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Secondary metabolites of soil Bacillus spp

Article  in  Biotechnology Letters · April 2011

DOI: 10.1007/s10529-011-0617-5 · Source: PubMed

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Biotechnol Lett (2011) 33:1523–1538
DOI 10.1007/s10529-011-0617-5

REVIEW

Secondary metabolites of soil Bacillus spp.

Estibaliz Sansinenea • Aurelio Ortiz

Received: 31 January 2011 / Accepted: 24 March 2011 / Published online: 29 April 2011
Ó Springer Science+Business Media B.V. 2011

Abstract Bacillus species produce secondary Introduction

metabolites that are the object of natural product
chemistry studies. The wide structural variability of In 1891, Kossel defined secondary metabolites by
these compounds has attracted the curiosity of exclusion (compounds that do not belong to primary
chemists and their biological activities have inspired metabolites), provoking criticism which has never
the pharmaceutical industry to search for lead struc- ceased. The current, generally accepted concept, in
tures in microbial extracts. Screening of microbial line with Kossel’s view, is that primary metabolites
extracts reveals the large structural diversity of are chemical components of living organisms that are
natural compounds with broad biological activities, vital for their normal functioning, while secondary
such as antimicrobial, antiviral, immunosuppressive, metabolites are compounds which are dispensable.
and antitumor activities, that enable the bacterium to Secondary metabolites are structurally highly diverse
survive in its natural environment. These findings and each compound is produced only by a small
widen the potential industrial importance of Bacillus number of species (Karlovsky 2008).
spp., particularly of B. thuringiensis, beyond insec- For many years, secondary metabolism was virtu-
ticidal usage and may help explain the role of ally ignored; study of this type of non-essential
Bacillus spp. in the soil ecosystem. metabolism was left to industrial scientists and
academic chemists and pharmacognocists. Today,
Keywords Antibiotics  Antimicrobials  the situation is different. The wide structural variabil-
Bacillus spp.  Bio-control  Secondary metabolites ity of these compounds has attracted the curiosity of
chemists and the biological activities possessed by
natural products have inspired the pharmaceutical
industry to search for lead structures in microbial
cultures and plant extracts. Many economically valu-
able microbial products are secondary metabolites
(Karlovsky 2008). Secondary metabolites serve: (i) as
competitive weapons used against other bacteria,
fungi, amoebae, plants, insects, and large animals; (ii)
E. Sansinenea (&)  A. Ortiz as metal transporting agents; (iii) as agents of
Facultad de Ciencias Quı́micas de la Benemérita
symbiosis between microbes and plants, nematodes,
Universidad Autónoma de Puebla, 72570 Puebla,
PUE, México insects, and higher animals; (iv) as sexual hormones;
e-mail: [email protected]; [email protected] and (v) as differentiation effectors (Demain and Fang

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1524 Biotechnol Lett (2011) 33:1523–1538

2000). The MALDI (Matrix-Assisted Laser Desorp- The metabolites produced by Bacillus spp. can also
tion Ionization) imaging can be used to observe affect the microflora in the rhizosphere, providing an
natural product production and chemical communi- environment antagonistic to pathogens, or they can
cation between microorganisms, providing an addi- trigger host defense responses (Velusamy and Gnana-
tional tool for exploration of interspecies interactions manickam 2008). There are two very recent reviews
and symbiotic or pathogen-host communication of about secondary metabolites of B. thuringiensis,
organisms grown in culture, and explaining the disappointingly only two secondary metabolites are
biological effects of secondary metabolites (Yang mentioned in these reviews (Bode 2009; Zhou et al.
et al. 2009). 2008). In this review we focus on the all secondary
The genus Bacillus consists of a large number of metabolites produced by Bacillus spp. in soil and
diverse, rod-shaped Gram-positive bacteria that are B. thuringiensis reviewing all literature currently
motile by peritrichous flagella and are aerobic. available on this topic.
Members of the genus are capable of producing
endospores that are highly resistant to unfavorable
environment conditions (Claus and Berkeley 1986). Secondary metabolites from Bacillus sp. in soil
The Bacillus species subtilis, licheniformis, amylo-
liquefaciens, and pumilus are closely related. There Bacteriocins, lantibiotics and miscellaneous
are several species of the genus that are known antibiotics
pathogens. These include B. anthracis, which is
pathogenic to humans and other animals, and The potential of B. subtilis to produce antibiotics has
B. cereus, which is a common cause of food poisoning been recognized for 50 years with peptide antibiotics
(Claus and Berkeley 1986). B. thuringiensis, and representing the predominant class. Bacteriocins are
some strains of B. sphaericus are pathogenic to certain proteins or ribosomal peptides with bactericidal
insects. Bacillus species have special characteristics activity towards species that are often closely related
that make them good candidates as biological control to the producer bacteria and display variable molec-
agents (Wulff et al. 2002). First, they are well known ular weights, biochemical properties, inhibitory spec-
as antibiotic producers with antagonistic activity tra, and mechanisms of action. Due to their potential
against fungal and some bacterial pathogens. Second, use as natural preservatives, bacteriocins produced by
they form spores that can be easily formulated, and lactic acid bacteria have been the subject of intensive
have high viability compared with vegetative cells. investigation in recent years. Bacillus spp. appears to
Third, they are commonly found in soils. be a relatively abundant source of antimicrobials,
B. thuringiensis is a Gram-positive, spore-forming, since many species of this genus synthesize antimi-
soil bacterium and is the most successful biological crobial peptides (Cherif et al. 2001; Lisboa et al.
control agent that produces distinctly shaped crystals 2006). The possibility of screening for a new Bacillus
during sporulation. These crystals are composed of bacteriocin producer is considered to be one of the
proteins, known as insecticidal crystal proteins major interests in bacteriocin research. B. subtilis, the
(ICPs), also called Cry proteins, which are selectively model system for Gram-positive organisms, is able to
toxic to different species of several invertebrate produce more than two dozen antibiotics with a
phyla. Much of the technology developed to study variety of structures (Stein 2005).
structure and function of Cry proteins has provided Several bacteriocins have been reported such as
the foundation for genetic engineering of this class lichenin produced by the B. licheniformis 26-103RA
of biopesticides (Sansinenea et al. 2010). Formerly, strain (Pattnaik et al. 2001), megacin produced by
only the insecticidal properties of B. thuringiensis strains of B. megaterium (Lisboa et al. 2006),
attracted extensive attention. However, B. thuringi- antilisterial coagulin produced by B. coagulans
ensis has also been shown to produce other active (Le Marrec et al. 2000), polyfermenticin SCD pro-
components that are secondary metabolites, some duced by B. polyfermenticus (Lee et al. 2001), and
only recently discovered. cerein produced by strains of B. cereus (Torkar and
Bacillus spp. secrete many secondary metabolites, Matijašić 2003; Bizani et al. 2005a, b). Moreover,
including antibiotics, antifungals and siderophores. some of these bacteriocins have been indicated as

