 genome with 14 other
genomes belonging to different taxonomic groups (Fig. 3). Even at a short distance
(up 0.22 of divergence of 16S rRNA), the total fraction of sequences which switched
their strand reaches almost 50%. But there is a very biased input of sequences
from the leading and the lagging DNA strands into this fraction. While the fraction
of sequences which stay at the leading strands in both compared genomes drops
to about 70% of the initial value in the most distant pair, the relative numbers for
the lagging strand are up to 40%. These results suggest that the sequences lying
on the lagging strand are much more prone to inversions than the sequences lying on
the leading strand.
252 Mackiewicz D. et al. 3

A 60

50
fraction of orthologs

40

30

20

10

0
0.0 0.1 0.2
phylogenetic distance

B 60

50
fraction of orthologs

40

30

20

10

0
0.0 0.1 0.2
phylogenetic distance

C 60

50
fraction of orthologs

40

30

20 Fig. 2. Relation between the fractions of

orthologs and the phylogenetic distance
measured by 16S rRNA performed for three
10
groups of orthologs: lying on the leading
strand (A), lying on the lagging strand (B)
0 and which changed DNA strand (C).
0.0 0.1 0.2 Data obtained from pairwise comparison of
phylogenetic distance 7 genomes belonging to g- Proteobacteria.
3 Rearrangements in asymmetric bacterial genomes 253

A 60

50

fraction of orthologs
40

30

20

10

0
0 0.1 0.2 0.3 0.4 0.5
phylogenetic distance

B 60

50
fraction of orthologs

40

30

20

10

0
0 0.1 0.2 0.3 0.4 0.5
phylogenetic distance

C 60

50
fraction of orthologs

40
Fig. 3. Relation between the fractions
of orthologs and the phylogenetic dis- 30
tance measured by 16S rRNA per-
formed for three groups of orthologs: 20
lying on the leading strand (A), lying
on the lagging strand (B) and which
10
changed DNA strand (C).
Data obtained from comparison of the
E. coli EDL933 genome with 14 other ge- 0
nomes belonging to different taxonomic 0 0.1 0.2 0.3 0.4 0.5
groups. phylogenetic distance
254 Mackiewicz D. et al. 3

This observation implies also that there are some sequences which “are used to”
staying on the leading DNA strand and they have lower probability of being inverted
than sequences which “are used to” staying on the lagging strand. As a consequence,
the set of coding sequences found on the leading strand should be not uniform. It
should consist of a set of sequences which permanently or preferentially stay on the
leading strand and a set of mobile sequences which are only transiently transferred
from the lagging strand. To test this hypothesis we analysed the sets of 233 orthologs
represented in all 15 genomes. In the first step we compared the most closely related
genomes in the analysed set – two E. coli strains – and we counted the fractions of
orthologs which stayed at the same DNA strands (leading or lagging) and the fraction
of orthologs which switched their strands. In the next step we added to the compari-
son the third genome (the closest to the E. coli EDL933 genome according to the 16S
rRNA phylogenetic distance) and again counted sequences which stayed at the same
DNA strand in all the three genomes and sequences which switched their strand at
least in one genome and so on, adding new, more distant genome to the analysed
group. In Fig. 4, in the diagram, we have presented the results of analysis; values on
y-axis correspond to the fraction of sequences of a given group of orthologs, while at
the bottom the name of a new genome added to the comparison is shown. The fraction

100

80
fraction of orthologs

60

the leading strand orthologs

40 the lagging strand orthologs

orthologs which changed strand

20

0
t

p
f

h
a

p
m

s
cK

p
j
m

+M
+X

+C

+H
+B
+V

+P
+P

+R

+C
+T
+N
+P
+E

Fig. 4. The fractions of three groups of orthologs counted for the comparisons of E. coli EDL933
with successively added genomes to the comparison.
The group of the leading strand orthologs contains sequences which stay on the leading strand in all analysed
genomes in a given comparison. Analogously for the lagging strand orthologs. The third group of orthologs
includes sequences which switched their strand at least in one genome in a given comparison. Data were obtained
for the sets 233 orthologs present in all 15 genomes. For genomes name abbreviations see Materials and Methods.
3 Rearrangements in asymmetric bacterial genomes 255

Table II
Orthologs found in all 15 analysed genomes
on the leading strand

COG number description

COG0051 Ribosomal protein S10
COG0087 Ribosomal protein L3
COG0088 Ribosomal protein L4
COG0090 Ribosomal protein L2
COG0091 Ribosomal protein L22
COG0092 Ribosomal protein S3
COG0093 Ribosomal protein L14
COG0094 Ribosomal protein L5
COG0096 Ribosomal protein S8
COG0097 Ribosomal protein L6
COG0098 Ribosomal protein S5
COG0185 Ribosomal protein S19
COG0186 Ribosomal protein S17
COG0197 Ribosomal protein L16/L10E
COG0198 Ribosomal protein L24
COG0200 Ribosomal protein L15
COG0256 Ribosomal protein L18

of sequences which stay in all analysed genomes on the lagging strand drops very fast
and after adding the eighth genome it reaches zero, which means that there are no
orthologous coding sequences located on the lagging strands in all compared genomes.
For this group of compared genomes, there are still some orthologs which stay on the
leading strand in all the genomes and this fraction seems to approximate asymptoti-
cally about 7% of all compared coding sequences, even after adding the most distant
genome belonging to Archaea. These orthologs code for ribosomal proteins commonly
considered highly conserved (Table II). The position of these genes on the leading
strand seems to be conserved even across the two kingdoms (Bacteria and Archaea).
It was observed that their operons are well preserved even in divergent species
(W a t a n a b e et al., 1997; I t o h et al., 1999; N i k o l a i c h i k and D o n a c h i e,
2000; T a m a m e s, 2001). Moreover, it was found that ribosomal genes are prefer-
entially located in many genomes on the leading strand (M c L e a n et al. 1998) prob-
ably (what is important for highly expressed genes) to avoid head-on collisions
between replication and transcription complexes (B r e w e r, 1988; F r e n c h, 1992).
In the next studies we have analysed the divergence measured by the mean num-
ber of amino acid substitutions per site in groups of sequences classified according to
their mobility between differently replicating DNA strands. Analyses were performed
with the sets of 1521 orthologs present in all 7 genomes belonging to (-Proteobacteria.
We compared the E. coli EDL933 genome with six other genomes.
256 Mackiewicz D. et al. 3

3
orthologs in all genomes on the leading strand
orthologs in all genomes on the lagging strand
2.5 orthologs on the leading strand in a given pair
orthologs on the lagging strand in a given pair
orthologs on different DNA strands in a given pair
2

1.5

0.5

0
EcE-EcK EcE-Vc EcE-Pm EcE-Hi EcE-Pa EcE-Xf

Fig. 5. The divergence measured by the mean number of amino acid substitutions per site according to WAG
model (Whelan and Goldman, 2001) in five groups of sequences classified according to their mobility
between differently replicating DNA strands for comparisons of E. coli EDL933 with other genomes.
Analyses were performed with the sets of 1521 orthologs present in all 7 genomes belonging
to (-Proteobacteria. For genomes name abbreviations see Materials and Methods.

We divided all orthologs into five sets: 1 – genes staying in all analysed genomes
on the leading strand, 2 – genes staying in all analysed genomes on the lagging strand,
3 – genes which are located in E. coli EDL933 and in the compared genome on
the leading strand but can be found in at least one of the other genomes of
(-Proteobacteria on the lagging strand, 4 – genes which are located in E. coli EDL933
and in the compared genome on the lagging strand but can be found in at least one
of the other genomes on the leading strand and, 5 – sequences which are located on
different DNA strands in the compared genomes. The divergence values between
genes of the E. coli EDL933 genome and other genomes of (-Proteobacteria are
shown in Fig. 5. We have found that there are statistically significant differences
in the relative divergence between genes classified according to their position and
mobility. The differences between set 1 and set 5 are statistically significant (with
p < 0.01) for all comparisons. It is clear that the divergence of the orthologs which
switched strand (set 5) is especially high for the closest genomes, which was already
reported (T i l l i e r and C o l l i n s, 2000c; S z c z e p a n i k et al., 2001; R o c h a
and D a n c h i n, 2001) and decreases for pairs of distant genomes. Differences in
divergence between set 5 and all other sets are statistically significant (with p <0.01)
for pairs: EcE-EcK, EcE-Vc and EcE-Pa.
In all compared pairs of genomes the lowest divergence is observed for the
orthologs which permanently stay at the leading strand and do not change their strand
3 Rearrangements in asymmetric bacterial genomes 257

even at long evolutionary distances (set 1). If we eliminate this set of conserved genes
from the set of all orthologs found on the leading strand (receiving set 3), the rest still
seems to be less prone to accumulate substitutions than the genes from the lagging
strand. However, we have found only one statistically significant difference in diver-
gence (5.6% of all comparisons) when we compared sets 2, 3 and 4 with each other
for all pairs of genomes. Furthermore, the divergence in these three sets is signifi-
cantly different (with p < 0.01) when analysed by the ANOVA Kruskal-Wallis test
only for one pair EcE-Pa. It indicates that these three sets form rather uniform group.

Conclusions

The observed rearrangements in bacterial chromosomes are not random. Muta-

tional pressure, responsible for the observed asymmetry in DNA composition, affects
especially the copies of genes translocated to other DNA strand. According to
the mobility (frequency of translocations between leading and lagging strand) it is
possible to classify genes into two groups: highly conserved genes permanently or
preferentially lying on the leading strand and genes switching their position between
the leading and lagging DNA strands.

Acknowledgments. The work was supported by the grant number 1016/S/IMi/03. M.K. was sup-
ported by Foundation for Polish Science.

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Acta Microbiologica Polonica
2003, Vol. 52, No 3, 261–269

Synthesis of Siderophores by Strains of Staphylococcus cohnii

Isolated from Various Environments
JADWIGA SZARAPIÑSKA-KWASZEWSKA and £UKASZ I. FARKAS

Department of Pharmaceutical Microbiology, Medical University of £ódŸ,

Pomorska 137, 90-235 £ódŸ, Poland

Received in revised form 2 June 2003

Abstract

Siderophore activity as the feature of microorganisms enabling colonization of human body and
the survival in inanimate environment was investigated in 108 strains of Staphylococcus cohnii:
S. cohnii ssp. cohnii (50 strains) and S. cohnii ssp. urealyticus (58 strains). Strains were isolated
from people, hospital and non-hospital environment. Highest siderophore activity was noted in
strains S. cohnii ssp. urealyticus particularly from the inanimate environments origin. In 86%
analyzed strains siderophores of hydroxamate class were detected. Larger amounts of these com-
pounds were synthesized in strains S. cohnii ssp. urealyticus. Strains belonging to both subspe-
cies from human origin showed lower activity of siderophores (total pool) and did not produce
hydroxamate class chelators or produced very small amounts of these compounds.

K e y w o r d s: siderophores, Staphylococcus cohnii, colonization of human body

Introduction

Staphylococcus cohnii species were described by S c h l e i f e r and K l o o s

(1975) in 1975. In IX edition of Bergey’s key (H o l t et al., 1994) two subspecies
were differentiated: S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Although
strains of Staphylococcus cohnii in comparison with other coagulase-negative staphy-
lococci were rarely noted as a cause of infection (R ó ¿ a l s k a et al., 1995),
S z e w c z y k et al. (2000) proved that S. cohnii recovered from the hospital envi-
ronment were predominant species of staphylococci (about 50%). Many isolated
Staphylococcus cohnii strains were characterized by resistance to numerous anti-
biotics, and nearly 90% of them showed resistance to methicillin (S z e w c z y k and
R ó ¿ a l s k a, 2000).
One of the conditions limitating survival or reproduction of microorganisms in
a specified environment is their ability to assimilate iron. In case where there is low
availability of this element, bacteria use siderophore systems (G u e r i n o t, 1994;
M i k u c k i and L i s i e c k i, 1998). Production of siderophores with high activity
262 Szarpiñska-Kwaszewska J., Farkas £.I. 3

could give preferences in iron competition, in a certain ecosystem, to strains which

synthesize them and conduct human organism, as well as environment colonization
(K u r e k and J a r o s z u k, 1993; M i k u c k i and L i s i e c k i, 1998).
Investigation of siderophore activity in numerous of Staphylococcus cohnii strains
isolated from various environments (hospital and non-hospital) may allow to deter-
mine whether it could play any role in bacterial survival in a specified environment.
It may also help in the evaluation of the possible danger of multiresistant S. cohnii to
the patients due to the common presence of this bacteria in hospital environment.

Experimental

Materials and Methods

Bacterial strains. The study comprised 108 strains of Staphylococcus cohnii (50 strains of S. cohnii
ssp. cohnii and 58 of S. cohnii ssp. urealyticus) from the collection of the Department of Pharmaceutical
Microbiology, Medical University of £ódŸ. S. cohnii strains were isolated in 1997–1999 in the Intensive
Care Unit of the clinical hospital from material samples (skin swab) taken from patients and personnel
– 40 strains, and hospital environment (swab from floors, walls and equipment surfaces) – 26 strains,
as well as material samples (swab from floors, walls and furniture surfaces in private houses) in non-
hospital environment – 42 strains. These strains were isolated and identified by S z e w c z y k and
R ó ¿ a l s k a (2000). Strains were stored at –70°C in 50% glycerol broth medium and cultivated on agar
plates suplemented with sheep blood for the experiment use.
Media. The NB with dipyridyl medium (iron poor medium with iron chelator added used for iron
starvation): Nutrient Broth (Difco) 0.8g, Agar No1 (Oxoid) 1.5g, ","’-bipyridyl (Sigma) 3.04 mg and
deionized water 100 mL. Nutrient Broth solutions and Agar No1 were combined and after sterilization
(20 min at 121°C) a sterile solution of ","’-bipyridyl was added. The BM-3 medium used for determin-
ing siderophore production was: stock solution of trace elements according to Lankford et al. (1966)
supplemented with 1% glucose, 0,2% casamino acids (Difco) and 0,05% yeast extract (Difco). The
solutions were sterilized separately (20 min at 121°C) and combined in appropriate proportions.
Siderophore production. For enhancement of siderophore production and reduction of intracellular
iron reserves strains were inoculated on NB medium with dipyridyl and incubated for 24 hours at
a temperature of 37°C (double passage). Next a small portion of bacterial mass (material from some
colonies was taken and inoculated in BM-3 medium),was incubated for 48 hours at 37°C with constant
shaking (75 strokes/min) and centrifuged (10000 rpm for 15 min) and after siderophores were deter-
mined in the supernatant culture.
Siderophore determination. Total siderophore chelating activity was tested by the Schwyn and
Neilands method with standard reagent containing Chrome Azurol S (CAS) (Sigma) (S c h w y n and
N e i l a n d s, 1987). Siderophore activity was expressed as :moles of desferrioxamine mesylate
(Desferal, Ciba-Geigy) per mL of culture supernatant; the standard curve for Desferal was used.
Hydroxamate class siderophores production was estimated by the periodic acid method (H o l z b e r g
and A r t i s, 1983) and also expressed as :moles of Desferal per mL of supernatant (standard curve for
Desferal). Phenol-catechol class siderophores production was assessed by the colorimetric assay of
A r n o w (1937) and siderophore activity was expressed as :moles of 2.3-dihydroxybenzoic acid
– DHBA (Fluka) per mL of culture supernatant (standard curve for DHBA). Obtained results were
recounted to mg bacterial protein contained in 1 mL of culture. Protein concentration was estimated
by Ehresmann et al. method (1973) in cell extract obtained after lysis (with 1 M NaOH) of cell sedimen-
tation after culture centrifuging. The standard curve for bovine albumin (Sigma) was used.
All solutions and media were prepared with deionized water. Glassware was washed with deionized
water, kept for 24 hours in 2 N hydrochloric acid (to remove remains of iron) and rinsed three times with
deionized water.
3 Synthesis of siderophores by S. cohnii 263

