Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum
© Chan et al; licensee BioMed Central Ltd. 2011
Received: 21 September 2010
Accepted: 12 May 2011
Published: 12 May 2011
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.
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 DH5α 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.
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.
The phytopathogenic enterobacterium, Pectobacterium carotovorum subsp. carotovorum, is a phytoparasitic, Gram-negative, facultative anaerobic bacterium . Pcc produces many extracellular pectic enzymes (pectate lyase, pectin lyase, exopolygalacturnoate lyase) and hydrolytic enzymes causing soft-rot disease, tissue maceration, and cell wall collapse [2, 3]. The only current strategy against soft-rot disease involves chemical agents that unavoidably contaminate the environment . Kikumoto et al. have demonstrated that mixed bacteriocin-producing avirulent strains of Pcc show high efficacy against soft-rot disease of Chinese cabbage .
Bacteriocins are bactericidal, extracellular toxins, produced by both Gram-positive and Gram-negative bacteria [6, 7]. These proteinaceous molecules kill closely related bacteria. The susceptible cell is recognized by specific target receptors on the membrane, and the producer cell evades lethality by expressing a cognate immune protein. The colicin family produced by Escherichia coli is divided into DNase (colicins E2, E7, E8 and E9), RNase (colicins E3, E4 and E6), tRNase (colicins D and E5), and pore-forming colicins (colicins A, E1, Ia and Ib) . Bacteriocins (especially nuclease bacteriocins) have a high amino acid sequence homology.
Natural bacteriocin molecules act via a number of mechanisms. For example, colicin E3 is a well-known ribonuclease that specifically cleaves 16S rRNA at the 3'-end of the coding sequence both in vivo and in vitro, which leads to the abolishment of protein synthesis resulting in death of the susceptible cell [9–12]. Previous reports indicate that colicin E3 consists of a killer protein with three domains (i.e., a translocation domain [T domain], receptor binding domain [R domain], and nuclease domain) and an immunity protein that retards antibiotic activity [13, 14]. The R domain recognizes a specific receptor, BtuB on the cell membrane and the T domain interacts with the TolB protein in the cell periplasm of the sensitive cell to facilitate entry of the killer domain through the cell membrane. In addition to the attack mechanism, the immunity mechanism has been extensively elucidated. Notably the immunity protein and the killer protein interact initially at very high affinity because of charge attraction, and are separated at the cell surface through energy generated from the proton motif force [15–17].
In general, the C-terminal domain determines the type of bacteriocin. The C-terminal nuclease domains are not only interchangeable but also lack species specificity . Strikingly, the tRNase type of bacteriocin may accelerate exhaustion of tRNA in the cytoplasmic pool and thereby impair protein synthesis in vivo. Ogawa et al. have demonstrated that particular tRNA molecules can be digested by colicin D as well as by colicin E5 [19, 20]. It has been suggested that phage-associated klebicin D is a tRNase type of bacteriocin based on similarity to the nuclease-like domain of colicin D .
Nguyen et al. reported production of a high-molecular-weight bacteriocin (carotovoricin Er) and Chuang et al. 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 inhibition 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 .
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 Carocin 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 introduced into an expression plasmid encoding two proteins, CaroS2K and CaroS2I. These proteins were purified and characterized and their primary activities of killing (CaroS2K) and immunity (CaroS2I) were investigated in vivo and in vitro.
Isolation of Transposon Insertion Mutants
To ascertain whether Tn5 was actually introduced into the genomic DNA of putative isolates, the nptII gene of isolates was amplified using two primers P3 and P4 . Southern blot technology showed that Tn5 had been inserted (Additional file 1, Figure S1).
Identification of Tn5-inserted DNA Structures
Transcriptional analysis and in vivo expression of carocin S2 gene
The presence of the 925-bp amplicon revealed that caroS2K was being transcribed in the cell (panel caroS2K in Figure 3). The TF1-2 strain, which is a Tn5 insertional mutant, could not transcribe caroS2K (lane 2), but the ability of TF1-2 to transcribe caroS2K was restored by introduction of pMS2KI (lane 3). It was apparent that the amount of caroS2K expression was dependent on the number of copies of plasmid pMS2KI (compare lane 1 to lane 3). Additionally, carocin S2 can be expressed in E. coli strain DH5α by introduction of pMS2KI (lane 4 and lane 5).