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Biotechnol Lett (2011) 33:1523–1538 1525

potential biopreservatives in food systems and bever- system. The genes encoding the biosynthesis of the
ages, as agents for biological control of phytopatho- dipeptide bacilysin 1 and its antibiotic constituent
gens (Bais et al. 2004), and as antibiotic precursors. anticapsin were isolated from several strains of
Bacteriocin production and secretion was observed B. subtilis as well as B. amyloliquefaciens and
in stationary phase, after 10–16 h of bacterial pop- B. pumilus (Steinborn et al. 2005). Bacilysin produc-
ulation growth in BHI broth at 30°C. As is well tion of B. subtilis 168 parallels active growth in a
known, bacteriocins of lactic acid bacteria, particu- synthetic medium and becomes inhibited by certain
larly lantibiotics, are usually produced in the mid- growth conditions, i.e. supplements and above 30°C.
growth phase. The production of bacteriocins takes Its synthesis seems to be under transcription regula-
place successfully in both broth media and on solid tion via the stringent response (Inaoka et al. 2003) as
media (Khalil et al. 2009). The biopreservation well as under feedback regulation, likely by a
capacity of a bacteriocin could be achieved either component of the global quorum-sensing control
by using a bacteriocin producing starter culture or by system. In contrast to B. subtilis 168, over-production
applying the bacteriocin itself as a food additive. of bacilysin in B. amyloliquefaciens GSB272 toler-
Therefore, a thorough study of the essential param- ated higher concentrations of yeast extract up to 0.2%
eters affecting the inhibitory activity, followed by (vs 0.01%) and was achieved even at 37°C (vs 30°C)
optimization of production that is usually dependent (Steinborn et al. 2005).
on multiple strain-specific factors is necessarily There are some other miscellaneous antibiotics
required for introducing a bacteriocin into foods as produced by B. subtilis such as bacilysocin 2
a potent biopreservative. For this purpose Khalil et al. (Tamehiro et al. 2002), 3,30 -neotrehalosadiamine
(2009) described the influence of cultural and phys- (NTD) 3 (Inaoka and Ochi 2007), and amicoumacin
ical conditions such as nutrients, nutrient concentra- 4 (Pinchuk et al. 2002) (Fig. 1) among some others.
tions, nutrient combinations/interactions, aeration, Bacilysocin 2 is an anti-microbial phospholipid that
and other physical factors, including heat, UV, pH, can be isolated from B. subtilis 168 cells by
and storage conditions, to obtain better and more extraction with butanol, and is derived from the
stable bacteriocin activity from a newly isolated major B. subtilis phospholipid phosphatidylglycerol
strain of B. megaterium. through YtpA-catalysed acyl ester hydrolysis (Tame-
Peptide antibiotics with an inter-residual thioether hiro et al. 2002). The aminosugar antibiotic
bonds as unique feature are called lantibiotics. The 3,30 -neotrehalosadiamine (NTD) 3, structurally 3,30 -
best-characterized is subtilin, a 32-amino-acid penta- diamino-3,30 -dideoxy-a,b-trehalose, was originally
cyclic lantibiotic of B. subtilis. Subtilin production is identified as an antibiotic produced by several
controlled both by culture density in a quorum- Bacillus species such as, B. pumilus and B. circulans
sensing mechanism in which subtilin plays a phero- (Tsuno et al. 1986). Inaoka et al. (2004) showed that
mone type role and in response to the growth phase the production of NTD, dormant in the wild-type
(Stein et al. 2002). There are some other lantibiotics, strain, can be induced by a rifampicin-resistant
such as ericin, mersacidin, sublancin and subtilolisin phenotype of the RNA polymerase. The aminosugar
A, that are produced by B. subtilis and are well NTD 3 inhibits growth of Staphylococcus aureus and
described in another review (Stein 2005). Klebsiella pneumoniae and functions as an autoin-
Bacilysin 1 (Fig. 1), a non-ribosomally synthe- ducer by activating its own biosynthetic operon
sized dipeptide, is composed of L-alanine and the (Inaoka and Ochi 2007). NTD functions as a glucose
unusual amino acid L-anticapsin and represents one uptake modulator in B. subtilis and probably in other
of the simplest peptide antibiotics known with NTD producing bacteria, with a gene arrangement
antifungal and antibacterial activities. Bacilysin 1 is identical to that in B. subtilis. Amicoumacins 4 are
effective as a biocontrol agent, notably by inhibiting produced by several B. subtilis strains excluding the
the growth of Erwinia amylovora (Arguelles-Arias 168 strain (Pinchuk et al. 2002). Their anti-bacterial
et al. 2009). Its antibiotic activity depends on the and anti-inflammatory activities, as well as their
anticapsin moiety, which becomes released by pep- action on Heliobacter pylori make the amicoumacins
tidases (Chmara et al. 1982) after bacilysin 1 uptake attractive for the treatment of chronic gastritis and
into susceptible cells by a distinct peptide permease peptic ulcer in humans.