Results and Discussion

Three classes of siderophores produced by staphylococci were noted: hydroxamate

class siderophores (L i s i e c k i et al., 1994), catechol class siderophores (L i s i e c k i
et al., 1994) and polycarboxylate (complexone type) siderophores – staphyloferrin A
and staphyloferrin B (D r e c h s e l et al., 1993; H a a g et al., 1994; K o n e t s c h n y -
R a p p et al., 1990; M e i w e s et al., 1990). The ability to utilise ketoacids as
iron-carriers was also presented by H e u c k et al., (1995). A single strain is able
to synthesize different chelators and total siderophore strain activity is the sum of
each chelator activity.
Siderophores of Staphylococcus cohnii species have not been well examined.
L i s i e c k i et al. (1994) studied the presence of hydroxamate and catecholate
siderophores in 180 staphylococci strains belonging to 26 species. Among them were
15 strains of Staphylococcus cohnii but no subspecies were assigned. All of them pro-
duced hydroxamate siderophores and one produced additionally catechol siderophore
(L i s i e c k i et al., 1994). These Staphylococcus cohnii strains were distinguished
from others staphylococci species by high activity of synthesized iron chelators.
Our investigation involved 108 strains: 50 strains of S. cohnii ssp. cohnii and 58 of
S. cohnii ssp. urealyticus. The number of set strains belonging to both subspecies varied
according to the source of their isolation. S. cohnii ssp. cohnii strains were more
frequently isolated in hospital (from patients, medical staff and from the hospital envi-
ronment), than in non-hospital environment. S. cohnii ssp. urealyticus strains occurred
more frequently in home environment and rarely were isolated from examined patients
and personnel (S z e w c z y k et al., 2000, S z e w c z y k and R ó ¿ a l s k a, 2000).
Siderophore activity of investigated strains was various – from very low, adequate
to 0,85 :mole desferrioxamine mesylate (Desferal) per mg of protein, to high
– 138.1 :mole desferrioxamine mesylate (Desferal) per mg of protein, but nearly half
of investigated strains in each subspecies were located in group ranged between
20–40 :mole Desferal per mg of protein (Table I). Figure 1 shows the distribution
percentage of strains Staphylococcus cohnii ssp. cohnii and Staphylococcus cohnii
ssp. urealyticus in groups of total siderophore activity expressed in :mole of Desferal
per mg of protein. In distribution analysis it was observed that most of Staphylococ-
cus cohnii ssp. cohnii strains were present in the group with the lowest activity and in
the group with highest activity were only Staphylococcus cohnii ssp. urealyticus
strains. Staphylococcus cohnii ssp. cohnii strains with least active siderophores, were
mainly isolated from humans (12 strains). Activity of the next twelve strains from that
same origin was a little higher, where six of them had an activity less than 30 :mole
Desferal per mg of protein. Only twelve Staphylococcus cohnii ssp. urealyticus strains
investigated in this study were isolated from humans. Eight of them had also low
activity, about 30 :mole Desferal per mg of protein.
Strains isolated from the environment, regardless of their subspecies status had
more active siderophores in comparison to those strains isolated from human. Although
the groups of studied strains were different in number, the proportional participa-
tion of strains in each group with specified siderophore activity showed with certain
264 Szarpiñska-Kwaszewska J., Farkas £.I. 3

Table I
Distribution of Staphylococcus cohnii strains isolated from various environments
(percentage of investigated environment) in groups of total siderophore activity

Siderophore Staphylococcus cohnii ssp. cohnii Staphylococcus cohnii ssp. urealyticus

activity
Source of isolation and number of strains Source of isolation and number of strains
expressed
in µmoles Environment Environment
of Desferal Human Non- Total Human Non- Total
Hospital Hospital
per mg 28=100% hospital 50 =100% 12=100% hospital 58=100%
8=100% 18 =100%
of protein 14 =100% 28=100%
< 20 42.85 12.5 14.3 30 0 11.1 7.1 6.9
20 – 40 42.85 50.0 50.0 46 66.7 38.9 39.3 44.82
40 – 60 14.3 37.5 35.7 24 25.0 22.2 42.8 32.76
60 < 0 0 0 0 8.3 27.8 10.8 15.52

regularity. Strains isolated from human were obviously different from strains isolated
from the environment but no distinct differences between strains isolated from hospi-
tal and non-hospital environment (Table I), especially in Staphylococcus cohnii
ssp. cohnii strains were observed. Staphylococcus cohnii ssp. urealyticus strains
were rarely isolated from human; most of them were from the environment and were
characterized by high siderophore activity (Table I, Figure 1). The differences
in siderophore activity in strains relating to “human” and “environment” could be
explained by lower iron accessibility in the environment, which leads to intensive
siderophore synthesis. There is a high supply of iron in human body, even though it is
binded with proteins, bacteria are capable to obtain it from such sources (M i k u c k i
and L i s i e c k i, 1998, W a l d o n et al., 2002).
Besides total siderophore activity determination, level of chelators synthesis in
two main siderophore classes: hydroxamate and catecholate was assayed. Catecholate
siderophores were not detected in supernatants culture of all investigated strains. That
result was not surprising, because rare strains of staphylococci are able to synthetize
catecholate siderophores. L i s i e c k i et al. (1994) found catecholate siderophores
only in 7,8% of 180 studied strains of staphylococci belonging to 26 species.
Hydroxamate synthesis is common among staphylococci but some strains also could
not produce these compounds (L i s i e c k i et al., 1994).
In our study hydroxamate siderophores were not detected in 15 (13,9%) of
108 analyzed Staphylococcus cohnii strains. Twelve of them belonged to Staphylo-
coccus cohnii ssp. cohnii (ten of them were isolated from human) and only three
belonged to Staphylococcus cohnii ssp. urealyticus (all isolated from non-hospital
environment). Staphylococcus cohnii ssp. urealyticus strains produced hydroxamate
in significant amount in contrast to Staphylococcus cohnii ssp. cohnii (Table II,
Figure 2). That regularity was also observed in “environmental” strains of Staphylo-
coccus cohnii ssp. urealyticus comparing to strains of other subspecies.
Analyzing the obtained results concerning the total siderophore activity in super-
natants culture and hydroxamates presence we observed that both the values were not
3 Synthesis of siderophores by S. cohnii 265

100,0%

80,0%
S. cohnii ssp. cohnii
60,0%

40,0% S. cohnii ssp.

urealyticus
20,0%

0,0%
<20 20-40 40-60 60<

100,0%

80,0%
S. cohnii ssp. cohnii
60,0%

40,0% S. cohnii ssp.

urealyticus
20,0%

0,0%
<20 20-40 40-60 60<

100,0%

80,0%
S. cohnii ssp. cohnii
60,0%

40,0% S. cohnii ssp.

urealyticus
20,0%

0,0%
<20 20-40 40-60 60<

100,0%

80,0%
S. cohnii ssp. cohnii
60,0%

40,0% S. cohnii ssp.

urealyticus
20,0%

0,0%
<20 20-40 40-60 60<

Fig. 1. Percentage of strains Staphylococcus cohnii ssp. cohnii and Staphylococcus cohnii ssp. urealyticus in
groups of total siderophore activity expressed in :moles of Desferal per mg of protein.
A – all studied strains in subspecies, B – strains isolated from human, C – strains from hospital environment,
D – strains from non-hospital environment.
266 Szarpiñska-Kwaszewska J., Farkas £.I. 3

100,0%

80,0%
S. cohnii ssp. cohnii
60,0%

40,0% S. cohnii ssp.

urealyticus
20,0%

0,0%
0 0-0,5 0,5-1,0 1,0-1,5 1,5<

100,0%

80,0%
S. cohnii ssp. cohnii
60,0%

40,0% S. cohnii ssp.

urealyticus
20,0%

0,0%
0 0-0,5 0,5-1,0 1,0-1,5 1,5<

100,0%

80,0%
S. cohnii ssp. cohnii
60,0%

40,0% S. cohnii ssp.

urealyticus
20,0%

0,0%
0 0-0,5 0,5-1,0 1,0-1,5 1,5<

100,0%

80,0%
S. cohnii ssp. cohnii
60,0%

40,0% S. cohnii ssp.

urealyticus
20,0%

0,0%
0 0-0,5 0,5-1,0 1,0-1,5 1,5<

Fig. 2. Percentage of strains Staphylococcus cohnii ssp. cohnii and Staphylococcus cohnii ssp. urealyticus in
groups of hydroxamate siderophore class activity expressed in :moles of Desferal per mg of protein.
A – all studied strains in subspecies, B – strains isolated from human, C – strains from hospital environment,
D – strains from non-hospital environment.
3 Synthesis of siderophores by S. cohnii 267

Table II
Distribution of Staphylococcus cohnii strains isolated from various environment
(percentage of investigated environment) in groups of hydroxamate siderophore class activity

Hydroxamate Staphylococcus cohnii ssp. cohnii Staphylococcus cohnii ssp. urealyticus

siderophore
Source of isolation and number of strains Source of isolation and number of strains
class activity
expressed in Environment Environment
µmoles of Human Non- Total Human Non- Total
Hospital Hospital
Desferal per 28=100% hospital 50 =100% 12=100% hospital 58=100%
8=100% 18 =100%
mg of protein 14 =100% 28=100%
0 35.7 25.0 0 24.0 0 0 10.7 5.2
0 – 0.5 17.85 0 28.6 18.0 0 22.2 7.1 10.3
0.5 – 1.0 17.85 50.0 57.1 34.0 16.7 38.93 39.3 34.5
1.0 – 1.5 25.0 25.0 14.3 22.0 66.6 33.3 35.7 41.4
1.5 < 3.6 0 0 2.0 16.7 5.6 7.2 8.6

always correlated. The occurence of siderophore activity and the simultaneous lack of
hydroxamates and catecholates compounds could be due to the presence of other
siderophores classes, e.g. staphyloferrin A and staphyloferrin B produced commonly
by staphylococci (D r e c h s e l et al., 1993; H a a g et al., 1994; K o n e t s c h n y -
R a p p et al., 1990) or ketoacids (H e u c k et al., 1995) or also not yet described
chelators. H e u c k et al. (1995) examined iron assimilation by coagulase – negative
staphylococci by ketoacids mediation. They noted a great amount of pyruvic and
"-ketoglutaric acid in Staphylococcus cohnii cultures growing in poor iron environ-
ment. W a l d o n et al. (2002) showed that S. cohnii strains from the same collection
were able to utilize numerous oxo-acids in iron assimilating system.
Strains, in which no hydroxamates have been detected, were characterized by low
total siderophore activity (except two). That proves the significant participation of
hydroxamates in all Staphylococcus cohnii siderophores, though it was not always the
same in all strains. Nor is it known if all hydroxamates issued from both subspecies
have the same structure. They could be different compounds with different chelating
activity. That may explain the differences between hydroxamate amounts and total
siderophore activity in some strains.
High correlation level between high siderophore activity and high hydroxamate
production was noted in strains of Staphylococcus cohnii ssp. urealyticus, especially
in those isolated from the environment; in a group of 19 strains which had over
1 :mole hydroxamates per mg of protein in supernatants cultures, 13 strains were
with high and 4 strains with the highest total siderophore activity, while in a group of
six strains which had under 0.5 :mole hydroxamates per mg of protein no strain with
high total siderophore activity was observed.
Most of Staphylococcus cohnii ssp. cohnii strains, which mainly synthesized
hydroxamates was characterized with low total siderophore activity. We may presume
that siderophore activity of these Staphylococcus cohnii ssp. cohnii strains depends
268 Szarpiñska-Kwaszewska J., Farkas £.I. 3

mainly on compounds from hydroxamate class. Staphylococcus cohnii ssp. urealyticus

strains isolated from human behaved similarly – with high amount of hydroxamates
synthesis, their total siderophore activity was not high either.
On the basis of the obtained results we conclude, that S. cohnii ssp. urealyticus
strains produce siderophores with higher chelating activity than strains of S. cohnii
ssp. cohnii. Strains S. cohnii of both subspecies isolated from hospital and non-
hospital environments present higher total activity of siderophores and synthesize
larger amounts of hydroxamate class chelators in comparison to strains of human
origin. This clear difference suggests, that strains of both subspecies forms on human
skin not only transient population, but are able to colonize it for longer time permit-
ting such characteristic for human strains adaptation.

Acknowledgements. This study was supported by the grant 503-406/2002 from Medical Univer-
sity of £ódŸ.

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2003, Vol. 52, No 3, 271–276

Differences in Inhibition of Apoptosis Depending

on the Virulence of Used Herpes Simplex Virus type 1 Strains.
Function of Interferon Alpha in Apoptotic Death
of Virus Infected Cells
MAGDALENA RECHNIO and BOGUMI£A LITWIÑSKA

National Institute of Hygiene, Department of Virology, Warsaw, Poland

Received 15 July 2003

Abstract

The purpose of the present study was to determine whether there is the relation between the
virulence of used HSV-1 strains and inhibition of apoptosis. HEp-2 cells were induced to
apoptosis by osmotic shock after infection by HSV-1 strains. HSV-1 ts, earlier described as less
virulent for mice inhibited apoptosis in smaller degree than native strain and HSV-1 tr. We sug-
gest that this is due to the hyperproduction of IFN alpha by human cells after the stimulation by
this strain. All strains of HSV-1 didn’t inhibit apoptosis in the presence of IFN alpha and apoptosis
was inhibited by anti IFN alpha antibodies. We confirm that IFN alpha plays an important func-
tion in controlling acute HSV-1 infection.

K e y w o r d s: HSV-1, interferon alfa, apoptosis

Introduction

Apoptosis, also known as programmed cell death, is a morphologically distinct

form of the death, and is known to be induced by various active cellular processes
under the genetic control of cells (G u m i e n n y et al., 1999).
Inhibition of apoptosis appears to be a mechanism used by several viruses to pre-
vent the premature death of infected cells in order to maximize production of infec-
tious virions. For example, cowpox virus and baculovirus contain antiapoptotic fac-
tors that inhibit proteases (caspases) involved in the induction of apoptosis (C r o o k
et al., 1993; T e w a r i et al., 1995). Epstein-Barr virus (H e n d e r s o n et al.,
1993), herpesvirus saimiri (D e r f u s s et al., 1998), and African swine fever viruses
(A l f o n s o et al., 1996) encode proteins that are homologues of the cellular
antiapoptotic protein Bcl-2. In addition, the large T antigen of polyomavirus
(R o d i e r et al., 2000) and the M11 gene product of murine gammaherpesvirus
(R o y et al., 2000) have also been shown to inhibit programmed cell death.
272 Rechnio M., Litwiñska B. 3

Recent studies indicate that HSV-1 also prevents apoptosis of infected cells
and that it is able to protect cells against apoptosis by various inducers (A u b e r t
and B l a h o, 1999; A u b e r t et al., 1999; G a l v a n et al., 1999; G a l v a n and
R o i z m a n, 1998; K o y a m a and M i w a, 1997; Z a c h o s et al., 2001). Several
other viral genes have been proposed to play antiapoptotic roles during HSV-1 infec-
tion. Studies indicate that apoptosis is inhibited at early times postinfection in Jurkat
cells by the action of two immediate-early genes, Us5 and Us3 (J e r o m e et al.,
1999). Furthermore, cultured human epithelial cells infected with an ICP27 deletion
virus exhibited the characteristic signs of apoptosis (A u b e r t and B l a h o, 1999).
Finally, it has been suggested that the viral glycoproteins gD (Us6) and gJ (Us5) are
involved in blocking the apoptotic pathway during productive infections in neuron-
like SK-N-SH cells (Z h o u et al., 2000). These studies suggest that several HSV-1
factors may have antiapoptotic functions that contribute to maintaining the viability
of the virus during its life cycle.
Furthermore, in cells infected with several mutant viruses, HSV-1 can activate
apoptosis (A u b e r t et al., 1999; G a l v a n and R o i z m a n, 1998; K o y a m a
and A d a c h i, 1997). However, the specific mechanisms by which HSV activates or
suppresses apoptotic pathways and the relative importance or contribution of these
pathways to the outcome of the infection are still unclear.
We induced apoptosis in HEp-2 cells by osmotic shock after infection by HSV-1
strains characterised by different pathogenicity to determine the effect of virulence on
inhibition of apoptosis. We used temperature sensitive mutant in 28°C (HSV-1 ts) and
temperature resistant in 39°C (HSV-1 tr) isolated by L i t w i ñ s k a et al., 1991, from
McIntyre strain. It was defined earlier, that HSV-1 ts is less virulent for mice and
establish latent infections rarely and HSV-1 tr is more virulent for mice than native
strain (L i t w i ñ s k a et al., 1996; L i t w i ñ s k a et al., 2001).

