Chan et al.

BMC Microbiology 2011, 11:99

http://www.biomedcentral.com/1471-2180/11/99

RESEARCH ARTICLE Open Access

Cloning, purification, and functional

characterization of Carocin S2, a ribonuclease
bacteriocin produced by Pectobacterium
carotovorum
Yung-Chieh Chan1, Jian-Li Wu1, Huang-Pin Wu2, Kuo-Ching Tzeng3 and Duen-Yau Chuang1*

Abstract
Background: Most isolates of Pectobacterium carotovorum subsp. carotovorum (Pcc) produce bacteriocins. In this
study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight
bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is
inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C.
Results: A carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A
5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to
the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized,
and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably,
carocin S2 could be expressed not only in the mutant TF1-2 but also in Escherichia coli DH5a after entry of the
plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of
colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and
that of CaroS2I was 10 kDa.
Conclusion: This study shown that another nuclease type of bacteriocin was found in Pectobacterium carotovorum.
This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity
of CaroS2I.

Background Bacteriocins are bactericidal, extracellular toxins,

The phytopathogenic enterobacterium, Pectobacterium produced by both Gram-positive and Gram-negative
carotovorum subsp. carotovorum, is a phytoparasitic, bacteria [6,7]. These proteinaceous molecules kill closely
Gram-negative, facultative anaerobic bacterium [1]. Pcc related bacteria. The susceptible cell is recognized by
produces many extracellular pectic enzymes (pectate specific target receptors on the membrane, and the pro-
lyase, pectin lyase, exopolygalacturnoate lyase) and ducer cell evades lethality by expressing a cognate
hydrolytic enzymes causing soft-rot disease, tissue immune protein. The colicin family produced by Escher-
maceration, and cell wall collapse [2,3]. The only cur- ichia coli is divided into DNase (colicins E2, E7, E8 and
rent strategy against soft-rot disease involves chemical E9), RNase (colicins E3, E4 and E6), tRNase (colicins
agents that unavoidably contaminate the environment D and E5), and pore-forming colicins (colicins A, E1, Ia
[4]. Kikumoto et al. have demonstrated that mixed bac- and Ib) [8]. Bacteriocins (especially nuclease bacterio-
teriocin-producing avirulent strains of Pcc show high cins) have a high amino acid sequence homology.
efficacy against soft-rot disease of Chinese cabbage [5]. Natural bacteriocin molecules act via a number of
mechanisms. For example, colicin E3 is a well-known
* Correspondence: [email protected] ribonuclease that specifically cleaves 16S rRNA at the
1
Department of Chemistry, National Chung-Hsing University, 250, Kuokuang 3’-end of the coding sequence both in vivo and in vitro,
Rd., Taichung, 402, Taiwan
Full list of author information is available at the end of the article which leads to the abolishment of protein synthesis

