Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Characterization of putative toxin/antitoxin systems in Vibrio parahaemolyticus M. Hino1, J. Zhang2, H. Takagi2,3, T. Miyoshi4, T. Uchiumi4, T. Nakashima2,3, Y. Kakuta2,3 and M. Kimura2,3 1 2 3 4

Department of Health and Nutrition Sciences, Faculty of Health and Nutrition, Nishikyushu University, Kanzaki-shi, Saga, Japan Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Higashi-ku, Fukuoka, Japan Department of Biology, Faculty of Science, Niigata University, Nishi-ku, Niigata, Japan

Keywords enteropathogen, stress response, toxin/ antitoxin, viable but nonculturable state, Vibrio parahaemolyticus. Correspondence Makoto Kimura, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. E-mail: [email protected] 2014/0427: received 28 February 2014, revised 16 March 2014 and accepted 27 March 2014 doi:10.1111/jam.12513

Abstract Aim: To obtain more information about the toxin/antitoxin (TA) systems in the Vibrio genus and also to examine their involvement in the induction of a viable but nonculturable (VBNC) state, we searched homologues of the Escherichia coli TA systems in the Vibrio parahaemolyticus genome. Methods and Results: We found that a gene cluster, vp1842/vp1843, in the V. parahaemolyticus genome database has homology to that encoding the E. coli TA proteins, DinJ/YafQ. Expression of the putative toxin gene vp1843 in E. coli cells strongly inhibited the cell growth, while coexpression with the putative antitoxin gene vp1842 neutralized this effect. Mutational analysis identified Lys37 and Pro45 in the gene product VP1843 of vp1843 as crucial residues for the growth retardation of E. coli cells. VP1843, unlike the E. coli toxin YafQ, has no protein synthesis inhibitory activity, and that instead the expression of vp1843 in E. coli caused morphological change of the cells. Conclusions: The gene cluster vp1842/vp1843 encodes the V. parahaemolyticus TA system; VP1843 inhibits cell growth, whereas VP1842 serves as an antitoxin by forming a stable complex with VP1843. Significance and Impact of the Study: The putative toxin, VP1843, may be involved in the induction of the VBNC state in V. parahaemolyticus by inhibiting cell division.

Introduction Vibrio parahaemolyticus, a seafood enteropathogen in coastal countries, causes acute gastroenteritis in humans (Nair et al. 2007). A characteristic feature of V. parahaemolyticus is that it can become nonculturable at a low temperature in a minimum medium while maintaining at least some metabolic activity but can be recovered by a temperature up-shift treatment (Xu et al. 1982). This phenomenon is termed ‘viable but nonculturable’ (VBNC). As Colwell and coworkers first reported on the VBNC state (Kaneko and Colwell 1975), this phenomenon has now been described for over 50 bacterial species using various criteria for viability (Oliver 2010). The VBNC state has been believed to be a survival strategy in response to certain harsh environmental stresses, but its

physiological roles in bacteria are yet to be elucidated and no specific factors have been identified because many environmental conditions induce the VBNC state in different bacterial species. Recently, Hayes and Low have described a possible role of the toxin/antitoxin system (TA system) in the VBNC state (Hayes and Low 2009). TA systems, which were initially characterized as plasmid-borne mediators of plasmid stability (Ogura and Hiraga 1983; Gerdes et al. 1986), have in recent years been identified within the chromosomes of numerous bacterial species (Masuda et al. 1993; Aizenman et al. 1996). Recent findings suggest that chromosomally encoded copies of toxins and antitoxins might function as metabolic stress response elements (Buts et al. 2005; Gerdes et al. 2005), although their biological function is still under debate (Tsilibaris et al. 2007). In the well-

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known type II TA systems, they are composed of two genes organized in an operon that encodes a stable toxin and a labile cognate antitoxin (Gerdes 2000). In a steady state, antitoxins neutralize the effects of toxins by direct protein/protein interactions (Galvani et al. 2001). Upon induction by environmental stresses, such as amino acid and carbon source limitation, labile antitoxins are degraded by a specific protease such as Lon, ClpXP, or ClpAP, leading to rapid growth arrest and cell death by the cellular effects of toxins (Christensen et al. 2001, 2004). The type II toxins inhibit cell growth by targeting a key molecule in any one of several essential cellular functions, including DNA replication, mRNA stability and protein synthesis (Yamaguchi and Inouye 2011; Yamaguchi et al. 2011). The Escherichia coli chromosome encodes at least 36 TA loci, and five TA systems, relBE, mazEF (chpA), chpBIK, yefM-yoeB and dinJ-yafQ, have been well studied; the five toxins inhibit translation by cleaving mRNA on ribosomes (Yamaguchi and Inouye 2011; and references therein). Recent biochemical and structural analyses indicated that toxins from the MazF, ChpB and YafQ families have intrinsic RNase activity; they are, therefore, referred to as mRNA interferases (Inouye 2006). By contrast, RelE cleaves ribosome-associated mRNA in a codon-specific manner positioned at the ribosome A-site (Pedersen et al. 2003). Thus, RelE is a ribosome-dependent RNase and preferentially cleaves stop codons (UAG > UAA > UGA) and sense codons (UCG and CAG) as well. Recently, the structural basis for the ribosome-dependent RNase of the E. coli RelE has been elucidated by structural analysis of the ribosome in complex with the E. coli RelE (Neubauer et al. 2009). As for TA systems in the Vibrio genus, V. cholerae has been reported to contain 13 loci, and one of the putative TA systems, higBA, has been characterized in terms of biochemical properties, although its involvement in the VBNC state has remained unknown (Budde et al. 2007). To obtain more information about the TA systems in the Vibrio genus and also to examine their involvement in the VBNC state, we searched for TA systems in the V. parahaemolyticus genome (RIMD2210633) and characterized them by comparison with those of the corresponding systems in E. coli. Materials and methods Materials The oligonucleotides used in this study were purchased from Sigma-Aldrich. KOD DNA polymerase and DNA ligation kit were purchased from TOYOBO (Tokyo, Japan) and NIPPON GENE (Osaka, Japan), respectively, 186