Experimental

Materials and Methods

Cell lines. CV1, a monkey kidney cell line, obtained from National Bacteriological Laboratory,
Department of Virology in Stockholm, was grown in MEM (Gibco BRL) supplemented with 10% FCS
(Sigma), 100 U/ml penicillin, and 100 :g/ml streptomycin (MEM – 10% FCS). HEp-2, a human epider-
moid carcinoma cell line was grown in MEM – 5% FCS.
Viruses. HSV-1 strain McIntyre, obtained from Institute of Hygiene in Freiburg, temperature sensi-
tive mutant in 28°C (HSV-1 ts), and temperature resistant in 39°C (HSV-1 tr) were grown and titered on
CV1 cells.
Induction of apoptosis. HEp-2 cells were seeded 1 day before infection in 24-well plates at 1×105
cells per well. Sheep antiserum to human leukocyte interferon alpha (aIFN alpha) and human leukocyte
interferon alpha (IFN alpha) (National Institute of Allergy and Infectious Diseases) were added to some
wells at concentration: 88 :g/ml (1 :500) – the first one, 500 IU/ml – the second one. The next day,
HEp-2 cells were exposed to 1×105 pfu of HSV-1, HSV-1ts, HSV-1 tr, and incubated at 37°C, 28°C, or
39°C for 1 h. Then culture medium was replaced with fresh MEM – 5% FCS, in some cases with aIFN
alpha and IFN alpha, and incubated at 37°C, 28°C, or 39°C for 4 h. Next, the cells were incubated in
medium containing 1 M sorbitol for 1 h at 37°C, 28°C, or 39°C and then maintained in sorbitol-free
medium (in some cases with IFN or aIFN) overnight.
3 Apoptosis and virulence of HSV-1 273

DNA fragmentation assay. The medium was removed, and attached cells were resuspended with
0.05% trypsin and added back to the removed medium. Cells were pelleted at 2000 xg for 20 min and
resuspended in 100 :l of phosphate buffered saline. Fragmented DNA was extracted by the Hirt method
(Hirt, 1967) with minor modifications. Briefly, the cell suspension was lysed by adding 400 :l of TE
buffer (10 mM Tris-HCl – pH 7,4; 10 mM EDTA) containing 0,6% sodium lauryl sulfate. The cell lysate
was gently mixed with 125 :l of 5 M NaCl and kept at 4°C overnight. The mixture was centrifuged at
14000 xg for 30 min and chromatin pellet was then removed. After treatment with RNase (0,1 mg/ml
– 1 h at 37°C) and proteinase K (0,1 mg/ml – 1 h at 50°C), DNA in the supernatant was precipitated with
ethyl alcohol and resuspended in TE buffer. Samples were analysed for a nucleosomal DNA ladder by
electophoresis on a 1,5% agarose gel.

Results

Inhibition of apoptosis by different strains of HSV-1. HEp-2 cells were treated

by solution with high osmotic concentration (1 M sorbitol) after infection by HSV-1,
HSV-1ts, and HSV-1 tr, to determine the effect of HSV-1 strains characterising dif-
ferent patogenicity on the inhibition of apoptosis induced by osmotic shock. After
incubation, low-molecular-weight DNA was isolated and samples were analysed for
a nucleosomal DNA ladder by electophoresis. The results are shown in Fig. 1.
The ladder of fragmented DNA was clearly observed in the sample from HSV
ts-infected cells and not observed in the samples from HSV-1 – and HSV-1 tr-
infected cells. These results indicate that HSV-1 and HSV-1 tr inhibit to a consider-
able degree apoptosis in infected HEp-2 cells, and HSV-1ts inhibits apoptosis in
a very small degree.
1 2 3 4 5

Fig. 1. Agarose gel containing electrophoretically separated, ethidium bromide-stained low-molecular-

weight DNA from HEp-2 cultures that were infected different strains HSV-1 and after osmotic shock.
Line 1: molecular ruler (Fermentas); line 2: low-molecular-weight DNA from HEp-2 cultures; line 3:
low-molecular-weight DNA from HEp-2 cultures that were infected HSV-1 ts; line 4: low-molecular-
weight DNA from HEp-2 cultures that were infected HSV-1; line 5: low-molecular-weight DNA from
HEp-2 cultures that were infected HSV-1 tr.
274 Rechnio M., Litwiñska B. 3

Role of IFN alpha in apoptosis of virus-infected cells. We earlier described that

HSV-1 ts stimulated hyperproduction of IFN alpha (R e c h n i o and L i t w i ñ s k a,
2002). HEp-2 cells were incubated with medium supplemented with IFN alpha or
anti-IFN alpha antibodies before infection by different strains of HSV-1 and before
induction apoptosis by osmotic shock in order to determine the effect of interferon
on induction of apoptosis. The results are shown in Fig. 2.

1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7

A B C D
Fig. 2. Agarose gel containing electrophoretically separated, ethidium bromide-stained low-molecular-
weight DNA from HEp-2 cultures that were infected different strains HSV-1: A – non-infected,
B – HSV-1 ts, C – HSV-1, D – HSV-1 tr. Line 1: molecular ruler (Fermentas); line 2, 3, 4: culture
exposed to sorbitol; line 5, 6, 7: culture not exposed to sorbitol; line 3, 6: culture exposed to IFN alpha;
line 4, 7: culture exposed to anti-IFN alpha antibodies.

Lysates from cells infected by HSV-1, HSV-1ts, and HSV-1 tr and exposed to IFN
alpha and sorbitol showed extensive DNA fragmentation (Fig. 2: A, B, C, D, lane 3),
whereas cells infected by HSV-1, HSV-1ts, and HSV-1 tr and exposed to aIFN alpha
and sorbitol showed no degradation of DNA (Fig. 2: A, B, C, D, lane 4). Lysates from
cells infected by HSV-1, HSV-1 tr and HSV-1 ts and exposed to IFN alpha but not
exposed to sorbitol showed no degradation DNA (Fig. 2: A, B, C, D, line 5, 6, 7). These
results indicate that HSV-1 doesn’t inhibit apoptosis in the presence of IFN alpha. We
also observed that apoptosis was inhibited by anti-IFN alpha antibodies.

Discussion

We studied how virulence of the used HSV-1 strains affects the inhibition of
apoptotic death. The temperature sensitive (ts) and temperature resistant (tr) HSV-1
mutants from the collection of Department of Virology, National Institute of Hygiene
were used. Current research has implemented characteristics of HSV-1 ts and HSV-1
tr. HSV-1 ts was described earlier as less virulent for mice but manifesting high im-
munogenic potency. Moreover, HSV-1 ts showed a significantly lower possibility to
cause the latent infection but the state of immunosuppression increased the frequency
3 Apoptosis and virulence of HSV-1 275

of latent HSV-1 ts infection (L i t w i ñ s k a et al., 1996; L i t w i ñ s k a et al., 2001).

The suspension of infectious and non-infectious HSV-1 ts induced PBMC to
hyperproduction of IFN alpha and we proved that there was no connection with virus
infectivity but there was one with the presence of glycoprotein D (gD) (R e c h n i o
and L i t w i n s k a, 2002).
The results presented in this report imply that HSV-1 and HSV-1 tr can protect the
infected cells from DNA fragmentation induced by osmotic shock, whereas HSV-1 ts
inhibits apoptosis in a very small degree. We suggest that it is due to the ability of
HSV-1 ts to induct IFN alpha in higher level than native HSV-1 strain and HSV-1 tr
can do. Our current results indicate that all strains of HSV-1 don’t inhibit apoptosis
in the presence of IFN alpha and apoptosis is inhibited by anti-IFN alpha antibodies.
It is known that IFN alpha/beta is essential mediator of apoptosis. Primary MEF
undergo apoptosis when infected with EMC virus (encephalomyacarditis virus), VSV
(vesicular stomatitis virus), and HSV, but apoptosis induced by these viruses is inhib-
ited by anti-IFN alpha/beta antibodies and in homozygous null cells lacking either the
IFN receptor or the Stat-1 signalling factor (T a n a k a et al., 1998).
Inhibition of apoptosis in a very small degree and induction of hyperproduction
of IFN alpha by PBMC can explain the decreasing of the pathogenicity of HSV-1 ts
for mice and declining of the possibility of latent infection. We suggest that presence
of IFN alpha essentially influences on controlling acute HSV-1 infection.

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T a n a k a N., M. S a t o, M.S. L a m p h i e r, H. N o z a w a, E. O d a, S. N o g u c h i, R.D.
S c h r e i b e r, Y. T s u j i m o t o and T. T a n i g u c h i. 1998. Type I interferons are essential
mediators of apoptotic death in virally infected cells. Genes to Cells. 3: 29–37.
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Z h o u G., V. G a l v a n, G. C a m p a d e l l i - F u i m e and B. R o i z m a n. 2000. Glycoprotein D or
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tant lacking intact genes expressing both glycoproteins. J. Virol. 74: 11782–11791.
Acta Microbiologica Polonica
2003, Vol. 52, No 3, 277–283

Enteropathogenic Activity and Invasion of HEp-2 Cells

by Aeromonas caviae Clinical Isolates
SYLWIA KRZYMIÑSKA, ADAM KAZNOWSKI*, KAROLINA LINDNER
and MAGDALENA MNICHOWSKA

Department of Microbiology, A. Mickiewicz University

Fredry 10, 61-701 Poznañ, Poland

Received 2 June 2003

Abstract

Twenty Aeromons caviae isolates from stool of children with diarrhea symptoms were examined
for virulence-associated properties: production of cytotoxic and cytotonic toxins, and invasive
ability. Most of A. caviae strains were cytotoxic to Vero and CHO cells and produced cytotonic
toxins which caused elongation of CHO cells. Moreover, five of A. caviae strains revealed inva-
sive ability towards HEp-2 cells.

K e y w o r d s: A. caviae, toxins, invasive ability

Introduction

Members of the genus Aeromonas are gram-negative, oxidase-positive rods widely

spread in water habitats (N a k a n o et al., 1990; A s h b o l t et al., 1995; H o l m e s
et al., 1996). Aeromonas spp. have been implicated in a wide range of human infec-
tions. They have been found opportunistic pathogens in skin and soft tissues infec-
tions, which may remain localized and mild with muscles necrosis or can lead to
septicemia (J a n d a and A b b o t t, 1996; A l t w e g g, 1999). Strains of Aeromonas
spp. are also responsible for other extraintestinal infections, including respiratory tract
infections, endocarditis, peritonitis, osteomyelitis and meningitis (M u r p h y, 1995;
J a n d a and A b b o t t, 1996; A l t w e g g, 1999).
Recently, most researches have concentrated on the role of these bacteria as poten-
tial etiological agents of gastroenteritis with diarrheal symptoms. Human Aeromo-
nas-associated diarrheal diseases range from a mild self-limiting, acute diarrhea to
cholera-like dysenteric illness or a more persistent diarrhea (G o s l i n g, 1996;

* corresponding author: Adam Kaznowski, Department of Microbiology, A. Mickiewicz Univ.,

Fredry 10, 61-701 Poznañ, Poland, e-mail: [email protected]
278 Krzymiñska S. et al. 3

T h o r n l e y et al., 1997; A l t w e g g, 1999). Although the genus Aeromonas

includes 16 species, only 3 of them, A. hydrophila, A. veronii biotype sobria and
A. caviae are responsible for 85% of Aeromonas-associated infections in humans
(T h o r n l e y et al., 1997). Of the three species, A. hydrophila and A. veronii biotype
sobria are considered the most virulent, whereas A. caviae is recognized as nonpatho-
genic (J a n d a and K o k k a, 1991). However, N a m d a r i and B o t t o n e
(1990) have regarded A. caviae as an important enteropathogen especially for very
young children. Moreover, A. caviae is the prevalent of Aeromonas species isolated
from fecal samples of European and American patients with diarrhea symptoms
(A l t w e g g, 1999).
The pathogenicity mechanisms of Aeromonas spp. are complex and multifactoral,
and may involve numerous putative virulence factors which role in disease etiology
has not yet been clearly defined. The first step of enteropathogenesis is colonization
of epithelial cells. Adhesion of Aeromonas spp. strains to intestine followed by inva-
sion to epithelial cells or the activity of other virulence factors as extracellular toxins
are probably the most common mechanism which results in diarrhea (N i s h i k a w a
et al., 1994). Some of A. hydrophila and A. veronii biotype sobria isolates produce
$-hemolysin which is a pore-forming toxin with hemolytic, cytotoxic, and entero-
toxin activity (G o s l i n g, 1996; T h o r n l e y, 1997; X i n - J. X U et al., 1998).
Strains of Aeromonas spp. have been also reported to produce cytotonic toxins
which cause elongation of CHO cells and increase the intracellular cyclic AMP level
(M c C a r d e l l et al, 1995; G o s l i n g, 1996; T h o r n l e y et al., 1997; J i a n
S h a et al., 2002). These toxins were isolated from A. hydrophila and A. veronii
biotype sobria (G o s l i n g, 1996).
This study was undertaken to examine cytotoxic and cytotonic activity, and inva-
sion of HEp-2 cells by A. caviae strains isolated from stool of young children with
diarrheal symptoms.