© 2011 Chan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Chan et al. BMC Microbiology 2011, 11:99 Page 2 of 12
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resulting in death of the susceptible cell [9-12]. Previous agar medium containing rifampin and kanamycin. In
reports indicate that colicin E3 consists of a killer protein bacteriocin assay, the size of the inhibition zone around
with three domains (i.e., a translocation domain [T each isolate was compared with that of F-rif-18. Mutant
domain], receptor binding domain [R domain], and colonies were identified by smaller inhibition zones. This
nuclease domain) and an immunity protein that retards evidence of mutation suggested that transposon Tn5 had
antibiotic activity [13,14]. The R domain recognizes a speci- been inserted into LMW bacteriocin-related genes. The
fic receptor, BtuB on the cell membrane and the T domain strain TF1-2, a putative insertion mutant, would no
interacts with the TolB protein in the cell periplasm of the longer produce LMW bacteriocin (Figure 1).
sensitive cell to facilitate entry of the killer domain through To ascertain whether Tn5 was actually introduced into
the cell membrane. In addition to the attack mechanism, the genomic DNA of putative isolates, the nptII gene of
the immunity mechanism has been extensively elucidated. isolates was amplified using two primers P3 and P4 [23].
Notably the immunity protein and the killer protein inter- Southern blot technology showed that Tn5 had been
act initially at very high affinity because of charge attrac- inserted (Additional file 1, Figure S1).
tion, and are separated at the cell surface through energy
generated from the proton motif force [15-17]. Identification of Tn5-inserted DNA Structures
In general, the C-terminal domain determines the type To identify Tn5-interrupted genes, genomic DNA from
of bacteriocin. The C-terminal nuclease domains are not TF1-2 was amplified with TAIL-PCR using an array of
only interchangeable but also lack species specificity specific primers (Additional file 1, Figure S8). A 2621-bp
[18]. Strikingly, the tRNase type of bacteriocin may DNA fragment, including two open reading frames
accelerate exhaustion of tRNA in the cytoplasmic pool (ORFs), was identified as the sequence containing the
and thereby impair protein synthesis in vivo. Ogawa et bacteriocin structural gene. This gene was designated the
al. have demonstrated that particular tRNA molecules carocin S2 gene. To characterize the carocin S2 gene,
can be digested by colicin D as well as by colicin E5 the TF1-2 probe was designed to hybridize in Southern
[19,20]. It has been suggested that phage-associated kle- blots with a Bam HI-digested DNA fragment from the
bicin D is a tRNase type of bacteriocin based on similar- genomic library of F-rif-18 (Figure 2A). A 5706-bp Bam
ity to the nuclease-like domain of colicin D [21]. HI-digested DNA fragment (Figure 2B), harboring two
Nguyen et al. reported production of a high-molecular- complete ORFs of carocin S2, was cloned into the plasmid
weight bacteriocin (carotovoricin Er) and Chuang et al. pMCL210 (Additional file 1, Figure S2). The carocin-
reported production of a low-molecular-weight bacteriocin
(LMWB; carocin) by Pectobacterium[22,23]. The former
has a bulky antenna-like tail, inner core, and contractile
cylindrical structure, and the carotovoricin-caused inhibi-
tion zone can be easily distinguished from that of carocin
by its low diffusibility. Carocin S1 is a deoxyribonuclease
type of LMWB (indicated by the letter S) and is secreted
by Pcc strain 89-H-4. Additionally, export of Carocin S1
utilizes the type III secretion system in Pcc, which also
controls the cell motility of the bacterium [24].
Pcc strain F-rif-18 is a spontaneous rifampin-resistant
mutant of the wild-type 3F-3. Ultraviolet radiation can
induce Pcc strain F-rif-18 to produce the LMWB Caro-
cin S2. One of several sensitive cells, SP33, was selected
as an indicator strain here. In the present study, the
chromosomal bacteriocin gene, carocin S2, was intro-
duced into an expression plasmid encoding two pro-
teins, CaroS2K and CaroS2I. These proteins were
purified and characterized and their primary activities of
killing (CaroS2K) and immunity (CaroS2I) were investi-
gated in vivo and in vitro.
Figure 1 Bacteriocin assays of Tn5 insertion mutants of Pcc
Results strains. Strain number: 1, 3F3 (wild type); 2, 1830 (E. coli); 3, F-rif-18
Isolation of Transposon Insertion Mutants (parent); 4, TF1-1 and 5, TF1-2 (insertion mutant). Other unlabelled
Conjugation between F-rif-18 and E. coli 1830 resulted in strains are Tn5 insertion mutants of F-rif-18 strain. The indicator is
Pcc strain SP33.
~3,500 colonies after selection on Modified Drigalski’s
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Figure 2 DNA library screening and scheme of carocin S2 gene. (A) The TF1-2 probe was used to screen DNA fragments from the genomic
DNA library of F-rif-18. The DNA was digested with various restriction enzymes as follows: 1. Hpy188I; 2. HindIII; 3 HpaI; 4. EcoRV; 5. EcoRI; 6. ClaI;
7. BsaAI; 8. BglII; 9. BamHI; 10. AhdI; M. DNA leader marker; C. The TF1-2 probe DNA. The arrowhead indicates the 5.7-kb carocin S2 fragment. (B)
Shown is the 5.7-kb segment of DNA containing the carocin S2. The location of TF1-2 probe and part amplicon of cDNA of caroS2K and caroS2I
were shown.

producing plasmid was designated as pMS2KI. The ampli- Transcriptional analysis and in vivo expression of carocin
con, comprising the predicted ORF2 of caroS2I, was S2 gene
subcloned into the pGEM-T easy vector, resulting in the To determine whether the carocin S2 gene is transcribed
plasmid pGS2I (Additional file 1, Figure S5). in a series of recombinant strains, reverse transcription-
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PCR was used to estimate RNA level. Two sets of inter- individual promoter for caroS2I gene is located behind
genic primers were designed to amplify parts of transcripts the Tn5 insertion site in the caroS2K gene. CaroS2I
from caroS2K or caroS2I, respectively (Figure 2B). Amplifi- transcripts were detected in strain SP33 with plasmid
cation of parts of 16S ribosomal RNA transcripts indicated pGS2I (lanes 6 and 7). Although both the SP33 strains
that RNA in these bacterial cells is expressed at normal (with or without pGEM T-easy) were susceptible to Car-
levels (Figure 3). ocin S2, SP33/pGS2I appeared to grow in the presence
The presence of the 925-bp amplicon revealed that of CaroS2K (Figure 4B).
caroS2K was being transcribed in the cell (panel car- To prove that pMS2KI contained the gene for Carocin
oS2K in Figure 3). The TF1-2 strain, which is a Tn5 S2, pMS2KI was introduced into TF1-2 and E. coli
insertional mutant, could not transcribe caroS2K (lane 2), DH5a. Both TF1-2/pMS2KI and DH5a/pMS2KI had
but the ability of TF1-2 to transcribe caroS2K was ability to express the activity of Carocin S2 (Figure 4A).
restored by introduction of pMS2KI (lane 3). It was The size of inhibition zone around strain TF1-2/
apparent that the amount of caroS2K expression was pMS2KI was equal to that around DH5a/pMS2KI but
dependent on the number of copies of plasmid pMS2KI still smaller than that around the wild-type strain F-rif-18.
(compare lane 1 to lane 3). Additionally, carocin S2 can On the other hand, the quantity of transcripts expressed
be expressed in E. coli strain DH5a by introduction of in vivo and in vitrodid not usually correspond.
pMS2KI (lane 4 and lane 5).
The presence of a 259-bp amplicon showed that Deduction of the amino acid sequence of Carocin S2
caroS2I was transcribed constitutively (panel caroS2I in The carocin S2 gene consists of two ORFs (Additional
Figure 3). The caroS2I gene was transcribed unexpect- file 1, Figure S7): one containing the 2352-bp caroS2K
edly in mutant strain TF1-2 even though the plasmid gene and the other containing the 273-bp caroS2I gene.
pMS2KI was introduced (lane 3). This demonstrated The stop codon (TGA) of caroS2K overlaps the first
that caroS2I is expressed constitutively regardless of start codon of caroS2I by 4-bp (ATGA). The amino
whether the gene caros2K is transcribed. Possibly an sequences were deduced from the nucleotide sequence