and used as recommended by the supplier. Restriction endonucleases and DNA-modifying enzymes were purchased from MBI Fermentas (Baltimore, MD). DNA gyrase was supplied as a reaction kit by New England Biolabs (Ipswich, England). Ciprofloxacin (CFX) was obtained as hydrochlorides from Enzo Life Sciences (Farmingdale, NY). Escherichia coli T7 S30 extract system for circular DNA and RiboMAXTM Large-Scale RNA Production Systems –T7 were obtained from Promega (Madison, WI). The plasmid vectors used in this work were as follows: pET-22b vector was from Novagen (Darmstadt, Germany) and pBAD/Myc-HisA was from Invitrogen (Waltham, MA). Escherichia coli strain JM109 was used as a host cell for cloning, and E. coli strains BL21(DE3) Codon Plus RIL (Stratagene, Santa Clara, CA) and LMG194 (Invitrogen) were used as host cells for the expression of recombinant proteins. All other chemicals were of analytical grade for biochemical use. RT-PCR-based analysis To determine whether vp1829 and vp1830, and vp1842 and vp1843 are cotranscribed, we performed RT-PCR. V. parahaemolyticus was grown in nutrient broth including 3% NaCl at 37°C with shaking. Bacterial cells at midexponential phase (turbidity was 0
3) were harvested by centrifugation (5000 g) for 10 min. Total RNA was extracted with the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacture’s method. The purified RNAs were eluted in RNase-free water, and total RNA was determined spectrophotometrically at 260 nm. cDNA was synthesized from total RNA with reverse transcriptase (Promega) and specific primers. Standard PCR was performed with KOD DNA polymerase using 375 ng of cDNA as template. Total RNA and genomic DNA were also used as templates for negative and positive controls, respectively. Expression of Vibrio parahaemolyticus genes in Escherichia coli The expression plasmid pBAD/Myc-HisA was used for the expression of V. parahaemolyticus genes in E. coli cells. Escherichia coli LMG194 strain harbouring expression plasmids, pBAD-vp1830, pBAD-vp1829/vp1830, pBAD-vp1843, pBAD-vp1842/vp1843, pBAD-yafQ, or pBAD-dinJ/yafQ, was grown at 37°C in RM medium with 0
2% D-glucose until an OD590 of 0
5. Then, the culture was divided into two equal parts; half was changed to fresh RM medium containing 0
2% D-glucose, while the other half was changed to fresh RM medium containing 0
2% L-arabinose. Incubation was further continued at 37°C, and bacterial growth was assessed by measuring