Experimental

Material and Methods

Bacterial strains. A total of 20 strains of A. caviae were used in this study. They were recovered
from fecal samples of patients suffering from diarrhea: 13 strains were isolated in Poland
(K a z n o w s k i, 1995), and seven isolates cultured in a hospital in Hong Kong were obtained from
dr R. Kong (Hong Kong City University). All strains were stored at –75°C in brain heart infusion broth
(BHI, Difco) containing 50% (v/v) glycerol.
Cell cultures. Chinese hamster ovary cells (CHO), African monkey kidney cells (Vero) and human
laryngeal epithelial cell line (HEp-2) were cultured in Minimum Essential Medium Eagle (MEM, Sigma)
supplemented with 5% fetal calf serum (FCS, Sigma), 2 mM glutamine, 80 IU penicillin per ml, 80 :g
streptomycin per ml, and 1 mg/ml of nystatin. The cell cultures were incubated at 37°C in atmosphere
containing 5% CO2 (G r a y et al., 1990; S c h i a v a n o et al., 1998).
Cytotoxic and cytotonic activity. Culture supernatants were prepared as described previously by
S c h u l t z and M c C a r d e l l (1998). A. caviae strains were grown on tryptic soy broth (TSB, Difco)
supplemented with 0.6% yeast extract at 37°C for 24 h in a water bath with agitation of 150 rpm. The
bacterial cultures were centrifuged at 700 g for 20 minutes and the supernatants were sterilized through
3 Enteropathogenic activity of A. caviae 279

0.22 :m-pore size filters Millex-GV (Millipore) of low protein binding. Sterile culture supernatants
were heated at 56°C for 20 min to destroy the activity of heat-labile toxins.
Cytotoxic activity of the supernatants was measured on Vero and CHO cells whereas cytotonic
activity was examined on CHO cells. The cells were seeded into 96-wells microtitre trays (Nunc) at
a concentration of 3×103 cells per well (CHO) and 1×104 cells per well (Vero cells). A 100-:l volume of
serial twofold dilutions of culture supernatants was added to the monolayer and the plate was incubated
at 37°C for 24 hours in atmosphere containing 5% CO2. The plates were read under an inverted micro-
scope. Cytopathic effects were identified as rounding and detachment of 50% of Vero or CHO cells
(S c h i a v a n o et al., 1998). Cytotonic activity was identified as elongation of CHO cells. Cytotonic
and cytotoxic titre was expressed as the reciprocal of the highest dilution yielding a positive result
(G r a y et al., 1990).
Invasion assay of HEp-2 cells. Invasion assay was performed according to W a t s o n et al. (1985)
with minor modifications. The cells were seeded at a concentration of 1×103 per well in 96-wells
microtitre tissue plates and incubated for 24 h in MEM supplemented with 5% FCS and antibiotics.
Then the cell culture medium was removed and replaced with MEM without serum and antibiotics, and
the plate was incubated for further 24 hours. A. caviae strains were cultured on BHI at 37°C for 24 h.
The bacterial inoculum was made to 1 McFarland standard and diluted 1:10 in Eagle MEM to give
a concentration of about 1×105 CFU per ml. After MEM removing, 100 :l of bacterial suspension was
added to each well and the monolayer was incubated at 37°C for 3 h. The cells were washed three times
with 200 :l of phosphate-buffered saline (PBS, Biomed), and 200 :l of MEM containing gentamycin
(0.1 mg/1 ml) was added to each well and the samples were incubated for 2 h at 37°C. After washing
three times with PBS, the integrity of the monolayer was checked and 200 :l of lysing solution contain-
ing 0.01 M NaH2PO4, 0.1% Tween 20 (v/v) and 0,025% trypsin (w/v) pH 8.0 was added to each well,
and incubation was continued for further 30 min at 37°C. Bacterial CFUs were determined by plating
100 :l of the lysates onto tryptic soy agar (TSA, Difco).
Invasion index was expressed as CFU×100 per 1×103 of HEp-2 cell according to L a w s o n et al.
(1985). The monolayer was infected separately with the invasive strain of Yersinia enterocolitica O:3 as
the positive control and E. coli K12C600 as the negative one.

Results

The results of the study concerning virulence factors like cytotoxic and cytotonic
toxins, and the invasive ability are listed in Table I.
Cytotoxic and cytotonic activity. Nineteen of twenty A. caviae strains were
found to be cytotoxic to Vero cells with cytotoxic titres ranging from 1 to 8, and
cytotoxic to CHO cells with titres ranging from 1 to 16. Preheating (56°C for
20 min) of the supernatants caused a decrease in cytotoxic activity of the isolates
to Vero cells and CHO cells. Cytotonic activity was observed in case of 15 strains
with titres ranging from 1 to16.
Invasion assay. Invasion of HEp-2 cells by A. caviae isolates was investigated in
a quantitative assay. Five strains (25%) gave invasion index greater than 10 CFU per
HEp-2 and these strains might be classified as invasive according to the criteria
of L a w s o n et al. (1985). However, the invasion index of Y. enterocolitica ser-
ving as a positive control was 68 CFU per HEp-2 cell. Twelve strains of A. caviae
demonstrated invasion index ranging from 1.1 to 6.8 CFU per HEp-2 cell. Strain
AK 390 showed invasion index 0.6 CFU per HEp-2 cell whereas AK 388 was
negative in this test. E. coli K12C600 used as negative control did not show invasion
ability towards HEp-2 cells.
280 Krzymiñska S. et al. 3

Table I
Cytotoxic, cytotonic activity, and invasion of HEp-2 cells by 20 clinical isolates of A. caviae

Cytotoxin titrea Cytotonic titreb

Strain No Vero CHO CHO Invasion
Indexc
d e d e d e
AK 375 4 – 2 1 – – 10.6’
AK 376 4 2 8 1 – – 3.8
AK 377 8 4 1 – 2 2 3.5
AK 378 4 2 8 1 – – 2.2
AK 379 4 2 4 1 4 – 31.6
AK 380 8 4 1 – 2 2 1.2
AK 383 4 2 4 2 2 1 1.1
AK 384 2 – 8 1 – – 4.7
AK 385 4 2 4 2 2 1 3.8
AK 386 – – – – 2 1 5.3
AK 388 1 – 4 2 16 – 0
AK 390 2 1 2 1 – – 0.6
AK 393 1 1 1 – 1 – 1.0
QM 77620 2 1 8 2 1 1 25.4
QM 66492 2 1 1 – 1 – 5.6
QM 65541 2 1 2 – 2 1 5.7
QM 27611 2 1 2 1 2 1 50.0
QM 25447 2 2 8 2 16 – 6.8
QM 220132 4 1 16 8 1 1 10.5
QM 217455 8 2 8 2 2 2 4.9
Y. enterocolitica ND ND ND 68.0
E. coli ND ND ND 0
a – the reciprocal of the highest dilution yielding rounding, detachment and destruction
of 50% of CHO or Vero cells
b – the reciprocal of the highest dilution causing elongation of CHO cells
c – CFU × 100 per 1 × 103 of HEp-2 cell
d – titre in unheated supernatant
e – titre in preheated supernatant (56°C for 20 min)
ND – not determined

Discussion

Strains of A. caviae have been considered as causative agents of human gastro-

enteritis (N a m d a r i and B o t t o n e, 1990). However, the exact mechanism of
enteropathogenicity has not been sufficiently understood. The association between
virulence factors and clinical symptoms will remain unproved until suitable animal
3 Enteropathogenic activity of A. caviae 281

models have been developed. One approach to this problem is the use of cell lines.
Studies have been conducted on various cell types including Vero, CHO, HEp-2 and
Y1. These systems resembling in vivo-like conditions are now being used to elucidate
mechanisms of bacterial enteropathogenesis (T h o r n l e y et al., 1997 ).
This study focused on explaining the contribution of cytotoxic, cytotonic toxins,
and invasiveness in gastrointestinal disease caused by A. caviae isolates. The analysis
of the incidence of virulence markers in A. caviae isolates revealed that 14 A. caviae
strains (70%) produced both cytotoxic and cytotonic toxins (Tab. I). G r a y et al.
(1990) reported that 2 of 22 A. caviae strains isolated from pig feces produced both
cytotoxic and cytotonic toxins with cytotoxic titre 32 and 1024, and cytotonic titre 1.
Moreover, four of environmental isolates of A. caviae produced cytotonic toxins with
a titre of 1. S c h i a v a n o et al. (2000) noticed that 1 of 3 A. caviae strains isolated
from stool of diarrheal patients was found to be cytotoxic to CHO cells with cytotoxic
titre equal 4. In our study, five isolates of A. caviae (25%) produced only cytotoxins
whereas one strain produced only cytotonic toxin. Previously, G r a y et al. (1990)
noted that 13.6% of A. caviae strains isolated from the environment produced
cytotoxin and 27% of these isolates possesed a heat-stabile cytotonic factor that
caused elongation of CHO cells. In research of G r e y and K i r o v (1993), none of
A. caviae isolates from clinical specimens produced cytotoxins. These differences may
be dependent on bacterial genetic determinants. C h a k r a b o r t y et al. (1984) noted
that the cytotoxic, cytotonic enterotoxin and hemolytic activities of Aeromonas spp.
strains were due to three different proteins resulting from the expression of three
distinct genes. However, R o s e et al. (1988) stated that hemolytic, cytotoxic and
enterotoxic activities as well as mice lethality were associated with only one protein.
C h o p r a et al. (1993) isolated cytotoxic enterotoxin gene from human diarrheal
isolate of A. hydrophila.
Gastrointestinal symptoms (dysentery, colitis) associated with some cases of
Aeromonas-linked diarrhea may be connected with an invasive mechanism. However,
there is little experimental evidence supporting this hypothesis. In our study, 5 of 20
A. caviae strains (25%) were invasive to HEp-2 cells with invasion index lower than
that of Y. enterocolitica positive control. This result is in agreement with the observa-
tions of S h a w et al. (1995) who reported that the majority of A. caviae strains
showed little or no invasive ability. W a t s o n et al. (1985), expressing invasion
index as CFU per 1 ml of lysate, suggested that strains which showed 5×106 CFU
per ml of lysate were invasive. They found that 26% of Aeromonas spp. isolates were
invasive but only 1 of 22 A. caviae strains was classified as invasive.
We compared our results of production concerning cytotoxic, cytotonic entero-
toxins and invasive ability of 13 A. caviae isolates with clinical manifestations of
infections previously described by M o k r a c k a et al. ( 2001). We found that
strains AK 375, AK 376 and AK 390, which caused intermittent diarrhea for 5 days
without dehydratation symptoms, revealed cytotoxic activity and low invasion
index. Strains AK 378 and AK 384, which produced cytotoxic enterotoxin and
possessed low invasion index, provoked hemorrhagic diarrhea in young children. The
isolate AK 388 which demonstrated cytotoxic and cytotonic activity without invasion
282 Krzymiñska S. et al. 3

ability was an etiological agent of diarrhea with toxicant course with vomiting and
dehydratation of the body.
The results of this study demonstrate that A. caviae strains isolated from fecal
samples produce cytotoxic and cytotonic toxins which may contribute to gastrointes-
tinal disease. Some of these strains showed invasive ability although the invasion
index was lower than that of Y. enterocolitica.

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Acta Microbiologica Polonica
2003, Vol. 52, No 3, 285–292

Amlodipine: a Cardiovascular Drug with

Powerful Antimicrobial Property
K. ASOK KUMAR, KUMKUM GANGULY, KAUSHIKI MAZUMDAR,
N.K. DUTTA, SUJATA G. DASTIDAR and A.N. CHAKRABARTY1

Division of Microbiology, Department of Pharmaceutical Technology,

Jadavpur University, Calcutta 700 032
1 Department of Medical Microbiology and Parasitology,

Calcutta University, Calcutta 700 020, India.

Received in revised form 15 May 2003

Abstract

Ten cardiovascular drugs were procured in pure form from their manufacturers in India and
screened for antimicrobial property against fifteen known bacteria belonging to both gram-positive
and gram-negative types. These bacteria were inhibited by the common antibiotics at 1–5 mg ml –1
level through our earlier studies. Since most of the bacteria were moderate to highly responsive
to amlodipine, this compound was further tested in vitro against 504 bacteria comprising 4 genera
of gram-positive and 15 genera of gram-negative bacteria. Most of these were inhibited by
the drug at 50–200 :g ml–1 level and few strains were sensitive even at lower concentrations
(10 :g ml–1). The bacteria could be arranged in the decreasing order of sensitivity towards
amlodipine in the following manner: Staphylococcus aureus, Vibrio cholerae, Vibrio para-
hemolyticus, Shigella spp., Salmonella spp., Bacillus spp., whereas Escherichia coli, Klebsiella
spp. and Pseudomonas aeruginosa were found to be resistant to the lower concentrations of the
drug. Amlodipine was found to be bactericidal in nature when its mode of action was studied
against S. aureus 6571, V. cholerae 14035 and Sh boydii 8 NCTC 254/66. The antibacterial activ-
ity of amlodipine could also be confirmed in vivo. When it was given to Swiss strain of white
mice at different dosages (30 and 60 :g/mouse), it could significantly protect the animals chal-
lenged with 50 MLD of Salmonella typhimurium NCTC 74. According to Chi square test the
in vivo data were highly significant (p < 0.001).

K e y w o r d s: anti-hypertensive, amlodipine, antimicrobial activity, non-antibiotic.

Introduction

Antibiotics are one of our most important weapons in fighting bacterial infections
and have greatly benefited the health-related quality of human life since their introduc-
tion. However, over the past few decades these health benefits are under threat as many

Correspondence to: Professor Sujata G. Dastidar, Division of Microbiology, Department of Pharma-

ceutical Technology, Jadavpur University, Calcutta 700 032,India. Telephone No.: 91-33-2414-6666;
Fax: 91-33-2414-6266. E-mail: [email protected]; [email protected]
286 Kumar K.A. et al. 3

commonly used antibiotics have become less and less effective against certain illness
not only because many of them produce toxic reactions but also due to emergence of
drug resistant bacteria. It is essential to investigate newer drugs with lesser resistances.
Systematic studies among various pharmaceutical compounds have revealed that any
drug may have the possibility of possessing diverse functions and thus may have useful
activity in completely different spheres of medicine. Drugs belonging to different phar-
macological classes such as antihistamines like diphenhydramine and bromodiphen-
hydramine (D a s t i d a r et al., 1976), methdilazine (C h a t t o p a d h y a y et al.,
1988) and promethazine (C h a k r a b a r t y et al., 1989), psychotropics, e.g., pro-
mazine (D a s h et al., 1977), chlorpromazine (M o l n a r et al., 1976), fluphenazine
(D a s t i d a r et al., 1995) and trifluoperazine (M a z u m d e r et al., 2001), anti-
hypertensives such as methyl-DOPA (D a s t i d a r et al., 1986), local anaesthetics
like procaine (D a s t i d a r et al., 1988) and antiinflammatory drugs e.g., diclofenac
(A n n a d u r a i et al.,1998), possess powerful antibacterial activity. Such chemo-
therapeutic agents have been grouped together and are now entitled as “Non- anti-
biotics” (C h a k a r b a r t y et al., 1998; K r i s t i a n s e n, 1992). The present paper
describes the detailed in vitro and in vivo activity of such a non antibiotic – the cardio-
vascular drug amlodipine.