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1526 Biotechnol Lett (2011) 33:1523–1538

Fig. 1 Chemical structures

of miscellaneous antibiotics
and surfactin (lipopeptide)
and its isoforms

Lipopeptides due to its high cost of production and recovery (Das

et al. 2008).
The lipoheptapeptide surfactin 5 (Fig. 1) is a power- Various strategies, such as strain improvement,
ful biosurfactant; it exerts a detergent-like action on medium optimization, bioreactor design or using
biological membranes (Carrillo et al. 2003), and is agroindustrial wastes for fermentation to reduce
distinguished by its exceptional emulsifying, foam- the raw material cost, have been implemented to
ing, anti-viral and anti-mycoplasma activities. A host achieve improved biosurfactant production. Several
of interesting features of surfactin has led to a wide approaches have been tried to improve surfactin 5
range of applications such as antibacterial, antiviral, yields both at the flask and fermentor level by
anti-adhesive and anti-inflammatory uses. In spite of changing environmental parameters, and optimizing
the immense potential for commercial, therapeutic medium components or trace elements. Both sub-
and environmental applications, its use is still limited merged (SMF) and solid-state fermentation (SSF)

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Biotechnol Lett (2011) 33:1523–1538 1527

have been tried for surfactin 5 production. Batch binding, bacterial pathogenesis, quorum sensing,
fermentation for surfactin 5 must be terminated at an motility, biofilm formation etc. The iturin family
appropriate time in order to avoid the utilization of (Fig. 2) encompasses the closely related cyclic lipo-
surfactin as a carbon source, suggesting that the heptapeptides mycosubtilin 6 (Moyne et al. 2004), the
supply of glucose is critical since it acts as a major iturines 7 and the bacillomycins 9, that contains one
carbon source. Surfactin production is the highest in a b-amino fatty acid and seven a-amino acids, and
defined medium with ammonium nitrate as nitrogen exhibit strong antifungal and hemolytic activities and
source. The recovery and concentration of biosurfac- a limited antibacterial activity (Stein 2005). Biolog-
tants account for a major portion of the total ical effects of the iturin family peptides are due to
production cost. The separation strategies for biosur- their capability of forming ion-conducting pores
factants vary according to the fermentation process (Maget-Dana and Peypoux 1994). Iturin A 7 and
and the physicochemical properties of the surfactant bacillomycin L 9 provoked hemolysis and released
in question (Shaligram and Singhal 2010). potassium from erythrocytes (Aranda et al. 2005).
Some biosurfactants may be used as alternatives to Iturin A 7 contains the heptapeptide Asn1-Tyr2-
synthetic medicines and antimicrobial agents (Shali- Asn3-Gln4-Pro5-Asn6-Ser7, whereas in the other
gram and Singhal 2010). Their production is widely members the amino acid residues in the heptapeptides
distributed among B. subtilis, B. pumilus, B. lichen- vary slightly; e.g. mycosubtilin 6, that was isolated
iformis (Tendulkar et al. 2007) and B. amylolique- from B. subtilis, has Asn1-Tyr2-Asn3-Gln4-Pro5-
faciens strains (Wulff et al. 2002) and thus, a variety Ser6-Asn7. Iturin A 7 was antagonistic to Fusarium
of surfactin 5 isoforms have been described under oxysporum, which cause potato diseases (Han et al.
different synonyms such as bacircine 5a, halo- and 2005). B. amyloliquefaciens strains B94 and FZB42,
isohalobacillin 5b, lichenysin A/G 5c, daitocidin 5d produce iturins that also inhibit fungal plant patho-
and pumilacidin 5e (Fig. 1) (Kalinovskaya et al. gens (Yu et al. 2002; Kilian et al. 2000). In
2002). The variations are due to changes in the lipid B. amyloliquefaciens FZB42, bacillomycin D 9 is
portion and/or the amino acid composition. Licheny- responsible for the main antifungal activity exerted
sin 5c belongs to the family of the lipopeptide by this bacterium and was shown to suppress growth
biosurfactants produced by Bacillus spp. In addition of such phytopathogenic fungi as Fusarium oxyspo-
to its surface activity, this compound exhibits various rum (Ramarathnam et al. 2007; Koumoutsi et al.
interesting biological properties making it attractive 2004). Fengycin 8 (synonymous to plipastatin) com-
for both industrial and biological applications (Gran- bines several exceptional structural properties: cycli-
gemard et al. 2001). The antibiotic pumilacidins 5e zation, branching and unusual constituents. Fengycin
A, B, C, D, E, F and G were isolated from the culture 8 is specifically active against filamentous fungi
broth of a strain of B. pumilus. They are cyclic (Stein 2005). Having in hand the whole genome
acylheptapeptide composed of a b-hydroxy fatty acid, sequences of environmental Bacillus strains, we can
two L-leucine, two D-leucine, L-glutamic acid, L- see that the gene clusters of many of these surfactants
aspartic acid and L-isoleucine (or L-valine). Pumilac- are found in some Bacillus genomes (Chen et al.
idin components were inhibitory to herpes simplex 2007, 2009), and we are in position to use the
virus type 1 and H?, K?-ATPase and demonstrated accumulated knowledge of Bacillus molecular biol-
antiulcer activity in rat (Naruse et al. 1990). ogy to investigate the different aspects of lifestyle,
B. amyloliquefaciens is closely related to B. including biofilm formation, of a motile low GC-soil
subtilis so many lipopeptides are produced by both bacterium in its natural environment.
microorganisms (Lisboa et al. 2006; Chen et al. 2007; Often the amount and type of a raw material can
2009) among them are iturin A 7 (Fig. 2) (Yu et al. contribute considerably to the production cost; it is
2002; Pyoung et al. 2010) and Fengycin 8 (Jacques estimated that raw materials account for 10–30% of
et al. 1999) that are cyclic lipopeptides (CLP). These the total production cost in most biotechnological
surfactants may also play different roles in the processes. Thus to reduce this cost it is desirable to
development and survival of Bacillus strains in their use low-cost raw materials for the production of
natural habitat: increasing bioavailability of hydro- biosurfactants. One possibility explored extensively
phobic water-insoluble substrates, heavy metal is the use of cheap and agro-based raw materials as

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1528 Biotechnol Lett (2011) 33:1523–1538

Fig. 2 Chemical structures

of lipopeptides and
polyketides

substrates for biosurfactant production. A variety of induce the maximum or optimum productivity.
cheap raw materials, including plant-derived oils, oil Similarly, efficient downstream processing tech-
wastes, starchy substances, lactic whey and distillery niques and methods are needed for maximum product
wastes have been reported to support biosurfactant recovery. Novel recombinant varieties of these
production. An efficient and economical bioprocess is microorganisms, which can grow on a wide range
the foundation for every profit-making biotechnology of cheap substrates and produce biosurfactants at high
industry. Hence bioprocess development is the yields, can potentially bring the required break-
primary step towards commercialization of all bio- through in the biosurfactant production process
technological products, including biosurfactants. Any (Muthusamy et al. 2008). This is possible only if
attempt to increase the yield of a biosurfactant the genetics of the microbial surfactant production is
demands optimal addition of media components and known in details. It is therefore desirable that the
selection of the optimal culture conditions that will future research on biosurfactants be focused on the

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Biotechnol Lett (2011) 33:1523–1538 1529