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OD590 every 30 min or 1 h. Cellular morphology was observed under a phase-contrast microscope (Olympus BX-PHD, Olympus Inc., Tokyo, Japan). Identification of essential amino acids Mutants, K14E, N37K, E43K, L45P, V54I, I72V and I79V, in which Lys14, Asn37, Glu43, Leu45, Val54, Ile72, and Ile79 in VP1830 were individually replaced with the corresponding residues Glu, Lys, Lys, Pro, Ile, Val and Val in VP1843, respectively, were generated by the PrimeSTAR mutagenesis basal kit (Takara Bio, Shiga, Japan) using pET-22b-vp1830 as a template according to the instructions provided by the manufacturer, and the resulting plasmids were introduced into E. coli JM109 cells. As for mutants, L83Q and M91T, the mutations were introduced by PCR using a specific back primer which contains the mutation. The PCR products were digested with NdeI and XhoI, cloned into pET-22b, and likewise, the resulting plasmids were introduced into E. coli JM109 cells. As no E. coli JM109 clones harbouring pET-22b-L45P were obtained, the E. coli cells transformed by pET-22b-vp1830 being mutated was grown at 37°C in liquid LB medium including 50 lg ml1 ampicillin, and the growth was assessed at the indicated times. Escherichia coli JM109 clones harbouring the other mutant genes were obtained, and mutations were identified by DNA sequence determination; the entire sequence of each mutant gene was determined to rule out that any additional mutations had arisen during the mutagenesis reaction steps. Then, E. coli JM109 cells were transformed by pET-22b-vp1830 or its mutant genes, grown at 37°C in liquid LB medium including 50 lg ml1 ampicillin, and bacterial growth was assessed by measuring OD590 at the indicated times. Viability assay Bacterial viability was determined using the the Live/Dead BacLight Bacterial Viability kit L-7007 (Molecular Probes, Waltham, MA). Escherichia coli LMG194 strain harbouring the expression plasmid pBAD-vp1843 was grown at 37°C in RM medium with 0
2% D-glucose until an OD590 of 0
5 as described above. Bacterial cells were harvested by centrifugation (5000 g) for 10 min and re-suspended in RM medium containing 0
2% L-arabinose. Incubation was further continued at 37°C for 3 h. A total of 1 ml of each culture was centrifuged and washed with filter-sterilized water. Equal volumes of reagents A and B from the kit were mixed and added to the cells (0
3 ll per 100 ll of cells). After thorough mixing, the suspension was incubated at room temperature in the dark for 15 min and 5 ll of the suspension placed on glass slide

with an 18-mm2 glass cover slip. Cells were viewed by using fluorescence microscopy. The LIVE/DEAD BacLightTM assay is based on cell membrane integrity. The fluorescent SYTO 9 stain viable bacteria cells with intact membranes green, while propidium iodide stains dead organisms with compromised membranes red. Overproduction of proteins Overproduction of VP1830, VP1829/VP1830, VP1842/ VP1843 and DinJ/YafQ was carried out using the expression plasmids pET22b-vp1830, pET22b-vp1829/vp1830, pET22b-vp1842/vp1843 and pET22b-dinJ/yafQ, respectively, in E. coli BL21 (DE3) codon plus RIL strain in a conventional manner. In addition, an expression plasmid encoding ΔN23VP1842/VP1843, in which the 23 N-terminal amino acids in VP1842 were deleted, was also constructed, and expression was induced in the same manner as described above. The E. coli lysate was loaded on a COSOMOGEL His-Accept column (Nacalai Tesque, Kyoto, Japan) equilibrated with binding buffer (50 mmol l1 Tris-HCl, pH 8
0, containing 0
5 mol l1 NaCl). After being washed with 50 mmol l1 Tris-HCl, pH 8
0, containing 0
5 mol l1 NaCl and 10 mmol l1 imidazole, the adsorbed proteins were eluted with binding buffer containing 0
4 mol l1 imidazole, and then further purified by gel filtration on a Sephacryl S-200 column (1
4 9 70 cm) equilibrated with 50 mmol l1 Tris-HCl, pH 8
0, containing 0
5 mol l1 NaCl. Subsequently, the complex, VP1842/ VP1843 was dissolved in a 0
1% trifluoroacetic acid (TFA) solution and applied to a reverse-phase high-performance liquid chromatography (RP-HPLC) column (4
6 9 250 mm, COSMOSIL 5C4-AR-300, Nacalai Tesque) equilibrated with 0
1% TFA. The proteins were eluted with a linear gradient of acetonitrile from 0% to 64% in the 0
1% TFA solution during 30 min at a flow rate of 1
0 ml min1. In this purification, VP1842 and VP1843 were eluted at 26 min and 31 min, respectively. The proteins were immediately dialysed against 50 mmol l1 TrisHCl buffer, pH 7
8, containing 0
5 mol l1 NaCl. Overproduction of V. parahaemolyticus homologues, VP2691 and VP0464, of the E. coli cytoskeletal proteins, MreB and FtsZ, respectively, was carried out in the same manner using an expression plasmid pET-22b, as described above. Protein synthesis inhibitory activity Prokaryotic cell-free system protein synthesis was carried out with Rapid Translation System RTS, E. coli HY (Roche, Basel, Switzerland). The reaction mixture consists of 3
0 ll of E. coli lysate, 2
5 ll of reaction mix, 3
0 ll of amino acid mixture, 0
25 ll of methionine, 1
25 ll of

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reconstitution buffer, 25 ng of green fluorescent protein (GFP) control plasmid and 20 lmol l1 toxin or toxinantitoxin complexes in a final volume of 12
5 ll. The reaction mixtures were incubated for 150 min at 37°C and then the synthesized GFP was detected by Western blotting using an anti-GFP. Model building A model of VP1843 was built with the Swiss-Model Server (http://swissmodel.expasy.org) using Caulobacter crescentus ParE (Protein Data Bank (PDB) code 3KXE) (Dalton and Crosson 2010) as a template. A structural representation was prepared with PyMOL (http://www. pymol.org).