Experimental

Materials and Methods

Drugs. The cardiovascular drugs clonidine and dipryridamole were obtained from German Rem-
edies, enalapril from Nicholas Piramol, lacidipine from Glaxo Pharma, nifidipine (Torrent), nitrendipine
(Concept), felodipine from Cipla, digoxin (Cadila) Pharma, benidipine from Stancare and amlodipine
from Pfizer Pharmaceuticals. All these drugs were obtained in pure dry powder form and dissolved in
either distilled water or DMSO depending on their solubility, and kept at 4°C upto 15 days.
Bacteria. A total of 504 strains of bacteria belonging to 19 genera comprising 172 gram-positive
and 332 gram-negative types were tested (Table I). These were of human origin, identified as described
by B a r r o w and F e l t h a m (1993) and preserved in freeze-dried state.
Media. Liquid media used for this study were peptone water (PW, Oxoid brand bacteriological
peptone 1% (w/v) plus Analar NaCl 0.5% (w/v), nutrient broth (NB, Oxoid), Mueller Hinton broth
(MHB; Difco). Solid media were: peptone agar (PA), bromothymol blue lactose agar media (BLA),
nutrient agar (NA) and Mueller Hinton agar (MHA), obtained by solidifying the liquid media with 1.2%
(w/v) agar (Oxoid No. 3). In case of BLA, bromothymol blue indicator 1.2% (w/v) and lactose 1% (w/v)
are added. The pH is maintained at 7.2–7.4 for all the media. NA was used for tests with gram-positive
bacteria and PA and BLA were used for the rest of the bacteria as needed.
Determination of minimum inhibitory concentration (MIC) of different drugs. The MIC of
clonidine, dipyridamole, enalapril, digoxin, benidipine, nitrendipine, nifidipine, lacidipine, felodipine and
amlodipine with respect to different test bacteria was accurately determined both by broth and agar dilution
methods. For broth dilution, 0.1 ml of standardized suspension of a strain (106 CFU/ml) was added to each
tube containing amlodipine at concentrations of 0 (control), 2, 5, 10, 25, 50, 100 and 200 :g ml–1 in
MHB.The tubes were incubated at 37°C for 24 h, and looked for, visible growth after vortexing the tubes
gently. For agar dilution the drug was added at concentrations of 0 (control), 10, 25, 50, 100, 200, 400 and
800 :g ml–1 in molten NA and poured in Petridishes (NCCLS, 1993). The organisms were grown in
PW, and the overnight culture was spot-inoculated on the NA plates such that each inoculum contained
2×106 CFU. The plates were incubated at 37°C, examined after 24 h and incubated further for 72 h, if
3 Antimicrobial activity of amlodipine 287

Table I
Source of bacterial strains

Bacteria Source
Bacillus pumilus NCTC 8241 S.P. Lapage, London
Staphylococcus aureus NCTC 6571, 8530,8531, 8532 S.P. Lapage, London
Escherichia coli K12 Row J.D. Abbott, U.K.
E. coli pBR 322 S. Palchaudhuri, USA
Salmonella typhimurium NCTC 11, 74, S. viballerup, J. Taylor, London
S. choleraesuis 37, S. uganda 101, S. paratyphi 85, S.typhi 57,59
Shigella boydii 5 NCTC 541/60, Sh. boydii 8 NCTC 254/66, K. Patricia Carpenter, London
Sh. boydii 9 NCTC 304/67, Sh. dysenteriae 3 NCTC 102/65,
Sh. dysenteriae 7 NCTC 519/66, Sh. dysenteriae 8 NCTC 599/52,
Sh. sonnei NCTC 5/59
Vibrio cholerae ATCC 14033, 14035 S. Mukerjee, Calcutta
V. cholerae 80, 540, 546, 566, 590, 738,7 64, 824, 838, 906, 1003, National Institute of Cholera
1021, 1023. & Enteric Diseases, Calcutta.
V. parahaemolyticus 4750, 9369, 72001, 72006 Y. Miyamoto, Japan
All the remaining organisms were available in the Department. They were clinical isolates collected from different
hospitals in Calcutta and identified by the methods described by Barrow and Feltham.

necessary. Since one solid agar medium containing amlodipine can be used for inoculation of a large
number of bacteria at a time, the results of this method are being presented here, as the total number of
test bacteria was 504. The lowest concentration of amlodipine in a tube or a plate that failed to show any
visible macroscopic growth was considered as its MIC. The MIC determination was performed in dupli-
cate for each organism, and the experiment was repeated where necessary. The MIC values for a given
isolate were either identical, or within ± one dilution with respect to different test bacteria.
Determination of mode of action of amlodipine. For this purpose S. aureus NCTC 6571,
V. cholerae ATCC 14035, Sh. boydii 8 NCTC 254/66 were grown in NB overnight at 37°C. 2 ml from
each of these were added to 4 ml of fresh NB and incubated for 2 h so that the cultures could attain the
logarithmic growth phase. To determine the number of colony forming units (CFU), 0.5 ml of each
culture was individually added to 4.5 ml fresh NB. This culture was subjected to serial dilutions in test
tubes containing 4.5 ml NB to produce 8-fold dilutions. A 0.025 ml aliquot was then removed from each
tube and pipetted onto a NA plate (K r o g s t a d and M o e l l e r i n g, 1980). Amlodipine was then
added to each culture at a concentration of 2×MIC value (50 :g ml–1) of the test bacterium. The CFU
counts were similarly determined upto 6 h at intervals of 2 h and then after 18 h. The number of colonies
appearing in each plate was counted after 24 h incubation for determination of CFU.
In vivo tests. Swiss strain of male white mice weighing 18–20 g were used for the in vivo studies.
Animals were maintained at standard conditions at 21 ±1°C and 50–60% relative humidity with a photo-
period of 14 : 10 h of light darkness. Water and a dry pellet diet were given ad libitum. The virulence of
the test strain S. typhimurium NCTC 74 was enhanced in the following manner: 0.1 ml of an overnight
grown NB culture of S. typhimurium NCTC 74 was given intraperitoneally as the challenge to 3 mice.
Next day, the animals were anaesthetized and dissected. Heart blood was aseptically collected from
them, added to fresh NB and incubated at 37°C. From this, the pure culture of S. typhimurium NCTC 74
was cultivated and given as challenge to another set of mice in the same manner. After 5 such passages,
the organism was administered as challenge to 4 batches of mice. The median lethal dose (MLD or
LD50) of the passaged strain corresponding to 0.95×109CFU/mouse suspended in 0.5 ml NB served as
the challenge dose for all the groups of animals (C r u i c k s h a n k et al., 1975). Reproducibility of the
288 Kumar K.A. et al. 3

challenge dose was ensured by standardization of its optical density in a Klett Summerson colorimeter at
640 nm and determination of the CFU count in NA.
Four batches of 20 mice each were taken for the study. The first two batches (constituting one group)
were administered 30 :g of amlodipine (by injecting i.p. 0.2 ml from a stock solution containing 150 :g
ml–1 of the drug) and the next two batches received 60 :g of amlodipine (0.2 ml from a stock solution of
300 :g ml–1 of the drug). After 3 h, one batch from each of the above two groups was challenged with
50 MLD of S. typhimurium 74. A control group comprising 60 animals was also injected with the same
organism and 0.1 ml of sterile saline in place of the drug. The number of animals dying upto 100 h was
recorded in each group to determine the protective capacity of amlodipine (Table IV).
In another in vivo experiment, the CFU counts in blood and organ homogenates of amlodipine treated
and untreated mice were determined. Two groups of mice (10 animals per group) were taken. All the
animals were given a 50 MLD challenge dose; of these 50% received the drug (60 :g/mouse) 3 h before
the challenge and the rest received saline (Table V). After 18 h all mice were sacrificed, blood was
collected individually from heart, and livers and spleens were removed aseptically and homogenized in
tissue homogenisers. CFU counts of individual organs were determined separately. Statistical analysis
of the data was performed using Student’s t-test.

Results

In vitro determination of antimicrobial action in cardiovascular drugs. All the

bacterial strains tested were found to be resistant to clonidine, dipyridamole, digoxin,
enalapril and nitrendipine while, felodipine, lacidipine, benidipine and nifidipine pro-
duced moderate inhibitory action. Amlodipine showed powerful antimicrobial action
against a large number of the bacteria (Table II).

Table II
Primary screening of cardiovascular drugs in vitro for presence of antibacterial action

Minimum inhibitory concentration (µg ml–1) of the drugs

clonidine, nifedipine,
Bacteria dipyridamole, lacidipine,
amlodipine
digoxin, enalapril, felodipine,
itrendipine benidipine
Bacillus pumilus NCTC 8241 200 50
Staphylococcus aureus NCTC 6571 200 – 400 25
S. aureus NCTC 8530 R 200 – 400 25
Escherichia coli K12Row E 200 – 400 > 800
Salmonella typhimurium NCTC 74 S 200 – 400 50
Salmonella typhi 59 I 100 – 200 50
Shigella dysenteriae 7 NCTC 519/66 S 25 – 200 25
Shigella sonnei 1NCTC 5/59 T 200 200
Shigella flexneri 4a24 A 100 – 200 25
Shigella boydii 8 NCTC 254/66 N 200 25
Klebsiella pneumoniae 14 T 200 – 400 400 – 800
Vibrio cholerae 569B, ATCC14033, 14035 100 – 200 25
Pseudomonas aeruginosa APC > 800 50 > 800
3 Antimicrobial activity of amlodipine 289

Bacterial inhibitory spectrum of amlodipine. 159 Staphylococcus aureus were

tested, of which 21 were inhibited at 10mg ml–1 of amlodipine (highly sensitive),
60 at 25 :g ml–1, 45 at 50 :g ml–1, 27 at 100 :g ml–1, 2 at 200 :g ml–1 and 4 by
400 :g ml–1 of the drug. Of 9 strains of Bacillus spp., 1 strain was inhibited at
25 :g ml–1, 2 strains at 50 :g ml–1, 5 by 100 :g ml–1 and 1 at 200 :g ml–1 of the drug.
Streptococcus spp. and Micrococcus spp. were also moderately sensitive to this drug.
In case of gram-negative bacteria tested, of 51 strains of Shigella spp., 8 were
inhibited at 25 :g ml–1, 16 between 50 to 400 :g ml–1 and 27 above 800 :g ml–1 of
amlodipine. In terms of sensitivity, the drug showed moderate activity towards strains
of Escherichia coli, Salmonella spp. and Klebsiella spp. Two strains of E. coli were
inhibited at 50 :g ml–1, 3 each at 100 :g ml–1 and 200 :g ml–1, 14 at 400 :g ml–1 and
10 strains stopped growing above 800 :g ml–1. In case of Salmonella spp., 3 strains
were inhibited at 25 :g ml–1, 1 each at 100 :g ml–1 and 200 :g ml–1 and 11 strains
could not be inhibited below 800 :g ml–1. Two strains of Klebsiella spp. were inhib-
ited at 50 :g ml–1, 2 at 100 :g ml–1, 4 at 400 :g ml–1 and 2 strains had MIC above
800 :g ml–1. Pseudomonas aeruginosa were found to be fairly resistant (> 400 :g ml–1)

Table III
In vitro activity of amlodipine on gram-positive and gram-negative bacteria

No No of strains inhibited by amlodipine (mg ml–1)

Bacteria
tested 10 10 10 25 50 100 >800
Bacillus spp. 9 1 2 5 1
Staphylococcus aureus 159 21 60 45 27 2 4
Streptococcus spp. 3 1 2
Micrococcus spp. 1 1
Escherichia coli 32 2 3 3 14 10
Salmonella spp. 16 3 1 1 11
Shigella spp. 51 8 3 1 6 6 27
Klebsiella spp. 10 2 2 4 2
Hafnia spp. 1 1
Proteus spp. 9 9
Providencia spp. 1 1
Citrobacter spp. 1 1
Arizona spp. 1 1
Pseudomonas spp. 13 1 3 9
Bordetella bronchiseptica 1 1
Pasteurella septica 136 1 1
Enterobacter cloaca 1 1
Vibrio cholerae 165 58 45 37 18 7
Vibrio parahaemolyticus 29 10 8 9 2
Total 504 21 143 108 87 34 39 72
290 Kumar K.A. et al. 3

to the drug. The MIC of 58 out of 165 V. cholerae were found to be 25 :g ml–1
(highly sensitive), 45 had MIC at 50 :g ml–1 (markedly sensitive), 37 at 100 :g ml–1,
18 at 200 :g ml–1 and 7 at 400 :g ml–1 (moderately sensitive). Similarly, of
29 V. parahemolyticus, 10 were inhibited at 25 :g ml–1, 8 strains at 50 :g ml–1,
9 could not grow at100 :g ml–1 and 2 at 200 :g ml–1 of the drug (Table III).
The drug also showed good inhibitory action (25–50 :g ml–1) against strains of
Pasturella and Hafnia, while Arizona (200 :g ml–1) and Bordetella bronchiseptica
(400 :g ml–1), were much less sensitive to this compound. Proteus, Citrobacter,
Providencia and Enterobacter cloaca were resistant to amlodipine.

9
8
Log viable cells in ml–1

7
6
5
4
3
2
1
0
0 2 4 6 8
Time in hours

Fig. 1. Mode of action of amlodipine on S. aureus NCTC 6571.

Bactericidal action of amlodipine. A killing curve was performed on S. aureus

NCTC 6571, V. cholerae ATCC 14035 and Sh. boydii 8 NCTC 254/66 by adding
2×MIC (50 :g ml–1) of amlodipine to a logarithmic phase of the organism that con-
tained 1.5×108 CFU/ml of S. aureus 6571, 4×108 CFU/ml of the organism V. cholerae
14035 and 2×108 CFU/ml for Sh. boydii 8. The viable count of the culture dropped
to 0 at 2 h proving bactericidal property of the drug (Fig. 1).
In vivo protection by amlodipine. The in vivo antibacterial effect was determined
by administering 50 MLD dosages (0.95×109 CFU in 0.5 ml of NB) of S. typhimurium
NCTC 74 into different groups of mice with or without administration of amlodipine
(Table IV). In the group that received the challenge and saline (in place of the drug),
49 of 60 mice died within 100 h. In the other groups, which were administered differ-
ent doses of amlodipine only; mortality rate of mice was very low.
The results of the experiment for the determination of the effect of amlodipine on
CFU/ml in blood and other organs of mice significantly reduced the CFU/ml counts
in mice 18 h after challenge (Table V) as compared with the control (p <0.001).
3 Antimicrobial activity of amlodipine 291

Table IV
Determination of protective capacity of amlodipine in vivo

Control group* Test group*

Drug injected Mice died Drug (µg) injected Mice died
per mouse (out of 60) per mouse (out of 20)
0.1 ml sterile saline 49 30 13
60 4

* Received a challenge dose of 0.95 × 109 CFU in 0.5 ml NB of S. typhimurium NCTC 74.
None of the animals died when 30 :g or 60 :g amlodipine was injected to 2 separate groups
of mice (20 mice in each), i.e., amlodipine was found to be non-toxic to mice.
p < 0.001, according to Chi-square test

Table V
Reduction in CFU/ml of S. typhimurium NCTC 74 in organ homogenates
of mice treated with amlodipine

Time CFU/ml counts in

Group Drug/mouse
of sampling Heart blood Liver Spleen
18 h 1 Amlodipine 1.5×104 5.5×103 1.6×103
60 :g to to to
7.2×10 5
2.0×10 5
3.9×104
18 h 2 Saline 3.2×10 8
2.0×10 8
8.9×107
(Control) 6.6×108 6.3×108 6.6×108

Viable counts between two groups highly significant; p < 0.001

Discussion

The antihypertensive calcium channel blocker amlodipine, a dihydropyridine de-

rivative, was seen to possess powerful antibacterial activity both through in vitro and
in vivo tests. While sensitive bacterial strains occurred among S. aureus, V. cholerae,
V. parahaemolyticus, Bacillus spp. and some enterobacteria, the drug was much less
active with respect to shigellae, salmonellae, E. coli, klebsiellae and pseudomonads.
Amlodipine is bactericidal in nature when tested in vitro against gram-positive and
gram-negative bacteria. The protection offered by the drug to mice challenged with
virulent bacterium was found to be statistically highly significant.
Search among the various classes of pharmacological agents has revealed that
the phenothiazines in general possess moderate to powerful antimicrobial action
(B o u r l i o u x et al., 1992). A detailed study of antimicrobial activity using in vitro
method on 18 derivatives of pheniothiazines revealed that such an activity is closely
linked to the halogen groups present in the basic tricyclic ring structure of pheno-
thiazines. In amlodipine, one benzene ring is attached to another, that may be
considered as an incomplete phenothiazine ring. Moreover, the presence of a halogen
292 Kumar K.A. et al. 3

(chlorine) moiety may be playing a key role in conferring antimicrobial activity to

this compound (A n n a d u r a i et al., 1998). Since this drug is in routine therapeutic
usage, in course of time it may be developed as the second or even the first line
antimicrobial agent in many infections.