development and use of hyperproducers. A detailed Streptococcus aureus and Streptococcus spp. Most
knowledge of the genetics of microbial surfactant Gram-negative organisms and yeasts are resistant.
production should be used to produce organisms The maximum activity of bacitracin was observed for
giving higher production with better product charac- S. aureus during 24 and 48 h of incubation at 30°C at
teristics (Das et al. 2008). an alkaline pH of 8.0–9.0 with 5% (w/v) glucose
(Awais et al. 2008).
Polyketides
Secondary metabolites of Bacillus thuringiensis
In addition to peptides, polyketides are the other
dominant family of secondary metabolites having Among Bacillus species, B. thuringiensis is the best-
relevant bioactivities. They are synthesized on known and best-studied entomopathogenic bacterium
modularly organized assembly lines starting from that produces parasporal protein crystals, which are
acyl-CoA precursors by decarboxylative Claisen selectively toxic to different species of several
condensations. In general their biosynthetic pathway invertebrate phyla (Bode 2009).
follows the same logic as in non-ribosomally synthe- Many isolates of B. thuringiensis also produce an
sized peptides and requires at least three domains. assortment of various other virulence factors that are
Essential domains of the modules harbored in bacte- secreted into the culture medium. These factors
rial type I polyketide synthases are acyl transferase include the vegetative insecticidal proteins (Vips),
(AT), ketosynthase (KS) and an acyl carrier protein and b-exotoxin I 13, also called thuringiensin 13
(ACP) which needs to be activated by Ppant-trans- (Fig. 3), a nonproteinaceous toxin (Espinasse et al.
ferase (Chen et al. 2009). 2002). Unlike the Vip and Cry toxins, b -exotoxin I
Three functional gene clusters directing the syn- 13 is not specific and thus, may have detrimental
thesis of difficidin 10, bacillaene 11 and macrolactin effects on nontarget organisms; it is particularly
12 (Fig. 2) were identified in FZB42 and GA1 strains active against dipteran species, but it is also active
of B. amyloliquefaciens. Difficidin 10 is an unsatu- against coleopteran, lepidopteran, and some nema-
rated 22-membered macrocyclic polyene lactone tode species. This toxin inhibits the synthesis of RNA
phosphate ester with broad spectrum antibacterial by competing with ATP for binding sites, thereby
activity. It inhibits protein biosynthesis and was affecting insect molting and pupation and causing
recently shown promising in its suppressive action teratogenic effects at sublethal doses (Espinasse et al.
against Erwinia amylovara, a devasting plant patho- 2002, 2004). The genetic determinants responsible
gen causing necrotrophic fire blight disease of apple, for b-exotoxin I production found on Cry-dependent
pear and other rosaceous plants (Arguelles-Arias plasmids are likely to be regulatory elements and
et al. 2009). The bae gene cluster was assigned to large amounts of b-exotoxin I can be produced in the
synthesis of the bacillaene polyketide 11 (Chen et al. absence of such plasmids. A putative ABC trans-
2009), an inhibitor of prokaryotic protein synthesis porter, encoded by berAB, is essential for b-exotoxin
with the empiric formula C35H48O7. The third I production. b-exotoxin I production can be acti-
polyketide with macrolid-like structure, macrolactin vated in response to particular environmental condi-
12, was originally detected in an unclassified deep- tions (Espinasse et al. 2002, 2004).
sea marine bacterium (Jaruchoktaweechai et al. The parasporal crystal proteins and spores, which
2000). Macrolactins 12, contain three separated diene are the primary toxic substances to insects, are
structure elements in a 24- membered lactone ring. At inactivated quickly after exposure to sunlight. It was
least 17 macrolactins have been described and one of shown that melanin is an excellent photoprotective
them, 7-O-malonyl macrolactin A, was effective agent and because of this photoprotection of the
against Gram-positive bacterial pathogens (Romero- mosquitocidal activity of B. thuringiensis israelensis,
Tabarez et al. 2006). Bacitracin is a mixture of related using melanin, has been studied. Several research
cyclic polypeptides produced by organisms of the groups have obtained B. thuringiensis mutants pro-
licheniformis group of Bacillus. Bacitracin inter- ducing melanin from B. thuringiensis (Espinasse
feres with bacterial cell wall synthesis and is et al. 2002; Saxena et al. 2002) but there are wild-
primarily active against the Gram-positive bacteria type strains of B. thuringiensis that produces melanin

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1530 Biotechnol Lett (2011) 33:1523–1538

Fig. 3 Bacillus
thuringiensis some
secondary metabolites

as a secondary metabolite (Che et al. 2004). Despite Zwittermicin A 14 has an unusual chemical structure
the appearance of melanin together with b-exotoxin that includes a D-amino acid and ethanolamine and
I during growth, regulation of the production of pig- glycolyl moieties, as well as having an unusual
ment is probably independent of that of b-exotoxin I. terminal amide that is generated from the modifica-
B. thuringiensis is also a producer of zwittermicin tion of the nonproteinogenic amino acid ureidoala-
A 14 (Fig. 3), a potent antibiotic and antifungal nine. The diverse biological activities and unusual
compound. This compound was firstly isolated in B. structure of Zwittermicin A 14 have stimulated
cereus for its ability to suppress damping-off disease interest in understanding how this antibiotic is
in alfalfa caused by Phytophthora medicaginis and its biosynthesized.
structure is a linear aminopolyol antibiotic (Silo-Suh The identification of the complete Zwittermicin A
et al. 1998). More importantly, zwittermicin A 14 biosynthesis gene cluster from B. cereus UW85
enhances the activity of the endotoxin from has been recently reported (Kevany et al. 2009). The
B. thuringiensis against insects (Zhou et al. 2008). biosynthesis of Zwittermicin A 14 does not originate

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Biotechnol Lett (2011) 33:1523–1538 1531