Figure 3 Reverse Transcription PCR of RNA. Shown are cDNA from the following strains: Lanes 1, F-rif-18; 2, TF1-2; 3, TF1-2/pMS2KI, 4, DH5a;
5, DH5a/pMS2KI.; 6, SP33; 7, SP33/pGS2I. The amplicons of caroS2K and caroS2I are 925 bp and 259 bp, respectively. The corresponding
amplicons of 16S rRNA from the examined strains (lower panel). All samples were loaded equally.
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amino acid segment between Asp677 and the C-terminus

of CaroS2K shares almost 60% similarity with the
minimal tRNase domain of colicin D and klebicin D
(Figure 5). Since the colicin D and klebicin D are
well-known tRNase family of bacteriocins, suggests that
Carocin S2 might therefore be a ribonuclease.

Purification and characterization of Carocin S2

E. coli BL21 (DE3) recombinants, which were trans-
formed with pES2KI or pES2I, were used to express
CaroS2K protein or CaroS2I protein individually. Coo-
massie blue stained SDS-PAGE gels of purified Carocin
S2 are shown in Figure 6. The band corresponding to
CaroS2K was purified. The gel indicates a relative mass
(M r ) of about 85 kDa (Figure 6A), enrichment of the
purified CaroS2K (arrowhead), and disappearance of
other bands. Purification of CaroS2I by the same proce-
dure resulted in a more intense band in the region of
Mr 10 kDa (arrowhead; Figure 6B).
The purified CaroS2K involved in the growth inhibi-
tion of the susceptible indicator strain SP33 was then
characterized. The number of viable cells decreased with
increasing concentration of CaroS2K (Figure 7). Almost
all cells were dead at the initial concentration of 4 μg
ml-1, indicating that about 90% of indicator strains are
killed at this concentration. However, the activity of
CaroS2K was inhibited by trypsin, but not inhibited by
CaroS2I.

Carocin S2 has ribonuclease activity

In order to confirm the role of carocin S2 as a ribonu-
clease type bacteriocin, we set up a RNA degradation
Figure 4 Recovery and immunity activity of carocin S2. (A)
Antibacterial activity of carocin S2 from different strains. The
assay. Northern blots of 5’-32P-labeled total RNA extract
indicator was Pcc strain SP33. Strain number: 1, F-rif-18; 2, TF1-2; 3, treated with increasing concentrations of CaroS2K
TF1-2/pMS2KI; 4, DH5a/pMS2KI; 5, DH5a. (B) Assay for caroS2I. The (Figure 8B) showed a markedly lower intensity of labeled
colony and inoculated strains were F-rif-18. The indicator strains RNA fragments compared to untreated extracted RNA
were: 1, SP33; 2, SP33/pGEM-T easy; 3, SP33/pGS2I. (Figure 8B, lane 1), suggesting that CaroS2K has ribonu-
clease activity.
of the carocin S2 gene using DNASIS-Mac software Surprisingly the RNA segments were larger when the
(HITACHI, Japan) and compared to other analogous RNA was 3’-32P-labeled compared with 5’-32P-labeling
proteins using the BLAST and FASTA search tools. (Figures 8B and 8C). As the concentrations of 23S
ORF1 was found to encode a 783-amino acid protein RNA and 16S RNA decrease on the addition of
with a high degree of homology to Pcc21 carocin D, increasing concentrations of CaroS2K, it is assumed
Escherichia coli colicin D and Klebsiella oxytoca klebicin D that more ribosomal RNA is degraded leaving material
(Figure 5); ORF2 was found to encode a 90-amino acid that is ostensibly the ribosome. When excess concen-
protein that shows homology to the immunity proteins trations of caroS2K (i.e 1 μg) are added then most of
of colicin D and klebicin D (Figure 5). Thus, caroS2K the ribosomal RNA is degraded leading to a destabili-
produces an antibiotic with a deduced molecular mass of zation and subsequent degradation of the ribosome
85 kDa. CaroS2I (a 10-kDa protein of 90 amino acids) (Figure 8C, lane 2). We hence consider that CaroS2K
was shown to confer resistance to CaroS2K. It is particu- (in sufficient amount) would degrade the ribosome.
larly noteworthy that the homology between CaroS2K CaroS2I inhibits the killing activity of CaroS2K because
and Colicin D and Klebicin D is at the C-terminal end of a mixture of equal quantities of CaroS2K and CaroS2I
these proteins where the catalytic center of a ribonu- prevented digestion of RNA segments by CaroS2K
clease is located. According to the FASTA program, the (Figure 8C, lane 6).
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Figure 5 Region similarity of the putative domains of carocin S2 with those of related bacteriocins. The related ORFs are shown.
Percentage values indicate the percent relatedness to the corresponding regions in carocin S2. The length of each domain is proportional to
the number of amino acids. Homologous domains are shaded similarly. Domain I is homologous with the N-terminal T domain of colicin E3 [27].
Domain II resembles the receptor binding domains of other bacteriocins, but has no significant homology to other sequences in the database
[8,30]. Domain III and ORF2 of carocin S2 are highly homologous to colicin D and klebicin D.