To prove that pMS2KI contained the gene for Carocin S2, pMS2KI was introduced into TF1-2 and E. coli DH5α. Both TF1-2/pMS2KI and DH5α/pMS2KI had ability to express the activity of Carocin S2 (Figure 4A). The size of inhibition zone around strain TF1-2/pMS2KI was equal to that around DH5α/pMS2KI but still smaller than that around the wild-type strain F-rif-18. On the other hand, the quantity of transcripts expressed in vivo and in vitrodid not usually correspond.
Deduction of the amino acid sequence of Carocin S2
The carocin S2 gene consists of two ORFs (Additional file 1, Figure S7): one containing the 2352-bp caroS2K gene and the other containing the 273-bp caroS2I gene. The stop codon (TGA) of caroS2K overlaps the first start codon of caroS2I by 4-bp (ATGA). The amino sequences were deduced from the nucleotide sequence of the carocin S2 gene using DNASIS-Mac software (HITACHI, Japan) and compared to other analogous proteins using the BLAST and FASTA search tools.
Purification and characterization of Carocin S2
Carocin S2 has ribonuclease activity
Surprisingly the RNA segments were larger when the RNA was 3'-32P-labeled compared with 5'-32P-labeling (Figures 8B and 8C). As the concentrations of 23S RNA and 16S RNA decrease on the addition of increasing concentrations of CaroS2K, it is assumed that more ribosomal RNA is degraded leaving material that is ostensibly the ribosome. When excess concentrations of caroS2K (i.e 1 μg) are added then most of the ribosomal RNA is degraded leading to a destabilization and subsequent degradation of the ribosome (Figure 8C, lane 2). We hence consider that CaroS2K (in sufficient amount) would degrade the ribosome. CaroS2I inhibits the killing activity of CaroS2K because a mixture of equal quantities of CaroS2K and CaroS2I prevented digestion of RNA segments by CaroS2K (Figure 8C, lane 6).
Subsequently, treatment of the genomic DNA of the indicator strain SP33 with the purified CaroS2K protein had no effect on deoxyribonuclease activity, as compared to the pattern of EcoRI-digested genomic DNA (Figure 8A and Additional file 1, Figure S4).
Nucleotide sequence accession number
The Genbank accession number of the sequence of the carocin S2 gene is HM475143.
In this study, a chromosome-borne gene encoding bacteriocin, carocin S2, in Pcc strain 3F3 was shown to possess ribonuclease activity. According to Bradley's classification, Carocin S2 is a low-molecular-weight bacteriocin . Two genes, caroS2K and caroS2I, encode the 85-kDa and 10-kDa components, respectively, of Carocin S2. The substrate and gene structure of carocin S2 were unlike those of other bacteriocins from Pcc.
On the basis of sequence analysis, carocin S2 comprises these two overlapping ORFs, caroS2K and caroS2I (Additional file 1, Figure S7). A putative Shine-Dalgarno sequence 5'-AUGGA-3', which has also been seen in the DNA sequence of carocin S1, is located upstream (-9 bp to -13 bp) of the start codon AUG, suggesting that it could be a ribosome binding site for caroS2K . Comparison of the upstream sequences of both caroS2K and caroS2I has shown that the two consensus sequences, 5'-TATAAAAA-3' (-34 bp to -41 bp) and 5'-GAAGT-3' (-61 bp to -65 bp), are both upstream from the start codon. Presumably, 5'-TATAAAAA-3' is the -10 promoter and 5'-GAAGT-3' is the -35 promoter for the carocin S2 gene, even though they differ from those of E. coli.
A putative -10 promoter is 33 bp upstream from the initiator ATG of the caroS2K gene, in which the SD sequence is embedded, while the -35 promoter is 19 bp upstream of the -10 promoter region. The putative promoter of the -35 box of caroS2I is located similarly near the -10 box, but the -10 box is just 24 bp upstream of the start codon where no SD sequence is apparent. Although those hypothesized promoters are located within the caroS2K structural gene, transcripts of caroS2I are routinely produced (Figure 3). This suggests that caroS2I RNA expression may be regulated posttranscriptionally, in spite of close neighboring genes downstream of the gene caroS2K; that is, core promoter elements may influence the expression of caroS2I gene.