The genetic organization of known type II TA systems is highly conserved. In general, the toxic gene is located directly downstream from the antitoxic gene so that they can be transcribed together. The database shows that the translational start codons, ATG, of vp1830 and vp1843 overlap with the stop codons, TGA, of the preceding vp1829 and vp1842, respectively (Fig. 1b). To examine whether vp1829/vp1830 and vp1842/vp1843 are organized as operons, PCR with reverse transcription (RT-PCR) was carried out using specific primers. The RT-PCR yielded single DNA fragments corresponding to their expected sizes, as shown in Fig. 1c. This result indicated that vp1829 and vp1830, and vp1842 and vp1843 are transcribed together as single mRNAs. These findings suggested that these gene clusters encode TA systems of V. parahaemolyticus, organized in an operon.

Results Identification of TA systems of Vibrio parahaemolyticus

Toxicity of the putative toxins of Vibrio parahaemolyticus

A search of the V. parahaemolyticus genome database (RIMD2210633) revealed that two gene clusters, vp1829/ vp1830 and vp1842/vp1843, have significant homology to that encoding DinJ/YafQ of E. coli (Yamaguchi and Inouye 2011). The amino acid sequences of the four V. parahaemolyticus proteins can be aligned to those of DinJ and YafQ, with 17–23% identical residues (Fig. 1a). The putative antitoxins, VP1829 and VP1842, are highly homologous with each other; VP1842 has 23 extra residues at the N-terminus when the residues are aligned so as to optimize the similarity, having only three amino acid replacements at positions 46, 72 and 80 in the VP1829 numbering. It should be noted that VP1842 has Met at position 24, which corresponds to the N-terminal Met in VP1829 (Fig. 1a), suggesting that the Met at position 24 is a true N-terminal amino acid in VP1842, even though TTG was assigned as an initiation codon in the V. parahaemolyticus genome database. As for the putative toxins, VP1830 and VP1843 are also highly homologous with only 9 amino acid replacements at positions 14, 37, 43, 45, 54, 72, 79, 83 and 91.

To evaluate the potential for the putative toxins to inhibit cell growth, the expression plasmid pBAD/Myc-HisA was employed because its expression is strictly controlled under the presence or absence of arabinose and glucose; that is, the presence of arabinose induces the expression whereas that of glucose suppresses it (Guzman et al. 1995). For this purpose, vp1830, vp1829/vp1830, vp1843, vp1842/vp1843, yafQ and dinJ/yafQ were placed under control of the araBAD promoter on the expression plasmid pBAD/Myc-HisA and their expressions were induced or suppressed by the addition of 0
2% arabinose or 0
2% glucose, respectively. The presence of glucose had no influence on the growth of cells harbouring the expression plasmids (Fig. 2a), whereas the growth of cells containing the expression plasmid for vp1843 or yafQ was inhibited in the presence of 0
2% arabinose; the inhibition by the vp1843 expression appeared to be stronger than that by yafQ (Fig. 2b). In contrast, the expressions of vp1830, vp1842/vp1843 and dinJ/yafQ in the presence of 0
2% arabinose had no influence on cell growth (Fig. 2b). These results suggested that vp1842/

Figure 1 Identification of toxin/antitoxin (TA) systems in Vibrio parahaemolyticus. (a) Sequence alignment of V. parahaemolyticus proteins with their homologues, DinJ/YafQ, in Escherichia coli cells. Amino acid sequences were aligned using the program CLUSTAL W (http://www.genome. jp/tools/clustalw/). Amino acids conserved completely in three proteins are indicated by grey boxes. Amino acid exchanges in the V. parahaemolyticus proteins are boxed. (b) Gene organization of vp1829/vp1830 and vp1842/vp1843 are schematically shown. Possible palindromic sequences upstream of the gene clusters are indicated by opposing arrows. The nucleotide sequence (ATGA) overlapping initiation (ATG) and termination (TGA) codons in intercistronic regions of the two clusters is highlighted by a straight line. The initiation codon (ATG) for vp1829 and those (TTG and ATG) for vp1843 are boxed. It should be noted that TTG is assigned as an initiation codon for vp1843 in the V. parahaemolyticus genome database (RIMD2210633). (c) RT-PCR of vp1829/vp1830 and vp1842/vp1843. V. parahaemolyticus was grown under conditions described previously, and total RNA was extracted. RT-PCR was carried out using specific primers for the genes. Lane 1, a 100 bp ladder marker, lane 2, RT-PCR for vp1829/vp1830; lane 3, RT-PCR for vp1829/vp1830 without RT; lane 4, RT-PCR for vp1842/vp1843; lane 5, RT-PCR for vp1842/vp1843 without RT; lanes 6 and 7, PCR for vp1829/vp1830 and vp1842/vp1843 using the V. parahaemolyticus genomic DNA as a template, respectively.