Subsequently, treatment of the genomic DNA of the the 85-kDa and 10-kDa components, respectively, of
indicator strain SP33 with the purified CaroS2K protein Carocin S2. The substrate and gene structure of carocin
had no effect on deoxyribonuclease activity, as com- S2 were unlike those of other bacteriocins from Pcc.
pared to the pattern of EcoRI-digested genomic DNA On the basis of sequence analysis, carocin S2 com-
(Figure 8A and Additional file 1, Figure S4). prises these two overlapping ORFs, caroS2K and caroS2I
(Additional file 1, Figure S7). A putative Shine-Dalgarno
Nucleotide sequence accession number sequence 5’-AUGGA-3’, which has also been seen in the
The Genbank accession number of the sequence of the DNA sequence of carocin S1, is located upstream (-9 bp
carocin S2 gene is HM475143. to -13 bp) of the start codon AUG, suggesting that it
could be a ribosome binding site for caroS2K [23]. Com-
Discussion parison of the upstream sequences of both caroS2K and
In this study, a chromosome-borne gene encoding bac- caroS2I has shown that the two consensus sequences,
teriocin, carocin S2, in Pcc strain 3F3 was shown to pos- 5’-TATAAAAA-3’ (-34 bp to -41 bp) and 5’-GAAGT-3’
sess ribonuclease activity. According to Bradley’s (-61 bp to -65 bp), are both upstream from the start
classification, Carocin S2 is a low-molecular-weight bac- codon. Presumably, 5’-TATAAAAA-3’ is the -10 promo-
teriocin [25]. Two genes, caroS2K and caroS2I, encode ter and 5’-GAAGT-3’ is the -35 promoter for the

Figure 6 SDS-PAGE analysis of purified protein. Shown are the CaroS2K (A) and CaroS2I (B). Samples were subjected to electrophoresis in
10% polyacrylamide gels, which were stained with Coomassie blue. Lane M, molecular weight standards (kDa); lane 1, cell lysate of E. coli BL21/
pET32a; lane 4, cell lysate of BL21/pET30b; lanes 2 and 5, IPTG-induced cell lysates of BL21/pES2kI and BL21/pES2I, respectively; lanes 3 and 6,
purified protein obtained after elution. The arrowheads indicate the killing protein of carocin S2K (A) and the immunity protein of carocin S2I (B).
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(a sequence recognition motif DTMTV) was found in