in carbohydrate metabolism, but from a nonriboso- produced by soil or pathogenic bacteria such as
mal peptide synthetase/polyketide synthase pathway Bacilli remains unclear. In addition to petrobactin 16,
(NRPS/PKS) involving two newly described type 1 B. anthracis, B. thuringiensis and B. cereus also
PKS extender units: hydroxymalonyl-acyl carrier secrete large quantities of the petrobactin precursor,
protein (ACP) and aminomalonyl-ACP. For industrial 3,4-DHB 17. Although the role of 3,4-DHB 17 as a
purposes a rapid method for producing the antibiotic siderophore was suggested for a magnetotactic bac-
is needed, and thus a large-scale purification method terium, Magnetospirillum magneticum (Calugay et al.
was developed based on ion-exchange chromatogra- 2006), it also appears to be able to serve as a
phy and HPLC. Zwittermicin A 14 was difficult to siderophore in Bacillus.
isolate in substantial quantities due to its highly polar, Bacillibactin 15 synthesis is dependent on func-
charged nature at physiological pH, and its sensitivity tional Ppant-transferase (Sfp) (Chen et al. 2009). The
to alkaline conditions. The difficulty in obtaining enzymes responsible for petrobactin 16 biosynthesis
the compound from natural sources underscores an are members of a family of proteins termed nonrib-
outstanding need for practical syntheses of zwitter- osomal peptide synthetase-independent siderophore
micin A 14, which has been recently reported (Rogers (NIS) synthases (Koppisch et al. 2008a). Biosynthe-
and Molinski 2007, 2009). sis of DHB 17 through early shikimate intermediates
One of the most widely utilized mechanisms of is favored over a mechanism involving late inter-
microbial iron acquisition is the production and mediates or aromatic amino acids (Koppisch et al.
secretion of siderophores, low molecular weight iron 2008b).
chelators that bind ferric iron in the environment with As above described, the genus Bacillus encom-
extremely high affinity and shuttle it into the cells. passes a number of bacteriocinogenic species. Sev-
Thus, the presence of siderophore-producing micro- eral bacteriocins have been partially characterized
organisms in the rhizosphere contributes to plant from B. thuringiensis: thuricin from strain HD2
health by complexing iron and making it less (Favret and Yousten 1989), tochicin from strain
available to phytophathogens that are generally not HD868 (Paik et al. 1997), kurstakin 18 (Fig. 3)
able to produce comparable Fe-transport systems (Hathout et al. 2000), thuricin 7 from strain BMG1.7
(Arguelles-Arias et al. 2009; Chen et al. 2009). All (Cherif et al. 2001), thuricin 439A and thuricin 439B
members of the B. cereus group, including B. subtilis from strain B439 (Ahern et al. 2003), entomocin from
and B. licheniformis, secrete bacillibactin 15 (Fig. 3), B. thuringiensis subsp. entomocidus HD9 (Cherif
a 2,3-dihydroxybenzoyl-Gly-Thr trilactone sidero- et al. 2003), bacthuricin F4 from B. thuringiensis
phore and is also produced by B. thuringiensis. In subsp. kurstaki strain BUPM4 (Kamoun et al. 2005),
addition, B. anthracis and B. cereus primarily thuricin S from B. thuringiensis subsp. entomocidus
produce petrobactin 16 and 3,4-dihydroxybenzoic HD198 strain (Chehimi et al. 2007), and thuricin CD
acid (3,4-DHB) 17 (Fig. 3) which were considered 19 (Fig. 3) from strain DPC 6431 (Rea et al. 2010).
virulence factors for these bacteria (Zawadzka et al. Thuricin exhibited a bactericidal and bacteriolytic
2009). Although it was recently shown that both are effect on a sensitive strain, and was active against
also produced by innocuous B. thuringiensis isolates, several Bacillus species (B. thuringiensis, B. mega-
indicating that by themselves are not indicative of terium, B. polymyxa, and B. sphaericus), but was not
virulence (Koppisch et al. 2008a). Petrobactin 16, active against B. licheniformis and B. macerans strain
which contains two 3,4-catecholate moieties and a (Favret and Yousten 1989). Tochicin showed a
citrate-based backbone, was first isolated from the narrow antibacterial spectrum of activity against
marine bacterium Marinobacter hydrocarbonoclasti- most of 20 typical B. thuringiensis strains and a
cus. The photoreactivity of ferric complexes formed strain of B. cereus, but not against other bacteria and
with citrate-based siderophores produced by marine yeasts tested (Paik et al. 1997). The kurstakin 18
bacteria, including petrobactin and aerobactin, was structure is closely related to the structure of other
suggested to be a mechanism for the production of lipopeptides synthesized by Bacillus species, fungi,
biologically available ferrous iron. However, the and yeasts. However, its amino acid composition
possible role of such photoreactivity in siderophores incorporates a His residue, which is rare in the

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bacterial lipopeptides known today. Its antifungal Conclusions and perspectives