In the present study, we attempted to separate CaroS2K from CaroS2I attached to (His)6-tag using a Nickel column (pEH2KI; Additional file 1, Figure S5), but a small amount of CaroS2I (Mr ~10 kDa) was observed in SDS-PAGE gels (Figure 6, bottom in lane 3), which had little influence on the activity of CaroS2K as the purified protein still had transient killing activity. Additionally, the activity of the Carocin S2 complex at 4℃ was long-lasting indicating good stability.
The C-terminal amino acid sequence of Carocin S2 had higher homology to those of colicin D and klebicin D, which are produced by E. coli and Klebsiella oxytoca, respectively, than to the amino acid sequence of carocin S1 from the same species (Additional file 1, Figure S6B).
The amino acid sequence of CaroS2K has three putative domains. Domain I (the N-terminal 314-residue sequence ending in Pro314) is regarded as the translocation domain and is homologous to the translocation domains of carocin D and colicin E3 (Figure 5). It is assumed to direct the cytotoxic domain to the periplasmic space [27, 28]. Additionally, the putative TonB box (a sequence recognition motif DTMTV) was found in the N-terminal domain of CarocinS2, which is thought to participate in bacteriocin translocation . 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 Val455 and is hypothesized to be the binding site that recognizes specific receptors on cell membranes. Additionally, domain III has no significant homology to carocin D, suggesting that carocin S2 and carocin D have different functions .
Finally, we showed that total RNA (whether labeled with radioactive phosphate at the 5'- or at the 3'- end) is sensitive to Carocin S2. Carocin S2 degraded 5'-labeled total RNA but not 5'-labeled CaroS2K-free RNA (Figure 8B), and the amount of degradation was not dose-dependent (arrowhead). However, the appearance of segments of unknown origin paralleled partial degradation of 23S and 16S rRNA (Figure 8C). These results suggest that the site of excision (either conformational or sequential) is close to the 5'-terminus of rRNA. Notably, the decrease in the amount of rRNA depended on the amount of Carocin S2 protein present, with complete degradation occurring in the presence of excess Carocin S2. Ogawa et al. reported that RNase type of bacteriocins, colicin E3 and colicin E5, catalyze the hydrolysis of the shorter RNAs from 16S rRNA [19, 32]. Moreover, colicin E5 was found to hydrolyze tRNA in vitro. Furthermore, it was previously reported that colicin E3 cleaved 16S rRNA completely, and even 30S rRNA [11, 33]. In our study, carocin S2 acted as an RNase that hydrolyzes rRNA (both 23S and 16S) in vitro. In terms of enzymatic function, Carocin S2 may act as an endo- and exo-ribonuclease simultaneously. Moreover, CaroS2I significantly inhibited nuclease activity in vitro but not in vivo (Figures 7, Figure 8 andAdditional file 1, Figure S3). We speculated that immunity protein CaroS2I might not be able to cross the cell membrane, as previously described . Although our in vitro experiment showed that carocin S2 was a ribonuclease, further investigation is needed to clarify its function in cells.
One of the other Tn5 insertional mutants, TF1-1, which disrupted the coding sequence of the fliC gene, was found to halt expression of Carocin S2 (Figure 1), indicating that Carocin S2 can also be secreted via the type III secretion system . The role of carocin S2 as an RNase in the cytoplasm is to prevent protein synthesis by cleaving either 23S rRNA or 16S rRNA. The role of the immunity protein, CaroS2I, is usually to stop the damage caused by CaroS2K in the cytoplasm. More details of the actual mechanism of carocin S2 remain to be elucidated.
As shown herein, the novel bacteriocin, Carocin S2, was characterized as a ribonuclease. It is the first bacteriocin with ribonuclease activity to be found in Pectobacterium strains. We suggested that Carocin S2 kills the indicator cell by exhausting its supply of some kinds of RNA, leading to inactivation of protein biosynthesis. It will be of interest to study the proteomics of Carocin S2 and its mechanism of action in the future.