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(a) Antitoxin VP1829 VP1842 DinJ

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-----------------------MDTRIQFRVDEETKRLAQQMAESQGRTLSDACRELTE MFVSCPSQSLVLTFVHTPTKEYIMDTRIQFRVDEETKRLAQQMAESQGRTLSDACRELTE ---------------------MAANAFVRARIDEDLKNQAADVLAGMGLTISDLVRITLT

40

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VP1829 VP1842 DinJ

QLAEQQRKSLSHDAWLTEQVNLAFEKFDSGKSVFVEHQTAKSQMEERKARIRNRGKQ QLAEQQRKTLSHDAWLTEQVNLAFEKFDSGKSVFLEHQTAKSRMEERKARIRNRGKQ KVAR--EKALPFDLREPNQLTIQSIKNSEAG--IDVHKAKDADDLFDKLGI------

Toxin

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VP1830 VP1843 YafQ

MILWEEESLNDREKIFEFLYDFNPDAAEKTDNLIEANVENLLEQLLMGVQ--RDGVRGRL MILWEEESLNDREEIFEFLYDFNPDAAEKTDNLIEAKVENLLKQPLMGVQ--RDGIRGRL MIQRDIEYSGQYSKDVKLAQKRHKDMNKLKYLMTLLINNTLPLPAVYKDHPLQGSWKGYR

VP1830 VP1843 YafQ

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LIIPEISMIVSYWIEGDIIRIMRVLHQKQKFPMD LIIPEISMIVSYWVEGDIIRVMRVQHQKQKFPTD DAHVEPDWILIYKLTDKLLRFERTGTHAALFG--

vp1830 vp1843

AATCGAGGTAAGCAATGATTTTATGGGAAGAA (b)

vp1829 vp1842 –40

–20

–1

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AGTACTAACATTTGTACACACACCAACAAAGGAATACATCATG vp1830

vp1829

vp1843

vp1842

–20

–1

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AATGCCACGCGTGCCGAATCACTCTTAAATTGTTTGTTAGTTGCCCATCTCAAAGCCTAGTGCTAACATTTGTACACACACCAACAAAGGAATACATCATG

(c)

1

2

3

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7

(kbp)

1·0 0·5

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Figure 2 Effect of the expression of Vibrio parahaemolyticus genes on the cell growth of Escherichia coli. (a, b) Effects of the expression of V. parahaemolyticus toxin genes and of E. coli dinJ/yafQ on the cell growth of E. coli in the presence of 0
2% glucose (a) or 0
2% arabinose (b). vp1843 (red); vp1842/vp1843 (cyan); vp1830 vp1829/vp1830 (violet); yafQ (green); dinJ/yafQ (blue); (orange); and pBAD (light blue). (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper).

tify essential amino acid residues in VP1843 for the growth retardation of E. coli cells, we prepared nine vp1830 mutants, in which codons encoding Lys14, Asn37, Glu43, Leu45, Val54, Ile72, Ile79, Leu83, and Met91 in VP1830 were individually replaced with those encoding the corresponding residues Glu14, Lys37, Lys43, Pro45, Ile54, Val72, Val79, Gln83 and Thr91 in VP1843, respectively, and examined their influence on cell growth when they were introduced into E. coli JM109 cells. The result clearly showed that mutation of Asn37 or Leu45 completely inhibited cell growth, while those of the other residues had no influence on the E. coli growth (Fig. 3). To corroborate this result, E. coli BL21 (DE3) codon plus RIL cells were transformed by pET-22b harbouring all mutant genes except for N37K and L45P, grown at 37°C in liquid LB medium including 50 lg/ml ampicillin, and bacterial growth was assessed in the presence of 1 mmol l1 isopropyl-b-D-thiogalacto-pyranoside (IPTG) by measuring OD590 at the indicated times. The result showed no influence on the E. coli growth when the mutant genes were overexpressed in E. coli cells. These results strongly suggested that Lys37 and Pro45 in VP1843 are crucial amino acids for the growth retardation of E. coli cells. Involvement of the putative TA systems in the VBNC state of V. parahaemolyticus It is known that the morphological change is a characteristic feature of the VBNC state (Mary et al. 2002; Pawlowski et al. 2011); the most apparent morphological

vp1843 at least serve as a TA system in V. parahaemolyticus.

2·5 2

In an initial stage of this study, we first attempted to construct inducible systems for putative TA systems using the expression vector pET-22b. However, this attempt failed; that is, no E. coli JM109 cells containing the expression plasmid for vp1843 were obtained, although the other genes were successfully cloned. This result suggested a high toxicity of the gene product of vp1843. Interestingly, we found during this study that a spontaneous mutation in the gene vp1843 suppressed the cell toxicity and produced E. coli clones containing the vp1843 variant. Sequence analysis of the mutant gene indicated that a codon encoding Pro45 in VP1843 was replaced by that encoding the corresponding amino acid Leu45 in VP1830. This observation suggested that Pro45 in VP1843 plays a crucial role in the growth retardation of E. coli cells. As described in Fig. 1a, there are 9 amino acid replacements between VP1830 and VP1843. To iden190

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Essential amino acid residues for the toxicity 1·5 1 0·5 0 0

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Time (h) Figure 3 Essential amino acid residues in VP1843 for the growth retardation of Escherichia coli cells. Site-directed mutagenesis of vp1830 was done, as described in Materials and methods, and mutations were identified by DNA sequence determination. , vp1830 (blue); , K14E (cyan); , N37K (green); , E43K (violet), , L45P (red); , V54I (orange); , I72V (light blue); , I79V (brown); , L83Q (light green); , M91T (pale violet). (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper).