activity was demonstrated against Stachybotrys cha-
ratum (Hathout et al. 2000). Thuricin 7, which is Bacillus spp. are a relatively abundant source of
bactericidal and bacteriolytic (Cherif et al. 2001), was antimicrobials since many species of this genus
active against several species of the genus Bacillus, synthesize antimicrobial peptides. These bacteria in
including three of the four known B. thuringiensis/ general represent a new and rich source of secondary
B. cereus bacteriocin producers, as well as against metabolites that need to be explored. These second-
Streptococcus pyogenes and Listeria monocytogenes ary metabolites exhibit strong antifungal and anti-
strains. The large spectrum of activity, biochemical bacterial activities and enable the bacterium to
and physical properties, molecular mass, and the survive in its natural environment (Stein 2005).
kinetics of production differentiate entomocin These findings widen the potential industrial impor-
from the other bacteriocins produced either by the tance of Bacillus spp. beyond just insecticidal usage
B. cereus group or by lactic acid bacteria (Cherif and may help explain the role of Bacillus spp. in the
et al. 2003). Thuricin S exhibits a large inhibitory soil ecosystem. The view that secondary metabolites
spectrum, which includes Listeria monocytogenes, act by improving the survival of the producer in
Salmonella enterica, and Pseudomonas aeruginosa competition with other living species has been
(Chehimi et al. 2007). Thuricin CD 19 consists of two expressed more and more in recent years in terms
distinct peptides, Trn-a and Trn-b, that act synergis- of having important biological effects (Demain and
tically to kill a wide range of clinical Clostridium Fang 2000), as MALDI imaging demonstrates (Yang
difficile isolates. However, this bacteriocin thuricin et al. 2009).
CD 19 has little impact on most other genera, Environmental factors affect microbial secondary
including many gastrointestinal commensals (Rea metabolism. Stress, starvation or supplementation
et al. 2010). with nutrients such as amino acids, phosphate,
Bacteriocins are generally isolated from cultures nitrogen, etc., have a plethora of effects on the
under laboratory conditions. During the long extrac- production of secondary metabolites. It has long been
tion, isolation, and purification process, they are standard practice in the pharmaceutical industry to
potentially subject to contamination by other bacteria develop specific and often complex culture media to
and related microorganisms. Like other compounds maximize the production yields of secondary metab-
used in research studies, they are stored prior to their olites such as antibiotics. Many mutations have been
use in research applications. Nevertheless, these introduced into so-called production strains to
bacteriocins must be free of other contaminating enhance the yield of a particular antibiotic and in
bacteria when used for research activities. There is a the majority of cases the genetic and biochemical
lack of information regarding their stability and nature of these strains is unknown. The growth of
activity following sterilization procedures. Bacterio- antibiotic-producing organisms in the presence of low
cins are completely degraded by autoclaving and concentrations of organic compounds (such as
were similarly affected by the tested filter mem- DMSO) results in qualitative and quantitative
branes. Polyvinylidene fluoride (PVDF), polyester- changes in secondary metabolite production. This
sulfone (PES), and cellulose acetate (CA) are could have value in natural product drug discovery
suitable for filter sterilization of these bacteriocins programs, since solvent-induced effects may reveal
(Woo-Jin et al. 2008). Unlike the previously the presence of secondary metabolites that are