the N-terminal domain of CarocinS2, which is thought
to participate in bacteriocin translocation [8]. Thus, we
suggested that Carocin S2 could be a TonB-dependent
bacteriocin.
Domain III (extending from Asp677 to the carboxyl
terminus) is the killer domain. Particularly noteworthy is
the resemblance of the killer domain to the tRNase
domain of colicin D and klebicin D (Figure 5), and thus
we suggested that carocin S2 might have tRNase activity
[29-31]. Domain II extends 141 residues from Ilu315 to
Figure 7 Survival of SP33 cells treated with Carocin S2. Aliquots Val455 and is hypothesized to be the binding site that
of indicator SP33 cells were treated with increasing concentrations recognizes specific receptors on cell membranes. Addi-
of CaroS2K (◆) and CaroS2K:CaroS2I in molar ratio of 1:1 (▲). The tionally, domain III has no significant homology to caro-
effect of trypsin on the CaroS2K was also assayed (■). The data are cin D, suggesting that carocin S2 and carocin D have
reported as means ± standard deviations.
different functions [28].
Finally, we showed that total RNA (whether labeled
carocin S2 gene, even though they differ from those of with radioactive phosphate at the 5’- or at the 3’- end)
E. coli[26]. is sensitive to Carocin S2. Carocin S2 degraded 5’-
A putative -10 promoter is 33 bp upstream from the labeled total RNA but not 5’-labeled CaroS2K-free RNA
initiator ATG of the caroS2K gene, in which the SD (Figure 8B), and the amount of degradation was not
sequence is embedded, while the -35 promoter is 19 bp dose-dependent (arrowhead). However, the appearance
upstream of the -10 promoter region. The putative pro- of segments of unknown origin paralleled partial degra-
moter of the -35 box of caroS2I is located similarly near dation of 23S and 16S rRNA (Figure 8C). These results
the -10 box, but the -10 box is just 24 bp upstream of suggest that the site of excision (either conformational
the start codon where no SD sequence is apparent. or sequential) is close to the 5’-terminus of rRNA. Nota-
Although those hypothesized promoters are located bly, the decrease in the amount of rRNA depended on
within the caroS2K structural gene, transcripts of car- the amount of Carocin S2 protein present, with com-
oS2I are routinely produced (Figure 3). This suggests plete degradation occurring in the presence of excess
that caroS2I RNA expression may be regulated posttran- Carocin S2. Ogawa et al. reported that RNase type of
scriptionally, in spite of close neighboring genes down- bacteriocins, colicin E3 and colicin E5, catalyze the
stream of the gene caroS2K; that is, core promoter hydrolysis of the shorter RNAs from 16S rRNA [19,32].
elements may influence the expression of caroS2I gene. Moreover, colicin E5 was found to hydrolyze tRNA in
In the present study, we attempted to separate Car- vitro. Furthermore, it was previously reported that coli-
oS2K from CaroS2I attached to (His)6-tag using a Nickel cin E3 cleaved 16S rRNA completely, and even 30S
column (pEH2KI; Additional file 1, Figure S5), but a rRNA [11,33]. In our study, carocin S2 acted as an
small amount of CaroS2I (Mr ~10 kDa) was observed in RNase that hydrolyzes rRNA (both 23S and 16S) in
SDS-PAGE gels (Figure 6, bottom in lane 3), which had vitro. In terms of enzymatic function, Carocin S2 may
little influence on the activity of CaroS2K as the purified act as an endo- and exo-ribonuclease simultaneously.
protein still had transient killing activity. Additionally, Moreover, CaroS2I significantly inhibited nuclease activ-
the activity of the Carocin S2 complex at 4℃ was long- ity in vitro but not in vivo (Figures 7, Figure 8 andAddi-
lasting indicating good stability. tional file 1, Figure S3). We speculated that immunity
The C-terminal amino acid sequence of Carocin S2 protein CaroS2I might not be able to cross the cell
had higher homology to those of colicin D and klebicin membrane, as previously described [14]. Although our
D, which are produced by E. coli and Klebsiella oxytoca, in vitro experiment showed that carocin S2 was a ribo-
respectively, than to the amino acid sequence of carocin nuclease, further investigation is needed to clarify its
S1 from the same species (Additional file 1, Figure S6B). function in cells.
The amino acid sequence of CaroS2K has three puta- One of the other Tn5 insertional mutants, TF1-1,
tive domains. Domain I (the N-terminal 314-residue which disrupted the coding sequence of the fliC gene,
sequence ending in Pro314) is regarded as the transloca- was found to halt expression of Carocin S2 (Figure 1),
tion domain and is homologous to the translocation indicating that Carocin S2 can also be secreted via the
domains of carocin D and colicin E3 (Figure 5). It is type III secretion system [24]. The role of carocin S2 as
assumed to direct the cytotoxic domain to the periplas- an RNase in the cytoplasm is to prevent protein synth-
mic space [27,28]. Additionally, the putative TonB box esis by cleaving either 23S rRNA or 16S rRNA. The role
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Methods
Bacterial strains, media, and growth conditions
Bacterial strains and plasmids used in the study are
listed in Table 1. Isolates of Pcc were grown at 28°C in
Luria-Bertani (LB) medium or IFO-802 medium. The
IFO-802 medium was supplemented with 1% polypeptin,
0.2% yeast extract, 0.1% MgSO4 (pH 7.0), and 1.5% agar.
Isolates of Pcc were distinguished from Escherichia coli
by their ability to grow on Modified Drigalski’s agar