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change in the cells was a shift from rod-shaped to either a more cocci-like cell shape or an elongated shape. To examine the involvement of the putative toxins in the VBNC state, we next examined the influence on the cell shape when vp1830, vp1843, or yafQ was expressed under control of the araBAD promoter on the expression plasmid pBAD/Myc-HisA, as described in Fig. 2. Interestingly, E. coli cells containing pBAD/Myc-HisA-vp1843 partially showed a filamentous shape without the induction (Fig. 4a). At 3 h after induction of vp1843 expression, the cell shape completely changed from a rod shape to a filamentous one, as shown in Fig. 4a. In contrast, the expression of vp1830 or yafQ had no influence on the cell shape for more than 3 h (Fig. 4a). Moreover, the coexpression of vp1843 with vp1842 neutralized the morphological change caused by the vp1843 expression alone, as shown in Fig. 4b. This result indicated that the observed phenotype was caused by the specific expression of vp1843, and that coexpression with vp1842 could suppress the morphological change caused by the vp1843 expression. We further examined if E. coli cells expressing vp1843 are viable by measuring membrane integrity using the Live/Dead BacLight Bacterial Viability kit L-7007 (Molec(a)

VP1830

ular Probes). Figure 4c shows a representative field of E. coli cells expressing pBAD/Myc-HisA-vp1843 stained with the Live/Dead BacLight and viewed using fluorescence microscopy. The figure shows most of the cells fluorescing green, indicating these E. coli cells an intact cellular membrane which is indicative of live cells. It was thus likely that vp1843 may be at least involved in the induction in the VBNC state of V. parahaemolyticus by inhibiting cell growth. Biological activity of VP1843 YafQ, the E. coli homologue of VP1830 and VP1843, is an endoribonuclease that associates with the ribosome and blocks translation through sequence-specific mRNA cleavage (Prysak et al. 2009). To examine the biological activity of VP1830 and VP1843 and also to examine whether they form a complex with their putative cognate antitoxins, the genes vp1830, vp1829/vp1830 and vp1842/ vp1843 were placed under control of the T7 phage promoter on the expression plasmid pET-22b and their expression was induced by the addition of 1 mmol l1 IPTG. The protein complexes, ΔN23VP1842/VP1843 and VP1829/VP1830, were purified to homogeneity, indicat-

VP1843

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pBAD

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2 µm Figure 4 Morphological changes by expression of Vibrio parahaemolyticus genes. Phase-contrast images of fixed Escherichia coli cells bearing the expression plasmids containing V. parahaemolyticus genes or E. coli genes grown in liquid cultures in the presence of 0
2% arabinose. (a) Cell morphology before (upper, 0 H) and after (lower, 3 H) the expression of V. parahaemolyticus genes, vp1830 and vp1843, and E. coli yafQ in the presence of 0
2% arabinose. (b) Cell morphology before (upper, 0 H) and after (lower, 3 H) the expression of V. parahaemolyticus genes, vp1829/vp1830 and vp1842/vp1843, and E. coli dinJ/yafQ in the presence of 0
2% arabinose. (c) LIVE/DEAD BacLightTM fluorescence microscopy of E. coli cells bearing pBAD-vp1843. Green cells were the healthy cells.

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rke

r

ing that VP1843 and VP1830 form a stable complex with ΔN23VP1842 and VP1829, respectively. In contrast, VP1842/VP1843 was overproduced as an inclusion body in E. coli cells, and it could not be refolded. Therefore, ΔN23VP1842/VP1843 was used for further characterization; it is henceforth referred to as VP1842/VP1843. Moreover, VP1830 was overproduced and purified to homogeneity, as shown in Fig. 5a, demonstrating little toxicity to E. coli cells. Subsequently, the putative toxin VP1843 was purified by RP-HPLC, as described in Materials and methods. Interestingly, it should be noted that VP1843 migrates slower in SDS-PAGE than VP1830 even though they have a molecular mass similar to each other (Fig. 5a). To define the biological activity, VP1830 and VP1843 were characterized with respect to a protein synthesis inhibitory activity, using YafQ as a positive control. VP1830 or VP1843, unlike YafQ, had little effect on in vitro GFP synthesis (Fig. 5b) as well as on poly(Phe) synthesis (data not shown). In addition, they appeared to have little RNase activity (data not shown). It is known that the type II toxins, such as ParE, target E. coli DNA

(kDa)