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Acta Microbiologica Polonica
2003, Vol. 52, No 3, 293–300

Comparative Delta-endotoxins of Bacillus thuringiensis against

Mosquito Vectors (Aedes aegypti and Culex pipiens)*
EL¯BIETA LONC, JOLANTA KUCIÑSKA and KATARZYNA RYDZANICZ

Departament of Parasitology, Institute of Genetics and Microbiology,

University of Wroc³aw, 63 Przybyszewski Street, 51-148 Wroc³aw, Poland

Received 28 April 2003

Abstract

Pure crystals of seven Bacillus thuringiensis field isolates from the Lower Silesia region (Poland)
were tested against larvae of Aedes aegypti L. and Culex pipiens L. (Culicidae, Diptera). The crys-
tals of OpQ3 phylloplane isolate (belonging to the first biochemical type of B. thuringiensis subsp.
japonensis, yoso, jinghongiensis) killed from 68 ± 7% to 84 ± 7% of the fourth instar larvae of
A. aegypti. The crystals of two other strains (KpF3 and KpC1) of this group caused mortality
between 3 ± 2% and 70 ± 7%. The LC50 ranged from 3.2 ± 0.4 to 34.1 ± 4.8 :g/ml. The effect of
B. thuringiensis wratislaviensis H-47 crystals was the lowest with larval mortality from 0% to
17 ± 3%. No significant (0% – 37 ± 6%) effect of B. thuringiensis crystals on the larvae of C. pipiens
was observed. Our results show that the delta-endotoxins of B. thuringiensis act very specifically.

K e y w o r d s: B. thuringiensis, delta-endotoxins, anti-mosquito activity

Introduction

Bacillus thuringiensis is one of the best known entomopathogene, rod-shaped, gram-

positive, spore forming bacterium. It is used first of all in biological control of many
lepidopteran leaf-feeding pests. Its insecticidal activity is connected with parasporal,
crystalline proteins (delta-endotoxins) produced during sporulation (A r o n s o n and
S h a i 2001). Important progress in mosquito biocontrol was made in 1977 when
Goldberg and Margalit discovered B. thuringiensis israelensis H-14 (B e c k e r and
M a r g a l i t 1993). Consequently, in recent years there has been increased interest in
expanding B. thuringiensis culture collections as well as analysing and characterizing
them in search for novel anti-dipteran toxins (L e c a d e t et al., 1994, R a g n i et al.,
1996, L o n c et al., 2001a, b). The aim of this study was to determine the suscepti-
bility of two mosquito species (A. aegypti and C. pipiens) to some B. thuringiensis
isolates from natural environment of the Lower Silesia Region (Poland).

* Study was carried out within project nr 2027/W/IGiM

294 Lonc E. et al. 3

Experimental

Materials and Methods

Bacillus thuringiensis strains. The susceptibility of mosquitoes (A. aegypti L. and C. pipiens L.) is
assessed against seven B. thuringiensis strains derived from the bacterial collection of the Microbiologi-
cal Institute of the Wroc³aw University. Two of them, designated as PO12 and PO13, were the only
Polish isolates (amongst 69 B. thuringiensis subspecies, each including several hundred strains from
more than 22 countries) which have been classified according to the H serotype based on flagellar
antigens registered at the International Entomopathogenic Bacillus Centre (IEBC) Collection at Institut
Pasteur, Paris, France (L o n c et al., 1997, L e c a d e t et al., 1999). They were isolated from soil
samples from garden, flower-bed and park areas in Wroc³aw, and designated as a new serovar Bacillus
thuringiensis wratislaviensis H-47. The remaining five strains (KpC1, KpF3, KsS1, OpQ1 and OpQ3)
were selected for bioassays from 26 phylloplane and 33 soil isolates originating from the Karkonosze
National Park, a part of the Sudety Mts, and Osola, vicinity of Wroc³aw (D o r o s z k i e w i c z and
L o n c 1999). The tree names and strains origins were used to designate isolates: K = Karkonosze
National Park, O = Osola (sampling sites for leaf and soil samples); sample source: p = phylloplane,
s = soil; C = Corylus avellana, F = Fagus silvatica, Q = Quercus robur, S = Sorbus aucuparia. The
numbers reflect the number of successive isolates. On the basis of their biochemical activities these five
strains, have been classified (L e c a d e t et al., 1999) into the first, third and fourth biochemical types
comprising the subspecies: japonensis, yoso, jinghongiensis (strains: KpC1, KpF3, OpQ3) – I type,
finitimus (KsS1) – III and tochigiensis (OpQ1) – IV.
Stock bacterial cultures were maintained on brain-heart infusion agar (Difco) slants and stored
at 4°C.
Separation of parasporal crystals. B. thuringiensis strains were grown overnight on nutrient
agar plates. The inoculum was introduced with a sterilized bent wire into 40 ml of sporulation medium
containing: glucose – 10 g, casamino acids – 7.5 g, KH2PO4 – 6.8 g, MgSO4 × 7H2O – 123 mg, MnSO4 ×
4H2O – 2.23 mg, ZnSO4 × 7H2O – 14 mg, Fe2SO4 – 20 mg, H2O – 1000 ml; pH 7.5 (K a e l i n et al.,
1994). Such cultures were incubated at 28°C for 144 hours with shaking, giving a spore content of about
108 ml–1. Crystals and spores were harvested by centrifugation (5000 rpm, 30 min). The pellet was
washed twice with cold double destilled water and finally resuspended in 3 ml of 50 mmol/l–1 Tris-Cl
buffer, pH 7.5, containing 10 mmol/l of KCl (N i c h o l s et al., 1989). From this suspension crystals
were separated by centrifugation (14 000 rpm, 30 min) through a discontinuous sucrose gradient (67, 72,
79 and 87%). The crystal-containing band was collected, observed under a phase contrast microscope
(S h a r i f and A l a e d d i n o g l u 1988), pelleted by centrifugation (14 000 rpm, 30 min), washed
several times with double distilled water and solubilized in 50 mmol l–1 Na2CO3HCl, pH 10,5 for 1 h at
37°C (G i l l and F e d e r i c i 1987). The crystal concentrations were measured by the B r a d f o r d
(1976) assay with bovine serum albumin as the standard.
Mosquito culture. Larvae of A. aegypti were obtained from the laboratory culture of the Institute of
Organic Industry in Warsaw, Pszczyna Branch (Poland). The insects were reared in the laboratory at the
room temperature of 28°C and 70% RH. They were maintained in porcelain cups and fed an artifical diet
medium (product of Pszczyna) according to B y r d y (1965) and ¯ ó ³ t o w s k i (1976). Larvae of
Culex pipiens were collected from water bodies of the Wroc³aw area.
Mosquito bioassay. The activity of B. thuringiensis strains against mosquitoes was assessed accord-
ing to the International Entomopathogenic Bacillus Centre’s formula (T h i é r y et al., 1997). Twenty
specimens of fourth instar larvae (in plastic cups with 10 ml of diet) were exposed to each of four
concentrations: 6.25 :g/ml, 12.5 :g/ml, 25 :g/ml or 50 :g/ml of pure crystals. Three replicates were
made at each concentration including two controls: one with the distilled water and another with
B. subtilis B003 suspensions. The percentage of larvae mortality was corrected according to A b o t t ’ s
(1928) formula. The concentrations of toxic proteins at which 50% of larvae were killed (LC50) were
determined according to the logaritmic-probit method (F i n n e y 1971).
3 Delta-endotoxins of B. thuringiensis 295

Results

The susceptibility of A. aegypti, evaluated as the percentage of dead larvae, varied

from 0 to 84 ± 7% (Table I). Crystals of phylloplane isolates OpQ3, KpF3 and KpC1
(belonging to the first biochemical type, comprising the subspecies: japonensis, yoso,
jinghongiensis) showed the highest insecticidal activity (3 ± 2 – 84 ± 7%). The most

Table I
LC50 values and corrected mortality (%) of Aedes aegypti and Culex pipiens larvae treated with
parasporal crystals

Corrected Corrected
Symbol of
Subspecies (serovar)/ Crystal LC50 mortality of mortality of
Bacillus
No Biochemical type dosage (:g/ml) A. aegypti C. pipiens
thuringiensis
(:g/ml) %±s %±s
strain
24 h 48 h 24 h 48 h
1. OpQ3 japonensis, yoso, 6.25 68 ± 7 79 ± 9 0 0
jinghongiensis / I 12.5 3.2 ± 0.4 74 ± 4 81 ± 7 5±4 11 ± 8
25 77 ± 9 84 ± 4 15 ± 4 17 ± 6
50 82 ± 7 84 ± 7 34 ± 6 37 ± 6
2. KpF3 japonensis, yoso, 6.25 4±2 35 ± 3 0 0
jinghongiensis / I 12.5 11.5 ± 2.2 14 ± 4 55 ± 4 0 0
25 37 ± 6 60 ± 7 5±4 5±3
50 56 ± 6 70 ± 7 22 ± 5 23 ± 6
3. KpC1 japonensis, yoso, 6.25 4±3 3±2 0 0
jinghongiensis / I 12.5 34.1 ± 4.8 10 ± 6 10 ± 5 2±3 2±3
25 32 ± 6 32 ± 6 4±2 4±2
50 66 ± 11 68 ± 7 8±4 8±4
4. KsS1 finitimus (H2) / III 6.25 0 1±2 0 0
12.5 – 3±3 4±3 0 0
25 7±4 6±4 3±3 3±2
50 13±4 17 ± 5 6±2 7±3
5. OpQ1 tochigiensis (H19) / IV 6.25 1±2 3±2 1±2 1±1
12.5 – 4±4 6±5 2±1 3±2
25 6±5 9±3 9±5 11 ± 5
50 19 ± 3 22 ± 5 12 ± 5 15 ± 6
6. PO12 wratislaviensis (H47) 6.25 0 0 0 0
12.5 – 0 2±3 0 0
25 5±3 7±4 1±2 1±1
50 11 ± 5 12 ± 5 1±2 1±2
7. PO13 wratislaviensis (H47) 6.25 0 1±1 0 0
12.5 – 0 0±1 0 0
25 1±2 6±4 0 0
50 5±6 17 ± 3 0 0

LC50 – concentrations of toxic proteins at which 50% larvae were killed.

s – standard deviation.
296 Lonc E. et al. 3

Table II
Comparision of A. aegypti and C. pipiens corrected mortality (PS), Student t-test (" = 0.05)

Mortality Mortality
Dosage Symbol
Subspecies of of
of crystals of t p
(serovar) / Biochemical type A. aegypti C. pipiens
:g/ml strain
PS ± s PS ± s
6.25 OpQ3 japonensis, yoso, jinghongiensis / I 79±9* 0* 42.0 0.00
KpF3 japonensis, yoso, jinghongiensis / I 35±3 0 33.5 0.00
KpC1 japonensis, yoso, jinghongiensis / I 3±2 0 2.6 0.03
KsS1 finitimus (H2) / III 1±2 0 1.6 0.15
OpQ1 tochigiensis (H19) / IV 3±2 1±1 1.4 0.19
PO12 wratislaviensis (H47) 0 0 – –
PO13 wratislaviensis (H47) 1±1 0 3.2 0.01
12.5 OpQ3 japonensis, yoso, jinghongiensis / I 81±7 11±8 16.4 0.00
KpF3 japonensis, yoso, jinghongiensis / I 55±4 0 30.2 0.00
KpC1 japonensis, yoso, jinghongiensis / I 10±5 2±3 3.3 0.01
KsS1 finitimus (H2) / III 4±3 0 3.6 0.01
OpQ1 tochigiensis (H19) / IV 6±5 3±2 1.4 0.20
PO12 wratislaviensis (H47) 2±3 0 1.6 0.15
PO13 wratislaviensis (H47) 0±1 0 1.6 0.15
25 OpQ3 japonensis, yoso, jinghongiensis / I 84±4 17±6 24.2 0.00
KpF3 japonensis, yoso, jinghongiensis / I 60±7 5±3 17.4 0.00
KpC1 japonensis, yoso, jinghongiensis / I 32±6 4±2 10.2 0.00
KsS1 finitimus (H2) / III 6±4 3±2 1.5 0.17
OpQ1 tochigiensis (H19) / IV 9±3 11±5 –0.9 0.39
PO12 wratislaviensis (H47) 7±4 1±1 3.5 0.01
PO13 wratislaviensis (H47) 6±4 0 3.4 0.01
50 OpQ3 japonensis, yoso, jinghongiensis / I 84±7 37±6 13.0 0.00
KpF3 japonensis, yoso, jinghongiensis / I 70±7 23±6 12.2 0.00
KpC1 japonensis, yoso, jinghongiensis / I 68±7 8±4 17.6 0.00
KsS1 finitimus (H2) / III 17±5 7±3 4.5 0.01
OpQ1 tochigiensis (H19) / IV 22±5 15±6 2.4 0.04
PO12 wratislaviensis (H47) 12±5 1±2 4.8 0.01
PO13 wratislaviensis (H47) 17±3 0 13.2 0.00

s – standard deviation, t – value of Student t-test, p – test probability.

* statistically significant (p < ") values indicated in bold.

active strain was OpQ3 (68 ± 7% – 84 ± 7%). LC50 values were 3.2 ± 0.4 :g/ml,
11.5 ± 2.2 :g/ml and 34.1 ± 4.8 :g/ml, respectively. Toxins of the remaining isolates
B. thuringiensis finitimus KsS1, B. thuringiensis tochigiensis OpQ1 and B. thurin-
giensis wratislaviensis PO12 and PO13 caused mortality from 0% to 22 ± 5%.
3 Delta-endotoxins of B. thuringiensis 297

The mortality of C. pipiens larvae was significantly lower than that of A. aegypti.
The most toxic crystals (0% – 37 ± 6%) were isolated from the OpQ3 strain. Crystals
of the other two isolates of the first biochemical type were less active (0% – 23 ± 6%).
No significant effect (0% – 1 ± 2%) was observed after exposure of C. pipiens larvae
to B. thuringiensis wratislaviensis PO12 and PO13 toxins.
In most cases, the insecticidal activity of B. thuringiensis crystals was positively
correlated with their concentration. Some toxins were pathogenic only at higher dos-
ages. High mortality (66 ± 11% after 24 hours and 68 ± 7% after 48 hours) of
A. aegypti larvae was observed only after exposure to 50 :g/ml of the toxins of iso-
late KpC1. However, they were not susceptible ( 3 ± 2% – 32 ± 6%) to lower concen-
trations. Only the crystals of OpQ3 strain were highly toxic (68 ± 7% – 84 ± 7%) for
A. aegypti at all four dosages.
The differences in mosquito larval mortality were evaluated with Student t-test
(" = 0.05). In most cases they proved to be statistically significant (Table II). At the
highest concentrations, the crystals of all B. thuringiensis strains were much (p < 0.05)
more toxic to A. aegypti than to C. pipiens. Less significant differences were observed
at the two lowest dosages of toxins of isolates B. thuringiensis finitimus KsS1,
B. thuringiensis tochigiensis OpQ1 and B. thuringiensis wratislaviensis PO12 and
PO13. C. pipiens larvae proved to be much less susceptible to parasporal crystals of
all B. thuringiensis strains examined.