Bacterial strains, media, and growth conditions
Bacteria and plasmids used in the study
Strain or plasmid
pro¯ met¯ Kan r Nmr, containing transposon Tn5 on the suicidal plasmid pBJ4JI
supE44ΔlacU169(Φ80lacZΔM15) hsdR17recA1 gyrA96thi-1relA1
hsdS gal(λcIts857 ind1 Sam7 nin5 lac UV5-T7 gene 1)
Pectobacterium carotovorum subsp. carotovorum
3F3, Rifr, wild-type
F-rif-18, fliC::Tn5, Rifr, Kanr
F-rif-18, CarocinS2::Tn5, Rifr, Kanr
p15A, Cmlr, Low copy number
Ampr; lacZ cloning vector
Ampr; expression vector with the N-terminal His-tag
Kanr; expression vector with the C-terminal His-tag
5.7-kb BamHI DNA fragment harboring carocin S2 gene from 3F3 genome, cloned into pMCL210
caroS2K subcloned into pET32a
Derived from pEN2K; deleted series of Tag element in front of expressed caroS2K
Derived from pES2KI; adding (His)6-Tag adjacent to caroS2I
caroS2I and its putative promoter from pMS2KI, subcloned into pGEM T-easy
caroS2I subcloned into pET30b, but the expressed fusion CaroS2I has no activity
Derived form pECS2I, the (His)6-Tag element was deleted
Overnight cultures of Pcc (recipient) and E. coli (donor) were mixed and spread onto 0.22-μm membrane filters placed on LB agar media and incubated overnight at 28°C . The progeny after conjugation were appropriately diluted and cultivated on Modified Drigalski's medium (with ampicillin and kanamycin [100 μg ml-1]) overnight at 28°C. All isolates were placed on IFO-802 medium and tested for bacteriocins. Bacteriocin was assayed using the double-layer method, and Pcc SP33 was used as indicator strain . The cells were incubated for 12 hours to form colonies, exposed to ultraviolet irradiation, incubated again for 12 hours, treated with chloroform to kill the cells, and then covered with soft agar containing indicator cells. The bacteriocin production was indicated by a zone of inhibition of indicator-cell (SP33) growth around the colony.
The procedures of plasmid preparation, genomic DNA isolation, and DNA manipulation were performed as described by Sambrook et al. . Oligonucleotide DNA primers were synthesized by MD Bio Inc. (Taipei, Taiwan). The PCR was amplified with Go-Taq DNA polymerase (Promega, USA). The thermal asymmetric interlaced PCR (TAIL-PCR) was performed as previously described .
Plasmids were introduced into Pcc strains using electroporation (1.25 kV/cm, 200 Ω, 25 μF) . For heat-shock transformation, the competent cells of E. coli were prepared according to the method of Hanahan .
Exponentially growing cells (OD595 of about 6.0) were harvested for RNA preparation. Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA was resuspended in diethylpyrocarbonate (DEPC)-treated water. The concentration of RNA was determined by OD260 absorption, and RNA was analyzed by electrophoresis on 1.5% formaldehyde-morpholinepropanesulfonic-agarose gel.
Reverse transcription-PCR (RT-PCR) was carried out with AMV Reverse Transcriptase (Promega Inc., Taiwan) according to manufacturer's instructions. RNA (1 μg) was subjected to RT-PCR containing CaroS2_re_1 used as a reverse primer in first-strand cDNA synthesis. The RT mixtures were diluted and used as templates in a PCR reaction with two primers CaroS2_re_1 and CaroS2_for_1 (Additional file 1, Table S1).
A 2621-bp BamHI-HindIII digested DNA fragment, including the caroS2K and caroS2I genes, was amplified from pMS2KI with primers of CarocinS2K_for2 and CarocinS2I_rev2 (Additional file 1, Table S1) and subcloned into pET32a to give the plasmid pEN2K (Additional file 1, Figure S5). The pES2KI was obtained by excision of the Tag element between the rbs (ribosome binding site) and start code (for CaroS2K) in pEN2K using the SLIM method as previously described [40, 41]. The 5IHT32a2KI_forT, 5IHTGT2KI_forS, 5IHT32a3KI_revT, and 5IHT32a4KI_revS primers were used. A 273-bp fragment of the caroS2I gene was amplified by PCR and ligated into the NdeI and XhoI site of pET30b to form the plasmid pEC2I. Similarly, the plasmid pES2I was obtained by deleting the (His)6-tag of pEC2I (carried out as described above with primers of X21_forT, X21_forS, X21_revT and X21_revS). Subsequently, pES2KI and pES2I were introduced into E. coli BL21 (DE3) cells, respectively.
Restriction DNA library screening and Southern blots
Southern blots were performed according to the DIG Application Manual (Roche, USA). A 543-bp DNA fragment (TF1-2 probe) was amplified with TF1-2P and TF1-2A2 primers (Additional file 1, Table S1), subcloned into pGEM-T Easy vector (Promega Inc., USA), and labeled using a Random Primed DNA Labeling Kit (Roche Diagnostics, USA).