27·0 20·0 14·3

gyrase and inhibit DNA replication (Critchlow et al. 1997; Jiang et al. 2002). We thus tested if VP1830 or VP1843 has potential to inhibit the DNA gyrase activity using plasmid pUC19 as a substrate. They had no inhibitory activity towards DNA gyrase (data not shown). Therefore, no clear biological activity of VP1843 to explain its specific toxicity towards cell growth could be defined from these analyses. Recently, one of the E. coli toxins, YeeV, was found to inhibit polymerization of bacterial cytoskeletal proteins, and its ectopic expression resulted in morphological change of the E. coli cells (Tan et al. 2011; Masuda et al. 2012). It has been further described that, when one of the essential cytoskeletal proteins, FtsZ, was inhibited by expression of the SOS gene SulA, the E. coli cells became filamentous (Tan et al. 2011). Hence, the present result has led to the speculation that VP1843, like E. coli toxin YeeV, might bind and inhibit the polymerization of cytoskeletal proteins, even though VP1843 has no sequence homology to YeeV. A BLAST sequence-similarity search with sequences of the E. coli cytoskeletal proteins, MreB and FtsZ, against the complete genome of V. parahaemolyticus identified two genes, vp2691 and vp0464, which probably encode homologues of MreB and FtsZ, respectively. Hence, we next examined whether VP1843 could interact with their gene products by pull-down assay. Namely, VP2691 and VP0464 were overproduced in E. coli cells using pET-22b, and the cell extracts were mixed with VP1843-His-tag in the presence of His-Accept to examine their potency to interact with VP1843. SDSPAGE analysis of elutes showed no significant interaction of VP1843 with VP2691 or VP0464 (data not shown), indicating that VP1843 may inhibit cell division in a distinct manner to the E. coli toxin YeeV.

82 9/V VP P1 83 18 0 30 VP 18 42 /VP VP 18 18 43 43

VP 1

fQ

Ya

ol

Din

ntr Co

Ma

rke

r

(b)

J/Y a

fQ

Discussion

-GFP

Figure 5 Biochemical characterization of putative toxins. (a) SDSPEGE of purified VP1830 and VP1843. (b) Green fluorescent protein (GFP) synthesis using Rapid Translation System RTS, Escherichia coli HY (Roche). The reaction mixtures were incubated as described in Materials and methods, and then the synthesized GFP was detected by Western blotting using an anti-GFP.

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A characteristic feature of V. parahaemolyticus is its entry into the VBNC state, which has attracted a great deal of interest as it plays a crucial role in the survival of human pathogens and possibly in their ability to produce disease (Oliver 2010). Although the expression of several genes related to VBNC has been characterized for Vibrio spp. (Vora et al. 2005; Mishra et al. 2012), factors involved in VBNC have remained unclear. Recently, possible involvement of TA systems in induction into the VBNC state has been proposed (Hayes and Low 2009). In the present study, we found that the gene products VP1829/VP1830 and VP1842/VP1843 derived from the gene clusters vp1829/vp1830 and vp1842/vp1843 in V. parahaemolyticus, respectively, had sequence homology to E. coli type II TA proteins, DinJ/YafQ. RT-PCR indicated that both gene clusters were cotranscribed together as single mRNAs.

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Furthermore, the expression of vp1843 completely inhibited the E. coli cell growth, and its inhibitory activity was neutralized by co-expression with vp1842. These findings suggested that the gene cluster vp1842/vp1843 serves as a TA system in V. parahaemolyticus. In contrast, the expression of vp1830 had no influence on cell growth and hence overproduced its gene product in E. coli cells, indicating that VP1830 has no ability to arrest cell growth, even though it is highly homologous to VP1843. It is, however, unlikely that the gene cluster vp1829/vp1830 is an evolutionary fossil of vp1842/vp1843 in V. parahaemolyticus because it is transcribed in the cell, as described above. One cannot thus exclude the possibility that vp1829/vp1830 may serve as a TA system in V. parahaemolyticus. On the basis of sequence similarity, the putative V. parahaemolyticus TA toxin VP1843 is classified into the RelE/ParE family, which is a broadly distributed superfamily of the type II TA toxins and encompasses several smaller families, including the RelE and ParE families (Roberts and Helinski 1992). YafQ, the E. coli homologue of VP1843, belongs to the RelE family and functions to inhibit translation by inducing cleavage of mRNAs in the ribosomal A-site (Prysak et al. 2009). The ParE family toxin, on the other hand, inhibits DNA gyrase and thereby blocks DNA replication (Jiang et al. 2002). The present study indicated that VP1843, unlike YafQ and ParE, has neither protein synthesis inhibitory activity nor DNA gyrase inhibitory activity. Instead, we found that the vp1843 expression in E. coli cells caused significant morphological change; E. coli cells became filamentous, and the co-expression of vp1843 with vp1842 neutralized this change. As it is known that morphological change is associated with incomplete cell division, VP1843 may be the first RelE/ParE family toxin which inhibits cell division, leading to rapid growth arrest. Lys37 and Pro45 in VP1843 were identified as essential amino acid residues for the growth retardation of E. coli cells. It is known that three-dimensional structure is conserved in the RelE/ParE family despite of sequence variation. Hence, to examine the functional implication of these amino acids in the activity, we developed a model of VP1843 using the structure of the C. crescentus ParDParE as a template in the Swiss-Model Server because the primary structure of ParE among the proteins deposited in PDB was shown to be the most similar to VP1843 (23
6% identity). ParE, originally identified as a stabilizing element on plasmid RK2 (Roberts and Helinski 1992; Roberts et al. 1994), inhibits DNA gyrase and thereby blocks DNA replication (Jiang et al. 2002). The crystal structure of a heterotetrameric ParD-ParE complex has been determined at a resolution of 2
6  A (Dalton and Crosson 2010). In our model, Lys37 is located at a2 and