Table 1 Bacteria and plasmids used in the study

Strain or plasmid Description Source
Escherichia coli
1830 pro¯ met¯ Kanr Nmr, containing [44]
transposon Tn5 on the suicidal
plasmid pBJ4JI
DH5a supE44ΔlacU169(F80lacZΔM15) [39]
hsdR17recA1 gyrA96thi-1relA1
BL21(DE3) hsdS gal(lcIts857 ind1 Sam7 nin5 [45]
lac UV5-T7 gene 1)
Pectobacterium
carotovorum subsp.
carotovorum
3F-3 Pcc, wild-type Laboratory
stock
F-rif-18 3F3, Rifr, wild-type This study
TF1-1 F-rif-18, fliC::Tn5, Rifr, Kanr This study
TF 1-2 F-rif-18, CarocinS2::Tn5, Rifr, Kanr This study
Figure 8 In vitro hydrolysis of DNA and RNA by Carocin S2. (A) SP33 Pcc, wild-type Laboratory
Analysis of the DNase activity of carocin S2. Lane M, the HindIII- stock
digested l DNA marker; lane 1, genomic DNA only; lanes 2 and 3, Plasmid
genomic DNA treated or untreated with carocin S2 in buffer,
pMCL210 p15A, Cmlr, Low copy number [46]
respectively; lane 4, equal quantity of EcoRI-digested genomic DNA.
The 5’-labeled total RNA (B) and 3’-labeled total RNA (C) (1 μg of pGEM T-Easy Ampr; lacZ cloning vector Promega
RNA per sample) were incubated without (lane 1) or with 1 μg pET32a Ampr; expression vector with the Novagen
(lane 2), 100 ng (lane 3), 10 ng (lane 4), or 1 ng (lane 5) of Carocin N-terminal His-tag
S2 and the result was assessed by autoradiography. The arrowhead pET30b Kanr; expression vector with the Novagen
indicates that the RNA segment digested from ribosome. Equal C-terminal His-tag
amounts of Carocin S2I and Carocin S2K mixed before RNA pMS2KI 5.7-kb BamHI DNA fragment This study
digestion (lane 6). harboring carocin S2 gene from
3F3 genome, cloned into
pMCL210
of the immunity protein, CaroS2I, is usually to stop the pEN2K* caroS2K subcloned into pET32a This study
damage caused by CaroS2K in the cytoplasm. More pES2KI Derived from pEN2K; deleted This study
details of the actual mechanism of carocin S2 remain to series of Tag element in front of
expressed caroS2K
be elucidated.
pEH2KI* Derived from pES2KI; adding This study
(His)6-Tag adjacent to caroS2I
Conclusion pGS2I caroS2I and its putative promoter This study
As shown herein, the novel bacteriocin, Carocin S2, was from pMS2KI, subcloned into
characterized as a ribonuclease. It is the first bacteriocin pGEM T-easy
with ribonuclease activity to be found in Pectobacterium pECS2I* caroS2I subcloned into pET30b, This study
but the expressed fusion CaroS2I
strains. We suggested that Carocin S2 kills the indicator has no activity
cell by exhausting its supply of some kinds of RNA, pES2I Derived form pECS2I, the (His)6- This study
leading to inactivation of protein biosynthesis. It will be Tag element was deleted
of interest to study the proteomics of Carocin S2 and its Kanr: Kanamycin; Cmlr: Chloramphenicol; Rifr: Rifampicin; Ampr: Ampicillin.
mechanism of action in the future. *: See Additional file 1, Figure S5.
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medium [34]. Antibiotics (final concentration, 100 μg from pMS2KI with primers of CarocinS2K_for2 and
ml-1 of media) were added when necessary. CarocinS2I_rev2 (Additional file 1, Table S1) and
subcloned into pET32a to give the plasmid pEN2K
Bacterial conjugation (Additional file 1, Figure S5). The pES2KI was obtained
Overnight cultures of Pcc (recipient) and E. coli by excision of the Tag element between the rbs (ribo-
(donor) were mixed and spread onto 0.22-μm mem- some binding site) and start code (for CaroS2K) in
brane filters placed on LB agar media and incubated pEN2K using the SLIM method as previously described
overnight at 28°C [23]. The progeny after conjugation [40,41]. The 5IHT32a2KI_forT, 5IHTGT2KI_forS,
were appropriately diluted and cultivated on Modified 5IHT32a3KI_revT, and 5IHT32a4KI_revS primers were
Drigalski’s medium (with ampicillin and kanamycin used. A 273-bp fragment of the caroS2I gene was ampli-
[100 μg ml -1 ]) overnight at 28°C. All isolates were fied by PCR and ligated into the NdeI and XhoI site of
placed on IFO-802 medium and tested for bacteriocins. pET30b to form the plasmid pEC2I. Similarly, the plas-
Bacteriocin was assayed using the double-layer method, mid pES2I was obtained by deleting the (His) 6 -tag of
and Pcc SP33 was used as indicator strain [35]. The pEC2I (carried out as described above with primers of
cells were incubated for 12 hours to form colonies, X21_forT, X21_forS, X21_revT and X21_revS). Subse-
exposed to ultraviolet irradiation, incubated again for quently, pES2KI and pES2I were introduced into E. coli
12 hours, treated with chloroform to kill the cells, and BL21 (DE3) cells, respectively.
then covered with soft agar containing indicator cells.
The bacteriocin production was indicated by a zone of Restriction DNA library screening and Southern blots
inhibition of indicator-cell (SP33) growth around the Southern blots were performed according to the DIG
colony. Application Manual (Roche, USA). A 543-bp DNA frag-
ment (TF1-2 probe) was amplified with TF1-2P and
Genetic-engineering technique TF1-2A2 primers (Additional file 1, Table S1), sub-
The procedures of plasmid preparation, genomic DNA cloned into pGEM-T Easy vector (Promega Inc., USA),
isolation, and DNA manipulation were performed as and labeled using a Random Primed DNA Labeling Kit
described by Sambrook et al. [36]. Oligonucleotide DNA (Roche Diagnostics, USA).
primers were synthesized by MD Bio Inc. (Taipei, Tai- The genomic DNA of the wild-type strain F-rif-18 was
wan). The PCR was amplified with Go-Taq DNA poly- digested with various restriction endonucleases, with
merase (Promega, USA). The thermal asymmetric sites located outside the putative open reading frame.
interlaced PCR (TAIL-PCR) was performed as pre- Samples were electrophoresed and analyzed with South-
viously described [37]. ern blotting. After detection using the TF1-2 probe, the
Plasmids were introduced into Pcc strains using elec- DNA from positive gel slices was purified and cloned
troporation (1.25 kV/cm, 200 Ω, 25 μF) [38]. For heat- into pMCL210 to give the carocin-producing plasmid
shock transformation, the competent cells of E. coli pMS2KI. The pMS2KI construct was isolated and
were prepared according to the method of Hanahan detected as above with the TF1-2 probe.
[39].
Exponentially growing cells (OD595 of about 6.0) were Protein purification
harvested for RNA preparation. Total RNA was isolated The transformant cells of BL21, harboring pES2KI or
using Trizol reagent (Invitrogen, USA) according to the pES2I, were grown in 500 ml to an OD595 of 0.4. The
manufacturer’s instructions. RNA was resuspended in cells were induced with isopropyl-b-D-thiogalactopyra-
diethylpyrocarbonate (DEPC)-treated water. The con- noside (IPTG; final concentration, 0.1 mM; at 25°C for 12
centration of RNA was determined by OD260 absorption, h). Subsequently, the cells were pelleted and the pellets
and RNA was analyzed by electrophoresis on 1.5% for- were sonicated (10 cycles of 9 s with 9-s intervals). BL21/
maldehyde-morpholinepropanesulfonic-agarose gel. pES2KI pellets were subjected to ammonium sulfate preci-
Reverse transcription-PCR (RT-PCR) was carried out pitation (30-40%), resuspended in buffer A (30 mM NaCl
with AMV Reverse Transcriptase (Promega Inc., Tai- and 20 mM Tris-Cl, pH 8.0), and applied to a Fractogel
wan) according to manufacturer’s instructions. RNA column (Merck, USA). The fraction was eluted by a NaCl
(1 μg) was subjected to RT-PCR containing CaroS2_re_1 gradient (30 mM-1.4 M). After purification through a
used as a reverse primer in first-strand cDNA synthesis. P-100 size-exclusion column (BioRad, USA), the CaroS2K
The RT mixtures were diluted and used as templates in fractions were pooled and concentrated using an Amicon
a PCR reaction with two primers CaroS2_re_1 and centriprep-50 column (Millipore, USA) and dissolved
CaroS2_for_1 (Additional file 1, Table S1). in buffer A. BL21/pES2I pellets were precipitated by
A 2621-bp BamHI-HindIII digested DNA fragment, ammonium sulfate (70-100%) and resuspended in buffer
including the caroS2K and caroS2I genes, was amplified A. CaroS2I purification involved a similar
Chan et al. BMC Microbiology 2011, 11:99 Page 10 of 12
http://www.biomedcentral.com/1471-2180/11/99