described B. cereus group bacteriocins, which show otherwise present in low concentrations under normal
a decrease in their antibacterial activities after the growth conditions (Chen et al. 2000).
induction of sporulation, entomocin bactericidal Lipopeptide antibiotics are among the most fre-
activity was constant for more than 50 h of incuba- quently produced Bacillus antibiotics that have
tion indicating its high stability and resistance to well-recognized potential biotechnology and biophar-
inactivation or degradation (Cherif et al. 2003). In maceutical applications due notably to their surface-
Table 1 secondary metabolites discussed in this active properties. Some possible roles for such
review have been summarized collecting some bioemulsifiers have been proposed in pollution and
information about them. environmental control (Muthusamy et al. 2008).

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Table 1 Secondary metabolites of soil Bacillus spp.

Bacteria Compound Structure class Biological effects Reference

B. licheniformis Lichenin Bacteriocin Bactericidal, bacteriolytic Pattnaik et al. (2001)

B. megaterium Megacin Bacteriocin Bactericidal, bacteriolytic Lisboa et al. (2006)
B. coagulans Coagulin Bacteriocin Bactericidal, bacteriolytic Le Marrec et al. (2000)
B. polyfermenticus Polyfermenticin Bacteriocin Bactericidal, bacteriolytic Lee et al. (2001)
B. cereus Cerein Bacteriocin Bactericidal, bacteriolytic Bizani et al. (2005)
B. subtilis Subtilin Lantibiotic Antibacterial Stein (2005)
B. subtilis Ericin Lantibiotic Antibacterial Stein (2005)
B. subtilis Mersacidin Lantibiotic Antibacterial Stein (2005)
B. subtilis Sublancin Lantibiotic Antibacterial Stein (2005)
B. subtilis Subtilolysin Lantibiotic Antibacterial Stein (2005)
B. subtilis, Bacilysin 1 Dipeptide Antifungal antibacterial Chmara et al. (1982)
B. amyloliquefaciens,
B.pumilus
B. subtilis Bacilysocin 2 Phospholipid Fungicidal antibacterial Tamehiro et al. (2002)
B. subtilis, B. pumilus, NTD 3 Aminosugar Antibacterial Tsuno et al. (1986)
B. circulans
B. subtilis, B. pumilus Amicoumacin 4 Isocoumarin Antibacterial, anti- Pinchuk et al. (2002)
inflammatory
B. subtilis Surfactin 5 Cyclic lipoheptapeptide Hemolytic, cytotoxic Carrillo et al. (2003)
B. subtilis, Bacircine 5a Cyclic lipoheptapeptide Hemolytic, cytotoxic Kalinovskaya et al. (2002)
B. amyloliquefaciens,
B.pumilus
B. licheniformis Halobacillin 5b Cyclic lipoheptapeptide Hemolytic, cytotoxic Kalinovskaya et al. (2002)
B. licheniformis Isohalobacillin Cyclic lipoheptapeptide Hemolytic, cytotoxic Kalinovskaya et al. (2002)
5b
B. licheniformis Lichenysin 5c Cyclic lipoheptapeptide Hemolytic, cytotoxic Grangemard et al. (2001)
Bacillus sp. Daitocidin 5d Cyclic lipoheptapeptide Hemolytic, cytotoxic Kalinovskaya et al. (2002)
B. pumilus Pumilacidin 5e Cyclic lipoheptapeptide Antiulcer activity in rat Naruse et al.(1990)
B. subtilis Mycosubtilin 6 Cyclic lipoheptapeptide Hemolytic, fungicidal Moyne et al. (2004)
B. subtilis, Iturin 7 Cyclic lipoheptapeptide Hemolytic, fungicidal Aranda et al. (2005)
B. amyloliquefaciens
B. subtilis, Fengycin 8 Cyclic lipoheptapeptide Antifungal Koumoutsi et al.( 2005)
B. amyloliquefaciens
B. subtilis, Bacillomycin 9 Cyclic lipoheptapeptide Hemolytic, fungicidal Aranda et al. (2005)
B. amyloliquefaciens
B. amyloliquefaciens Difficidin 10 Polyketides Antibacterial Arguelles-Arias et al. (2009)
macrolactone
B. amyloliquefaciens Bacillaene 11 Polyketides Antibacterial Chen et al. (2009)
macrolactone
B. amyloliquefaciens Macrolactin 12 Polyketides Antibacterial Jaruchoktaweechai et al.
macrolactone (2009)
B. thuringiensis b-exotoxin 13 Adenine nucleotide Insecticidal Espinasse et al. (2002)
analog
B. thuringiensis Melanin Polyacetilene derivative Photoprotective Espinasse et al. (2002)
B. thuringiensis, Zwittermicin 14 Aminopolyol Antibiotic, antifungal Silo-Suh et al. (1998)
B. cereus