The genomic DNA of the wild-type strain F-rif-18 was digested with various restriction endonucleases, with sites located outside the putative open reading frame. Samples were electrophoresed and analyzed with Southern blotting. After detection using the TF1-2 probe, the DNA from positive gel slices was purified and cloned into pMCL210 to give the carocin-producing plasmid pMS2KI. The pMS2KI construct was isolated and detected as above with the TF1-2 probe.
The transformant cells of BL21, harboring pES2KI or pES2I, were grown in 500 ml to an OD595 of 0.4. The cells were induced with isopropyl-β-D-thiogalactopyranoside (IPTG; final concentration, 0.1 mM; at 25°C for 12 h). Subsequently, the cells were pelleted and the pellets were sonicated (10 cycles of 9 s with 9-s intervals). BL21/pES2KI pellets were subjected to ammonium sulfate precipitation (30-40%), resuspended in buffer A (30 mM NaCl and 20 mM Tris-Cl, pH 8.0), and applied to a Fractogel column (Merck, USA). The fraction was eluted by a NaCl gradient (30 mM-1.4 M). After purification through a P-100 size-exclusion column (BioRad, USA), the CaroS2K fractions were pooled and concentrated using an Amicon centriprep-50 column (Millipore, USA) and dissolved in buffer A. BL21/pES2I pellets were precipitated by ammonium sulfate (70-100%) and resuspended in buffer A. CaroS2I purification involved a similar chromatographic procedure using the Amicon centriprep-3 column (Millipore, USA). The concentration of protein was determined by the Bradford assay (Amresco, USA).
In vitro determination of Carocin S2 activity
Total RNA was treated with calf intestinal alkaline phosphatase (Promega, USA) at 55°C for 30 min as recommended by the manufacturer. The reaction was arrested by adding 5 mM nitrilotriacetic acid, and RNA was extracted with equal volumes of phenol/chloroform. An aliquot of phosphatase-treated RNA was 5'-32P-labeled at 37°C for 30 min by incubation with a mixture of [γ-32P]ATP, T4 polynucleotide kinase (Promega Inc, USA), and reaction buffer in nuclease-free water . [5'-32P]Cytidine 3',5'-bisphosphate (pCp) and T4 RNA ligase (Promega, USA) were used for 3'-labeling of RNA . Subsequently, the mixture was purified by MicroSpin G-25 columns (GE Healthcare, USA). The purified labeled RNA was divided into aliquots and incubated without or with Carocin S2 at 28°C for 60 min, respectively. To measure its activity, CaroS2I was pre-mixed with an equal amount of CaroS2K. The mixtures were subjected to electrophoresis on a 9% polyacrylamide gel (19:1) containing 7M urea, 50 mM Tris, 50 mM boric acid, and 1 mM EDTA, pH 8.3. All samples were electrophoresed at 15℃ by PROTEIN II xi (BioRad, USA).
To confirm DNase activity, 1 μg of genomic DNA from SP33 in solution containing buffer A was incubated with or without Carocin S2 at 28°C for 90 min. An equal quantity of genomic DNA was digested with EcoRI at 28°C for 90 min. Samples were then subjected to electrophoresis on 1% agarose gel.
Antibiotic activity of Carocin S2
Overnight cultures of SP33 were diluted (1:100) with LB medium and grown at 28°C to a density of approximately 105 CFU ml-1. The activity of increasing concentrations of Carocin S2 on cells in suspension incubated at 28°C for 60 min was assessed. CaroS2I was pre-mixed with an equal molar ratio of CaroS2K. All reaction mixtures were spread onto LB agar plates and incubated at 28°C for 16 h. The experiment was performed three times. Colonies growing on a series of plates were respectively counted.
Computer analysis of sequence data
Sequencing of the DNA fragments was carried out using an ABI automated DNA sequencer 373S. The nucleotide sequence data were compiled by DNASIS-Mac software (Hitachi, Japan). Amino acid sequences were compared using international BLAST and FASTA servers. Also, the putative domains of Carocin S2 were predicted using the PSI/PHI-BLAST.
The support of this work by grants from the National Science Council (grants NSC-97-2313-B-005-027-MY3) of Taiwan (R.O.C.) is gratefully acknowledged.
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