chromatographic procedure using the Amicon centriprep- Additional material

3 column (Millipore, USA). The concentration of protein
was determined by the Bradford assay (Amresco, USA). Additional file 1: Figure S1. Analysis of Tn5 insertional mutants by
southern blotting. Lane M, the HindIII-digested l DNA marker; the
genomic DNA of strains were loading as follows: lane 1, TF1-2; lane 2, F-
In vitro determination of Carocin S2 activity rif-18; lane 3, 3F3; lane 4, TF1-1. Lane 5, the construct pGnptII that
Total RNA was treated with calf intestinal alkaline phos- contain the detect probe DNA nptII. The result shows that TF1-2 and
TF1-1 was a Tn5 insertional mutant. Figure S2. The construct pMS2KI
phatase (Promega, USA) at 55°C for 30 min as recom- was cloned from genomic DNA library and screening by southern
mended by the manufacturer. The reaction was arrested blotting with TF1-2 probe. By southern blotting, it showed that the
by adding 5 mM nitrilotriacetic acid, and RNA was carocin S2 has been cloned to form pMS2KI. Figure S3. The total RNA
of SP33 were digested with Carocin S2 and electrophoresis as
extracted with equal volumes of phenol/chloroform. An follows: lane 1, RNA (1 μg); lane 2, RNA and CaroS2K (20 μg); lane 3, RNA
aliquot of phosphatase-treated RNA was 5’-32P-labeled at and CaroS2I (4 μg); lanes 4 to 6 are RNA (1 μg) and CaroS2K (20 μg) with
37°C for 30 min by incubation with a mixture of [g-32P] gradient concentration of CaroS2I, which were added with 4 μg (lane 4);
20 μg (lane 5); 100 μg (lane 6). All reactions were performed at 28℃ for
ATP, T4 polynucleotide kinase (Promega Inc, USA), and 3 hours. Figure S4. Metal effect of In vitro hydrolysis of DNA by
reaction buffer in nuclease-free water [42]. [5’-32P]Cytidine Carocin S2. Lane M, the HindIII-digested l DNA marker; lane 1, the
3’,5’-bisphosphate (pCp) and T4 RNA ligase (Promega, genomic DNA of SP33 only; lane 2, the EcoRI-digested genomic DNA; the
genomic DNA was incubated with Carocin S2 (lane 3 to 5), or not.
USA) were used for 3’-labeling of RNA [43]. Subsequently, Magnesium acetate, nickel acetate and zinc acetate was added in buffer
the mixture was purified by MicroSpin G-25 columns (GE A (pH = 7), respectively. The reactions were performed at performed at
Healthcare, USA). The purified labeled RNA was divided 28℃ for 1 hour. Figure S5. Schematic representation of the cloning
strategy used in this study. (1) A 543-bp amplicon was cloned into the
into aliquots and incubated without or with Carocin S2 at vector pTF1 to form the pTF1-2-probe. (2) The TF1-2 probe was
28°C for 60 min, respectively. To measure its activity, Car- prepared. (3) The multi-enzyme-digested DNA fragments were obtained
oS2I was pre-mixed with an equal amount of CaroS2K. from F-rif-18 genomic DNA, and they were detected on southern blots.
(4) Positive cDNA was cloned into the carocin-producing plasmid
The mixtures were subjected to electrophoresis on a 9% pMS2KI. (5) A 2621-bp amplicon, from pMS2KI, was subcloned into
polyacrylamide gel (19:1) containing 7M urea, 50 mM pET32a to form pEN2K. (6) The 5’-transcriptional element, which would
Tris, 50 mM boric acid, and 1 mM EDTA, pH 8.3. All be translated into the Flag tag, was deleted from pEN2K using the SLIM
method [40]. (7) By using SLIM method, an element encoding a stretch
samples were electrophoresed at 15℃ by PROTEIN II xi of six histidines was inserted into caroS2I to form pEH2KI. (8) A 484-bp
(BioRad, USA). amplicon was subcloned into pGEM T-easy vector to form pGS2I. (9)
To confirm DNase activity, 1 μg of genomic DNA A273-bp fragment of the caroS2I gene was amplified from pGS2I and
subcloned into pET30b to form pECS2I. (10) The 3’-transcriptional
from SP33 in solution containing buffer A was incu- element, which would be translated to (His)6-Flag, was deleted from
bated with or without Carocin S2 at 28°C for 90 min. pES2I using the SLIM method. Figure S6. Alignment of the deduced
An equal quantity of genomic DNA was digested with amino acid sequences of carocin S2 with those of homologous
domains of bacteriocins. The potential TonB-binding motif is shown by
EcoRI at 28°C for 90 min. Samples were then subjected red underline. (A) The N-terminal translocation domain of CaroS2K (Met1
to electrophoresis on 1% agarose gel. to Pro314) has homology to carocin D and colicin E3. (B) The killing
domain of CaroS2K (Asp677 to carboxyl terminus) has homology to the
minimal tRNase domain of colicin D and klebicin D. (C) The deduced
Antibiotic activity of Carocin S2 amino acid of immunity protein of CaroS2I has homology to colicin D
Overnight cultures of SP33 were diluted (1:100) with LB and klebicin D. Figure S7. The gene and deduced amino acid
sequence of carocin S2 shows in the study. The sequence was
medium and grown at 28°C to a density of approxi- truncated form pMS2KI. The underline shows the putative promoter.
mately 105 CFU ml-1. The activity of increasing concen- Figure S8. Schematic representation of thermal asymmetric
trations of Carocin S2 on cells in suspension incubated interlaced PCR (TAIL-PCR) were manipulated according to the method
of Liu and Whittier, but the annealing temperature was decreased from
at 28°C for 60 min was assessed. CaroS2I was pre-mixed 63℃ to 60℃ for specific primers [37,23]. Amplifying the unknown DNA
with an equal molar ratio of CaroS2K. All reaction mix- fragment are the specific primers which are complementary to the
tures were spread onto LB agar plates and incubated at known sequence (Tn5) and the arbitrary degenerate primers which could
be complementary to the opposite unknown site. The specific primers
28°C for 16 h. The experiment was performed three (SP) are PR1, PR2, PR3, PF1, PF2, PF3, and TF1-2S1 to TF1-2A6 primers for
times. Colonies growing on a series of plates were opposite direction (Additional file 1, Table S1). In addition, the arbitrary
respectively counted. degenerate primers (AD) N1, N2, and N3 were respectively used as
simultaneous PCR amplification (see above).