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Table 1 continued
Bacteria Compound Structure class Biological effects Reference

B. subtilis, Bacillibactin 15 Catecholate siderophore Iron acquisition Arguelles-Arias et al. (2009)

B. licheniformis,
B. thuringiensis,
B. cereus,
B. anthracis
B. thuringiensis, Petrobactin 16 Catecholate siderophore Iron acquisition Zawadzka et al. (2009)
B. cereus,
B. anthracis
B. thuringiensis, DHB 17 Catecholate siderophore Iron acquisition Zawadzka et al. (2009)
B. cereus,
B. anthracis
B. thuringiensis Thuricin Bacteriocin Bactericidal Favret and Yousten (1989)
Bacteriolytic
B. thuringiensis Tochicin Bacteriocin Bactericidal Paik et al. (1997)
B. thuringiensis Kurstakin 18 Bacteriocin Fungicidal Hathout et al. (2000)
B. thuringiensis Thuricin 7 Bacteriocin Bactericidal Cherif et al. 2001
Bacteriolytic
B. thuringiensis, Thuricin 439A/ Bacteriocin Bactericidal Ahern et al. (2003)
anthracis B Bacteriolytic
B. thuringiensis Entomocin Bacteriocin Bactericidal Cherif et al. (2003)
B. thuringiensis Bacthuricin Bacteriocin Fungicidal Kamoun et al. (2005)
B. thuringiensis Thuricin S Bacteriocin Bactericidal Chehimi et al. (2007)
Bacteriolytic
B. thuringiensis, Thuricin CD 19 Bacteriocin Bactericidal Rea et al. (2010)
anthracis Bacteriolytic

These surfactants may also play different roles in the the synthesis of a diverse array of elaborate chemical
development and survival of Bacillus strains in their structures, which have remarkable biological activi-
natural habitat (Das et al. 2008). One vital factor ties (Stein 2005).
determining their biosynthesis is the genetic makeup Due to the importance of PKS and NRPS derived
of the producer organisms. Studies on molecular compounds, the next frontier was to investigate the
genetics and biochemistry of the synthesis of several corresponding gene clusters. As evidenced by
biosurfactants have revealed the operons, the genome sequencing, bacteria dedicate up to 20% of
enzymes and the metabolic pathways required for their genome to the biosynthesis of secondary
their extracellular production. metabolites, underscoring the importance of these
Many of the therapeutically most useful and best small molecules to the fitness of the organism in its
understood natural products are synthesized princi- native environment (Gross 2007). B. subtilis produces
pally, by two different biosynthetic pathways for more than two dozen antibiotics. If all pathways are
peptides which allow the incorporation of unusual considered, their production requires more than
(non-proteinaceous) constituents: (i) the non-ribo- 350 kb, corresponding to a remarkable 10% of the
somal synthesis of peptides by large megaenzymes, annotated ORFs. It should be emphasized that all
the non-ribosomal peptide synthetases (NRPSs) and investigated B. subtilis strains produce individual
the polyketide synthases (PKSs) and (ii) the ribo- antibiotic cocktails encompassing only a portion of
somal synthesis of linear precursor peptides that are the compounds depicted above; the average of a
subjected to post-translational modification and pro- B. subtilis genome that is devoted to antibiotic
teolytic processing. Both molecular systems result in production is about 4–5%. The potential of a given

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Biotechnol Lett (2011) 33:1523–1538 1535

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