Discussion

The insecticidal activity of B. thuringiensis body inclusions was confirmed against

larvae of A. aegypti (0% – 84 ± 7%) and C. pipiens larvae (0% – 37 ± 6%). They
showed a high insecticidal specificity (expressed by corrected mortality) of delta-
endotoxins. In most cases, the insect mortality depended on the protein concentration
and exposure time. Most toxic for A. aegypti larvae were crystals of the isolates be-
longing to the first biochemical group (japonensis, yoso, jinghongiensis): OpQ3
(68 ± 7% – 84 ± 7%), KpF3 (4 ± 2% – 70 ± 7%) and KpC1 (3 ± 2% – 68 ± 7%). LC50
values (3.2 ± 0.4 :g/ml, 11.5 ± 2.2 :g/ml and 34.1 ± 4.8 :g/ml, respectively) were sig-
nificantly higher than those recorded by R a g n i et al. (1996) for five subspecies
highly pathogenic to A. aegypti: i.e. B. thuringiensis israelensis 1884 (20 ± 3 ng/ml),
B. thuringiensis morrisoni PG14 (26 ± 1 ng/ml), B. thuringiensis canadensis 11S2-1
(96 ± 40 ng/ml), B. thuringiensis thompsoni B175 (18 ± 1 ng/ml) and B. thuringiensis
malayesiensis IMR 81–1 (16 ± 1 ng/ml).
M i s z t a l et al. (1996) explained the connection between insect mortality and
exposure time. He reported that highly susceptible insects stopped feeding within
60 min and died within 1–7 hours after ingestion of toxin. Less susceptible ones
ceased to feed after 3–4 hours and are dead after 2–7 days. Insects which were only
slightly susceptible, stopped feeding after 10–15 hours and died after 2 or 3 weeks.
In this case, the reason for larva’s death was spore germination in its gut. This
observation would confirm the high susceptibility of A. aegypti to the toxins (at the
298 Lonc E. et al. 3

Table III
Anti-dipteran activity of the Wroc³aw isolates of B. thuringiensis

Corrected mortality of larvae [%]

Symbol of strain
L.p. (biochemical Aa Cp
group/serotype)
S+K1) K S+K2) K
1. OpQ3 (I) 54–66 68–84 23–45 0–37
2. KpF3 (I) 17–60 4–70 13–44 0–23
3. KpC1 (I) 30–61 3–68 26–34 0–8
4. KsS1 (III) 48–66 0–17 2–15 0–7
5. OpQ1 (IV) 41–54 1–22 3–12 1–15
6. PO12 (H47) 7–25 0–12 <1–2 0–1
7. PO13 (H47) 26–30 1–17 0–4 0

Aa – Aedes aegypti, Cp – Culex pipiens, Dm – Drosophila melanogaster, Md – Musca domestica.

S + K – spore/crystal suspensions (1) L o n c et al. 2001a, 2) R y d z a n i c z 2001, K – pure
crystals.

concentrations examined) of isolates OpQ3 and KpC1, because over 90% of A. aegypti
larvae died within 24 hour exposure time.
L o n c et al. (2001b) examined susceptibility of A. aegypti to spore/crystal
formulations of these B. thuringiensis strains (Table III). The most insecticidal iso-
lates to A. aegypti were OpQ3 (55–66%) – I biochemical type (representing sub-
species: japonensis, yoso, jinghongiensis) and B. thuringiensis finitimus KsS1
(48–66%). High mortality was observed also after exposure of larvae to three other
isolate suspensions: KpC1 (30–61%), KpF3 (17–60%) – I group and B. thuringiensis
tochigiensis OpQ1 (41–54%). The pure crystals of B. thuringiensis finitimus KsS1
and B. thuringiensis tochigiensis OpQ1 were not toxic to A. aegypti larvae; they
caused mortality from 0% to 17 ± 5% (KsS1) and from 1 ± 2% to 22 ± 5% (OpQ1).
According to E l l a r (1994) spores of B. thuringiensis kurstaki HD-1 significantly
increased the potency of crystals. The mixture of spores and crystals killed lepi-
dopteran larvae, Plutella xylostella sooner than the toxins alone, with 2.8-fold
decrease in the time required to reach 50% mortality. LC50 value decreased 146-fold
after the addidion of 108 ml–1 B. thuringiensis kurstaki HD-1 spores. Such results
suggest that although viable spores play little or no role in the action of lethal doses
of toxin against highly susceptible insects, they are important in enhancing the action
of sublethal doses of delta-endotoxin. This observation would explain the significant
differences in A. aegypti mortality caused by spore/crystal and pure crystals of isolate
B. thuringiensis finitimus KsS1 and B. thuringiensis tochigiensis OpQ1. The pure
crystals of B. thuringiensis wratislaviensis H-47 (PO12 and PO13) were not active
against A. aegypti. Mixture of spores and crystals of these isolates caused a higher
(7–30%) mortality (L o n c et al., 2001b).
No significant effect (0% – 37 ± 6%) on C. pipiens treated with crystals of the
Wroc³aw B. thuringiensis isolates was observed. The highly alkaline mosquito gut pH
3 Delta-endotoxins of B. thuringiensis 299

plays the principal role in delta-endotoxin activation. After ingestion by the insect,
crystals dissolve in the gut juice and gut proteases clip the protoxins off. The resulting
activated toxin binds to receptors on the epithelial cell membrane, inserts into the
membrane, and forms pores that disturb ion stability and finally cause the death of the
insect. For this reason, slightly alkaline pH of C. pipiens (7.2) could have influenced
the low mosquitocidal activity of B. thuringiensis crystals. At such pH they were only
partially or not dissolved. The activity of spore and crystal suspensions of the Wroc³aw
B. thuringiensis isolates against C. pipiens larvae was examined by R y d z a n i c z
(2001). She observed 0–45% mortality which in most cases was higher than that
caused by crystals alone (0% – 37 ± 6%). Larvae of the other dipteran, Musca
domestica, proved to be the most susceptible (41–81%) to B. thuringiensis spore/
crystal formulas (L o n c et al., 2001a). However, these suspensions were signifi-
cantly less toxic (10–45%) to the fruit fly, Drosophila melanogaster.

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Acta Microbiologica Polonica
2003, Vol. 52, No 3, 301–313

Improvement of Rifemycins Production

by Amycolatopsis mediterranei in Batch and Fed-batch Cultures
HESHAM A. EL-ENSHASY1*, USAMA I. BESHAY1, AHMED I. EL-DIWANY2,
HODA M. OMAR3, ABDEL GHANY E. EL-KHOLY 3 and RABAB EL-NAJAR1

1 Bioprocess Development Dept., Mubarak City for Scientific Research

and Technology Applications, New Burg Al-Arab, Alexandria, Egypt
2 National Research Centre, Tahrir Street, Dokki, Cairo, Egypt
3 Microbiology Dept., Faculty of Pharmacy, Alexandria University,

Alexandria, Egypt

Received in revised form 11 August 2003

Abstract

The production of rifamycins B and SV using glucose as main C-source by Amycolatopsis

mediterranei in batch and fed-batch culture was investigated. Fed-batch culture using glucose
as mono feeding substrate either in the form of pulse addition, in case of shake flask, or with
constant feeding rate, in bioreactor level, proved to be an alternative production system with
a significant increase in both volumetric and specific antibiotic production. The maximal concen-
trations of about 1146 mg/l and 2500 mg/l of rifamycins B and SV, respectively, was obtained
in fed-batch culture in bioreactor level under non-oxygen limitation. On the other hand, the rate
of rifamycins production was increased from 6.58 to 12.13 mg/l×h for rifamycin B and from
9.47 to 31.83 mg/l×h for rifamycin SV on the bioprocess transfer and improvement from the
conventional batch cultivation in shake flask to fed-batch cultivation in stirred tank bioreactor.

K e y w o r d s: rifamycin, A. mediterranei, fed-batch cultivation

Introduction

The rifamycins are a family of ansamycins antibiotics produced by Amycolatopsis

mediterranei in submerged fermentation either in free cell cultivation system
(G h i s a l b a et al., 1984; K r i s h n a et al., 1999; Ve n k a t e s w a r l u et al.,
2000) or immobilized cell cultivation system (A b u - s h a d y et al., 1995; F a r i d
et al., 1995). Rifamycins are characterized by wide spectrum activity against many
groups of bacterial cells with pronounced anti-mycobacterial action through the inhi-
bition of the initiation step of RNA synthesis by binding to beta-subunit of RNA
polymerase (H a r t m a n n et al., 1967). Therefore, they are extensively used in the

* Corresponding author: Dr. Hesham A. EL-Enshasy, E-mail: [email protected]

302 El-Endhasy H.A. et al. 3

clinical treatment of tuberculosis, leprosy and AIDS-related mycobacterial infections

(S e p k o w i t z et al., 1995; R o l a n d et al., 1997). For the production of
rifamycins, the antibiotic yield is highly influenced by three main factors. First is the
producer strain, the selection of most productive colony of the producer strain, even
in wild type strain, is critical factor in the production process since a large intra popu-
lation variation was observed in colony morphology of the producer strain when grow
in solid culture (F a r i d et al., 1996). Second is the cultivation condition. Therefore,
several studies have been done related to the effect of cultivation conditions such as
aeration, agitation, temperature, amount of inoculum and pH of cultivation medium
on the production of rifamycins (V i r i l i o et al., 1964; L e e et al., 1983;
K r i s h n a et al., 1998; Ve n k a t e s w a r l u et al., 1999). Moreover, the cultiva-
tion medium shows significant influence on the yield of antibiotic. Of different
nutrients tested for many years, glucose and yeast extract were the most effective
nutrients in the rifamycins production process. For most antibiotics by actinomycetes,
glucose exerts an inhibitory effect on antibiotic production when used at high concen-
tration through the catabolite repression effect (M a r t i n and D e m a i n, 1980;
E s c a l a n t e et al., 1999). Therefore, fed-batch cultivation strategy was preferred
for most of secondary metabolite production processes (G o m e s and M e n a w a t,
1998). For example, slow feeding of glucose increases the yield of chloramphenicol
by Str. venezuelae (B h a t n a g a r et al., 1988). Similarly, the yield of actinorhodin
production by Str. coelicolor was increased by the intermittent or continuous addi-
tions of glucose (A t e s et al., 1997).
On the other hand, the importance of the presence of yeast extract in the produc-
tion medium due to the presence of some growth factors such as B-factor has been
well studied (K a w a g u c h i et al., 1984, 1988; A z u m a et al., 1990). However,
the degree of influence of these nutrients was varied and strain dependent. Therefore,
the present work was undertaken to investigate the influence of some key nutrients,
namely glucose and yeast extract, on the process of rifamycins production by
Amycolatopsis mediterranei. Moreover, the kinetic characteristics of the rifamycin
production process were completely investigated in both shake flask and bioreactor
cultures during batch and fed-batch cultivation.

Experimental

Materials and Methods

Microorganism. Amycolatopsis mediterranei ATCC 21789 was obtained from The American Type
Culture Collection, USA. It was maintained on BENNET´s medium containing (g/l): 10.0 glucose, 1.0 yeast
extract, 1.0 beef extract, 2.0 N-Z amine, 20.0 agar. The pH was adjusted to 6.8 before sterilization. Agar
slants were incubated at 28°C for 5–7 days.
Inoculum preparation. Inoculum was prepared in vegetative culture medium contained (g/l): glu-
cose, 20.0; KH2PO4, 3.0; K2HPO4, 1.5; MgSO4 × 7H2O, 0.016; Zinc acetate, 0.001 and yeast extract, 5.0.
The pH was adjusted to 7.0 with 1 M NaOH. Cultivation was carried out for 48 h at 30°C on an incuba-
tor shaker at 200 rpm (Infors Co., Switzerland). The obtained vegetative cells were used to inoculate
either the shake flask or the bioreactor with a concentration of 8% (v/v).
3 Rifamycins production by A. mediterranei 303

Medium for rifamycins production. Unless otherwise mentioned, the medium used in both shake
flask and bioreactor experiments was composed of (g/l): glucose, 40.0; KH2PO4, 3.0; K2HPO4, 1.5;
MgSO4 × 7H2O, 0.016; Zinc acetate, 0.001 and yeast extract, 5.0. The pH was adjusted to 7.0 before
sterilization. Glucose was sterilized separately and added to the cultivation medium before inoculation.
Cultivation conditions. In case of shake flask, cultivation was carried out in 250 ml Erlenmeyer
flasks containing 50 ml liquid medium. Inoculum was in the form of 48 h old vegetative culture as
described previously and the inoculated flasks were incubated at 30°C on a rotary shaker at 200 rpm for
144 h. In case of bioreactor experiments, cultivation was carried out in a 3 l stirred tank bioreactor
Bioflow III (New Brunswick Scientific Co., New Brunswick, NJ, USA) with a working volume of 2.2 L.
Agitation was performed using a three 4-bladed rushton turbine impellers (di(impeller diameter) = 65 mm;
dt(tank diameter) = 135 mm, didt–1 = 0.48) at 600 rpm. Aeration was performed by filtered sterile air
[1 v/v × m]. Dissolved oxygen concentrations were analyzed by polarographic electrode (Ingold, Germany).
Foam was suppressed, when necessary, by the addition of silicon antifoam reagent (Fluka, Switzerland).

Analysis

Sample preparation and cell dry weight determination. During cultivation in shake flask, samples
in form of three flasks each were withdrawn intermittently for analysis. In case of bioreactor cultivation,
aliquots (in form of 20 ml) of the culture were taken from the bioreactor vessel through a sampling system.
Samples were filtered using dry and pre-weighed filter paper (Whatman filter paper No. 1). The superna-
tant was taken for determination of antibiotic activity and glucose concentration. The filtered biomass was
washed twice by distilled water and subsequently dried in an oven at 100°C for a constant weight.
Determinations of rifamycins activity. Rifamycins were determined spectrophotometrically ac-
cording to the method of P a s q u a l u c c i et al. (1970).
Determination of glucose. Glucose was determined in the fermentation media by enzymatic
method using a glucose determination kit (Glucose kit Cat. No. 4611, Biocon Diagnostic GmbH,
Burbach, Germany).

Results and Discussion

Effect of glucose concentration on rifamycins production. To investigate

effects of glucose concentration on production of rifamycins, batch cultures at various
concentration of glucose (0–120 g/l) in fermentation medium were performed in
shake flasks containing 50 ml medium for 120 h. Final cell dry weight, rifamycins
production and yield coefficients of rifamycins per cell mass are shown in Figure 1.
The cell growth was increased gradually with the increase of glucose concentration
from 0–50 g/l. Further increase in glucose concentration did not exhibit any further
significant increase in cell growth. The production of rifamycins was also increased
gradually with the increase of glucose concentrations from 0–40 g/l reaching the
maximal values of about 630 mg/l and 900 mg/l for rifamycin B and SV, respectively.
The rifamycins production was decreased gradually for further increase in glucose
concentration beyond 40 g/l. However, for better understanding of cell efficiency for
rifamycins production the specific rifamycins production [YrifaB/X and YrifaSV/X] in
(mg/g) was calculated. The maximal values of specific rifamycins production was
obtained on using glucose in a concentration of 40 g/l. From these results together, we
can conclude that the decrease in rifamycins production with the increase of glucose
more than 40 g/l was due to the catabolite repression effect. V e n k a t e s w a r l u
304 El-Endhasy H.A. et al. 3

110
100

YRifaSV/X
90
80
[mg/g] 70
60
YRifaB/X ,

50
40
30
20
10
0
rifamycin SV

1000

800
[mg/L]

600
rifamycin B,

400

200

0
18
16
14
CDW [g/L]

12
10
8
6
4
2
0
0 20 40 60 80 100 120
Glucose [g/L]
Fig. 1. Effect of initial glucose concentration on cell growth, rifamycins production
and yield coefficients of rifamycins based on cell dry weight.
Error bars correspond to samples taken from two independent shake flask experiments.

et al. (1999) reported that glucose was the best carbon source for production of
rifamycins by A. mediterranei MTCC14, but the maximal antibiotic production, about
1080 mg/l, obtained at 25 g/l glucose. D e m a i n et al. (1980) also reported that
carbon source, which support high specific growth, such as glucose, lead to the sup-
pression of antibiotic synthesis. Therefore, the development of fed-batch cultivation
strategy is necessary to increase the antibiotic yield to overcome the glucose inhibi-
tion effect in batch culture.
3 Rifamycins production by A. mediterranei 305

110

YRifaSV/X
100
90
80
[mg/g]
YRifaB/X, 70
60
50
40
30
20
10
0
rifamycin SV

1000

800

600
[mg/L]
rifamycin B,

400

200

0
18
16
14
CDW [g/L]

12
10
8
6
4
2
0
0 1 2 3 4 5 6 7 8 9
Yeast extract [g/L]

Fig. 2. Effect of initial yeast extract concentration on cell growth, rifamycins production
and yield coefficients of rifamycins based on cell dry weight.