Computer analysis of sequence data

Sequencing of the DNA fragments was carried out using
an ABI automated DNA sequencer 373S. The nucleotide Acknowledgements
The support of this work by grants from the National Science Council
sequence data were compiled by DNASIS-Mac software (grants NSC-97-2313-B-005-027-MY3) of Taiwan (R.O.C.) is gratefully
(Hitachi, Japan). Amino acid sequences were compared acknowledged.
using international BLAST and FASTA servers. Also,
Author details
the putative domains of Carocin S2 were predicted 1
Department of Chemistry, National Chung-Hsing University, 250, Kuokuang
using the PSI/PHI-BLAST. Rd., Taichung, 402, Taiwan. 2Division of Pulmonary Medicine, Department of
Chan et al. BMC Microbiology 2011, 11:99 Page 11 of 12
http://www.biomedcentral.com/1471-2180/11/99

Internal Medicine, Chang Gung Memorial Hospital, Keelung, 204, Taiwan. 21. de Zamaroczy M, Mora L, Lecuyer A, Géli V, Buckingham RH: Cleavage of
3
Department of plant pathology, National Chung-Hsing University, 250, Colicin D Is Necessary for Cell Killing and Requires the Inner Membrane
Kuokuang Rd., Taichung, 402, Taiwan. Peptidase LepB. Mol Cell 2001, 8:159-168.
22. Nguyen AH, Tomita T, Hirota M, Sato T, Kamio Y: A simple purification
Authors’ contributions method and morphology and component analyses for carotovoricin Er, a
YC participated in the discovery and characterization of Carocin S2, and he phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora
wrote this manuscript. JL participated in protein purification. HP participated Er. Biosci Biotechnol Biochem 1999, 63:1360-1369.
in manuscript preparation. KC supported the Pcc strain SP33 and for 23. Chuang DY, Chien YC, Wu HP: Cloning and Expression of the Erwinia
insightful discussion and guidance. DY conceived of the study, participated carotovora subsp. carotovora Gene Encoding the Low-Molecular-Weight
in its design, and corrected the manuscript. All authors read and approved Bacteriocin Carocin S1. J Bacteriol 2007, 189:620-626.
the final version of the manuscript. 24. Chan YC, Wu HP, Chuang DY: Extracellular secretion of Carocin S1 in
Pectobacterium carotovorum subsp. carotovorum occurs via the type III
Received: 21 September 2010 Accepted: 12 May 2011 secretion system integral to the bacterial flagellum. BMC Microbiol 2009,
Published: 12 May 2011 9:181.
25. Bradley DE: Ultrastructure of bacteriophage and bacteriocins. Bacteriol Rev
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doi:10.1186/1471-2180-11-99
Cite this article as: Chan et al.: Cloning, purification, and functional
characterization of Carocin S2, a ribonuclease bacteriocin produced by
Pectobacterium carotovorum. BMC Microbiology 2011 11:99.

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