Effect of yeast extract concentration on rifamycins production. Since yeast

extract is also an important nutrient in the process of rifamycins production, the effects
of initial yeast extract concentration on antibiotic production was studied. Figure 2
shows the effect of yeast extract concentrations on cell growth and rifamycins pro-
duction after 120 h cultivation in shake flask culture. An increase of the maximal
cell mass is observed as the initial yeast extract concentration increases up to 9 g/l
and reached a maximal concentration of about 14 g/l. On the other hand, it was
found that the rifamycins production was enhanced with the addition of yeast
306 El-Endhasy H.A. et al. 3

extract. Both of rifamycins B and SV concentrations were increased gradually as

the concentration of yeast extract increase from 0–5 g/l. Further increase in yeast
extract concentrations resulted in significant decrease in both of volumetric and
specific rifamycins yields. These results agree with those of K a w a g u c h i et al.
(1984) who studied the effect of yeast extract on the production of rifamycin by
Nocardia sp. They reported that an active substance purified from yeast extract, named
B-factor, is an inducer for rifamycin production. B-factor or its analogue seems to
play an essential regulatory role for rifamycins production in Nocardia strain
(K a w a g u c h i et al., 1988). From above results, it seems that yeast extract is
necessary for rifamycins production but may also exhibit an inhibitory effect when
applied at higher concentration than 5 g/l.
Production of rifamycins in batch and fed-batch cultures in shake flask level.
The growth pattern of A. mediterranei, glucose consumption and rifamycins produc-
tion in shake flasks were observed for 166 h in optimized fermentation medium con-
taining 40 g/l glucose and 5 g/l yeast extract (Figure 3). It can be seen that cell grew
exponentially with time with growth rate of about [µ= 0.016 h–1] reaching maximal
cell mass, about 10 g/l, after 124 h and kept more or less constant for the rest of
cultivation time. Whereas, the production of rifamycins started after a small lag phase
of about 20 h and increased gradually during the first 100 h of cultivation and reached
629 mg/l and 914 mg/l for rifamycins B and SV, respectively. The rates of rifamycins
production were of about 6.58 mg/l ×h for rifamycin B and with higher rate of about
9.47 mg/l ×h for rifamycin SV. During the rifamycins production phase, the first 100 h
cultivation, the rate of glucose consumption was about 0.30 g/l × h and this rate was
decreased to about only 0.075 g/l × h for the rest of cultivation time. The yield of
rifamycins production based on the cell mass for rifamycin B [YRifaB/X] was of about
68 mg/g and with a higher yield for rifamycin SV [YRifaSV/X] of about 98 mg/g.
Based on these results attempts were done to increase rifamycins production by
pulse feed of glucose during cultivation. The time course of pulse fed fermentation is
shown in Figure 4. After 96 h of cultivation, when the glucose level decreased to 5 g/l,
a pulse of glucose (5 ml containing 1 g glucose) was fed under sterile condition. This
increased the glucose concentration to about 25 g/l in the cultivation medium. With
the addition of glucose, a significant increase in both rifamycins B and SV was
observed without any effect on cell growth. One more pulse of glucose was added
after 144 h. After the addition of second pulse further increase in both volumetric and
specific rifamycins production was observed. The maximal volumetric yield of
rifamycins reached about 820 mg/l and 1200 mg/l for rifamycins B and SV, respec-
tively. Since the glucose feeding did not show any significant influence on the cell dry
weight, thus the yield of rifamycins based on cell mass increased significantly and
reached about 84 mg/g and 124 mg/g for rifamycins B and SV, respectively.
Production of rifamycins in bioreactor in batch and fed-batch cultures. Culti-
vation was carried out in 3 l bioreactor. Inoculum was in form of vegetative cells of
48 h and inoculum size was 10%. As shown in Figure 5, cells grew exponentially
during the first 96 h reaching 7.3 g/l with growth rate of 0.02 [h–1]. After that time, the
cell dry weight decreased again with time due to cell degradation. However, during
3 Rifamycins production by A. mediterranei 307

100
90 50
YrifaSV/X

YrifaSV/S
80
70 40
60
[mg/g]

[mg/g]
30
50
YrifaB/X ,

YrifaB/S ,
40 20
30
20 10
10
0 0
rifamycin SV

1000

800
[mg/L]

600
rifamycin B,

400

200

0
18 45
16 40
14 35
glucose [g/L]
CDW [g/L]

12 30
10 25
8 20
6 15
4 10
2 5
0 0
0 20 40 60 80 100 120 140 160 180

time [h]

Fig. 3. Growth, glucose consumption and rifamycins production during batch cultivation
of A. mediterranei in shake flask.

this growth phase the dissolved oxygen decreased reaching the minimal of about 40%
and increased again during the cell degradation phase. Glucose was consumed during
this phase with a rate of about 0.35 g/ l × h and the concentration was about 5 g/l after
96 h. On the other hand, the production of rifamycins reached 650 mg/l and 1190 mg/l
for rifamycins B and SV, respectively. The specific production of antibiotic was higher
in the bioreactor compared to the corresponding batch cultivation in shake flask
cultures. This was due to the higher mixing and better oxygenation in the bioreactor
308 El-Endhasy H.A. et al. 3

120 50
YrifaSV/X

YrifaSV/S
100
40
80
30
[mg/g]

[mg/g]
60
YrifaB/X ,

YrifaB/S ,
20
40
20 10

0 0
1400
rifamycin SV

1200
1000
800
[mg/L]

600
rifamycin B,

400
200
0
14
50
12

glucose [g/L]
10 40
CDW [g/L]

8 30
6
20
4
10
2
0 0
0 50 100 150 200
time [h]

Fig. 4. Effect of pulse feed of glucose on rifamycins production during cultivation

of A. mediterranei in shake flask.

which reflect on the cell physiological activities and secondary metabolite production
since also the production process of rifamycins could be increased with higher oxy-
gen supply (C h u n g et al., 1987). Based on this data of batch culture, fed-batch
cultivation strategy was developed with a constant feeding rate of 0.35 g glucose/l × h
after 90 h to keep the glucose concentration at the level of 5–7 g/l (Figure 6). As
feeding started, cells grew exponentially again with a specific rate of 0.054 [h–1] and
the cell dry weight reaching its maximum of about 14.4 g/l after 132 h. After
3 Rifamycins production by A. mediterranei 309

220
200
YrifaSV/X 180
[mg/g] 160
140
YrifaB/X ,

120
100
80
60
40
20
0
rifamycin SV

1400
1200
1000
[mg/L]

800
rifamycin B,

600
400
200
0
100
90 10 50
80
70 8 40
glucose [g/l]
CDW [g/L]
DO [%]

60
50 6 30
40
4 20
30
20 2 10
10
0 0 0
0 20 40 60 80 100 120 140 160 180 200
time [h]
Fig. 5. Cell growth, glucose consumption and rifamycins production during batch cultivation
of A. mediterranei in stirred tank bioreactor.
Data are the average of values taken from two bioreactor cultivations. The standard error based
on these two cultivations was calculated and expressed as the error bar in the figure.

the feeding phase, the cell concentration decreased again with a specific degradation
rate of 0.035 [h–1]. The cell degradation rate was almost the same in both of batch and
fed-batch cultures after the growth phase. This indicates that, the cell degradation
310 El-Endhasy H.A. et al. 3

Phase I Phase II Phase III

220
200
YrifaSV/X
180
160
140
120
[mg/g]

100
YrifaB/X ,

80
60
40
20
0
rifamycin SV

2500

2000
[mg/L]

1500
rifamycin B ,

1000

500

0
100 18 45
90 16 40
80 14 35
CDW [g/L]

glucose [g/L]
70 30
12
DO [%]

60
10 25
50
8 20
40
30 6 15
20 4 10
10 2 5
0 0 0
0 50 100 150 200
time [h]
Fig. 6. Cell growth, glucose consumption and rifamycins production during fed-batch cultivation
of A. mediterranei in stirred tank bioreactor.

was mainly as a result of mechanical shear stress in the stirred tank bioreactor more
than substrate deficiency effect. This phenomenon of cell autolysis as a result of
shear stress was also reported by other authors in case of cultivation of filamentous
microorganisms producing antibiotics in stirred tank bioreactor such as in case of
3 Rifamycins production by A. mediterranei 311

Table I
Kinetic parameters of cell growth and rifamycins production by A. mediterranei
during shake flask and bioreactor cultivations in batch and fed-batch cultures

Type of cultivation vessel

Parameter Shake flask Bioreactor
Batch Fed-batch Batch Fed-batch
Xmax [g/l] 10.0 9.7 7.3 14.4
Rifa Bmax [mg/l] 629 820 650 1146
Rifa SVmax [mg/l] 914 1204 1190 2500
µ [h ]
–1
0.016 0.018 0.020 0.054
–µ [h–1] nil – 0.030 0.035
Yx/s [g/g] 0.28 0.26 0.20 0.32
YRifaB/X [mg/g] 68 84.5 84.1 80.1
YRifaSV/X [mg/g] 98 124.2 165.1 172.8
QS [mg/l × h] 0.30 0.30 0.35 0.26
QRifa B [mg/l × h] 6.58 7.20 8.44 12.13
QRifa SV [mg/l ×h] 9.47 10.04 12.66 31.83
– QRifa B [mg/l × h] 0.60 – 4.38 –
– QRifa SV [mg/l × h] nil – 7.58 18.24
Abbreviations:
Xmax: maximal cell dry weight; Rifa Bmax: maximal rifamycin B production, Rifa SVmax:
maximal rifamycin SV production; µ: specific cell growth rate, – µ: specific cell degradation
rate, QS: glucose consumption rate; Qp: volumetric antibiotic production rate, – Qp: volumet-
ric antibiotic degradation rate.
Yield coefficients:
Yx/s: [g] of cell dry weight / [g] glucose consumed.
YRifaB/X: [mg] rifamycin B produced / [g] cell dry weight.
YRifaSV/X: [mg] rifamycin SV produced / [g] cell dry weight.

erythromycin (H e y d a r i a n et al., 1996), penicillin (H a r v e y et al., 1998) and

streptomycin (E l - E n s h a s y et al., 2003).
On the other hand, the production of rifamycins in fed-batch culture is almost
doubled compared to the batch culture (Figure 6 and Table I). In order to investigate
the reason for the increase in the volumetric production in the fed-batch culture
compared to the batch culture under the same cultivation conditions, the specific pro-
duction of rifamycins (Figure 6) was also calculated from the data of volumetric
production and cell dry weight of the same figure. As shown, the specific production
of rifamycins was almost the same for batch and fed-batch cultures (Figure 5 and
Figure 6) during either growth phase or glucose feeding phase. However, the signifi-
cant increase in specific production values [YRifa B/X and YRifaSV/X] after 140 h in
case of batch culture was as a result of cell degradation. Since the specific produc-
tion rates in case of batch and fed-batch cultures were almost the same in both
cultures, the increase in volumetric production of rifamycins in fed-batch culture with
312 El-Endhasy H.A. et al. 3

glucose feeding was not due to the specific production, but to the increased cell
concentration. Supplemental glucose in the fed-batch culture prevented glucose
deficiency for certain extent and thus increased the cell mass, resulting in an increase
in the volumetric production.
In general, the different stages of bioprocess development for the production of
rifamycins are summarized in Table I. This show clearly the significant increase in
the total volumetric antibiotic production through the process transfer from shake
flask to bioreactor culture and switching the process from batch to fed-batch manner.

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Acta Microbiologica Polonica
2003, Vol. 52, No 3, 315–316

BOOK REVIEW

A.R. Ronald / D.E. Low (Eds)

Fluoroquinolone Antibiotics
2003, 272 pages. Hardcover
CHT 178 / EUR 112
ISBN 3-7643-65919-9

The book “Fluoroquinolone Antibiotics” received for review is a consecutive title in

the very interesting, informative and important series “Milestones in Drug Therapy”.
Edited by A.R. Ronald and D.E. Low, the volume embraces 15 contributions written
by authorities on the mechanism of action of the fluroquinolone antibiotics, their phar-
macokinetics and clinical applications and bacterial resistance to these agents. Every
chapter is furnished with an extensive list of literature references which allow the
interested reader to further pursue her or his interest.
The first modern fluoroquinolone antibiotic to be developed (in the late 1970s)
and launched in 1984, was norfloxacin. The drug was very active against several
gram-negative pathogens and also showed some activity against streptococci and
staphylococci.
The history of the quinolones in general and of course modern-day fluoro-
quinolones is described in the first very interesting chapter of the reviewed volume,
written by Sheehan and Chew. This is followed by a chapter on the structure-activity
relationships of quinolones (by Mitscher and Ma) which gives, amongst others,
a description of the various modifications to the original (classical) structures that
have resulted in broad-spectrum drugs as well as those targeted, for instance, at
streptococcal infections. This chapter also points towards future developments which
may be efficient at bacteria that develop resistance to the currently used drugs.
The third chapter of the book, written by Jo and Cheng, deals with quinolone
resistance among specific bacteria and brings data from many countries, including
Poland. The authors also discuss the problem of the contribution of animals use and
suboptimal human use to the development of bacterial resistance to the fluoroquino-
lones. The mechanisms of resistance are discussed very briefly, maybe somewhat too
generally in this reviewer’s opinion. Also, lacking is a discussion of the “two-edged
sword” action of the quinolones which being potent antibacterials at the same time
contribute to the emergence of resistance to themselves. The next two chapters deal,
respectively, with the safety profile of the newer fluoroquinolones (Mandell) which in
this regard are much better than the earlier drugs that had many, frequently unpleasant
316 Book Review 3

side effect and with the pharmacokinetics and pharmacodynamics (Zhanel and
Noreddin) of this group of drugs.
The following nine chapters of the book are devoted to the use of the fluoro-
quinolones in specific infections, such as urinary tract infections (Johnson), sexually
transmitted infections (Wyllie and Ridgway), pneumonia and other respiratory tract in-
fections (Marrie and Low, respectively). These chapters should be of particular interest
to physicians dealing with these particular infections since besides general information
they also present specific data frequently collected from various in-depth studies.
Finally, the last, very short chapter by the editors of the volume (Ronald and Low)
discusses the future prospects of the quinolones, with special emphasis on the fact
that these antibiotics are not readily destroyed biologically in the environment and
their persistence may therefore generate additional resistance. The authors predict
that by 2010 there should be 5–6 additional fluoroquinolones that will augment cur-
rent therapeutic regimens.
The book brings a lot of important and up-to-date information on this interesting
group of antibiotics and is highly recommended to clinicians as well as researchers in
pharmacology, clinical medicine or epidemiology. I am not sure whether researchers
in molecular biology will find it equally useful, this in view of the mentioned scant
data on the mechanism of action of these drugs as well as the mechanism of bacterial
resistance to them.

Zdzis³aw Markiewicz
Acta Microbiologica Polonica
2003, Vol. 52, No 3, 317

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and methods should be described in the text, not in figure legends or table footnotes. Present
acknowledgments as in full-length papers.
Minireviews are published in areas of particular interest and importance. They are usu-
ally invited, but authors wishing to submit a minireview should contact the scientific editor
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Before writing a manuscript authors are advised to consult a current issue of Acta Micro-
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tables and the rules concerning chemical, biochemical, genetic etc. nomenclature recommended.
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