C Thr91

Ile54

α4

β1

Gln83 β2 Glu14

Val72

β3 Val79 α1

Lys37

N

α3 Pro45 Lys43

α2

Figure 6 Three-dimensional model of VP1843. Three-dimensional model of VP1843 was constructed with the Swiss-Model Server (http://swissmodel.expasy.org) using Caulobacter crescentus ParE (PDB code 3KXE) as a template (Dalton and Crosson 2010). The secondary structures are labelled, and N and C termini are indicated. The side chains of amino acids exchanged between VP1843 and VP1830 are indicated. The figure was drawn with PyMol (http://pymol.sourceforge.net).

its side chain appears to be exposed into solution, and that Pro45 lies between a2 and a3 and appears to interrupt continuous helix formation of a2 (Fig. 6). Hence, the N-terminal helix (a2) in VP1843 may play an essential role in the interaction with target proteins involved in cell division. In this study, the putative toxin gene vp1843 could not be used to obtain E. coli JM109 clones using the expression plasmid pET-22b, probably because of its toxicity. The toxin variant that could be isolated from surviving clones has the spontaneous mutation at the codon encoding Pro45. A similar observation was reported for the epsilon/zeta TA family (Mutschler et al. 2011). Zeta toxins phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG), and the resulting phosphorylated UNAG inhibits Mur, the enzyme catalysing the initial step in bacterial peptidoglycan biosynthesis. Thus, they arrest the cell growth through global inhibition of peptidoglycan synthesis. Zeta toxins consist of around 250 amino acids and fold into phosphotransferase-like structures with a Walker A motif (Meinhart et al. 2003). Although VP1843 has no structural homology to zeta toxins, the molecular basis for growth arrest by zeta toxins may provide a clue to identify target proteins for VP1843. In conclusion, the gene cluster vp1842/vp1843 encodes the V. parahaemolyticus TA system; VP1843 arrests cell growth, whereas VP1842 serves as an antitoxin by form-

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ing a stable complex with VP1843. VP1843, unlike the E. coli toxin YafQ, has no protein synthesis inhibitory activity, and that instead the expression of vp1843 in E. coli caused morphological change of the cells. It is known that morphological change is a characteristic feature of the VBNC state of V. parahaemolyticus cells and associated with incomplete cell division. It is thus likely that VP1843 arrests cell growth, probably by inhibiting cell division. A homology search of the genome database revealed that the gene cluster vp1842/vp1843 is highly conserved in the genus Vibrio, including V. alginolyticus, V. anguillarum, V. cholerae, V. harveyi and V. splendidus. We hence proposed that the gene cluster vp1842/vp1843 may be one of the factors which are involved in the induction into the VBNC state. Further studies for the identification of target proteins for VP1843 as well as for the molecular mechanism as to how VP1843 interacts with target proteins should provide more insight not only into the physiological function of TA systems but also into the molecular basis for the VBNC state. Acknowledgements We are grateful to M. Mori and T. Ueda for their useful helps and suggestions. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (no. 22380062 to M.K.). J. Zhang was sponsored by CSC (China Scholarship Council). Conflict of Interest No conflict of interest declared. References Aizenman, E., Engelberg-Kulka, H. and Glaser, G. (1996) An Escherichia coli chromosomal ‘addiction module’ regulated by guanosine 30 ,50 -bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci USA 93, 6059–6063. Budde, P.P., Davis, B.M., Yuan, J. and Waldor, M.K. (2007) Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae. J Bacteriol 189, 491–500. Buts, L., Lah, J., Dao-Thi, M.H., Wyns, L. and Loris, R. (2005) Toxin-antitoxin modules as bacterial metabolic stress managers. Trends Biochem Sci 30, 672–679. Christensen, S.K., Mikkelsen, M., Pedersen, K. and Gerdes, K. (2001) RelE, a global inhibitor of translation, is activated during nutritional stress. Proc Natl Acad Sci USA 98, 14328–14333. Christensen, S.K., Maenhaut-Michel, G., Mine, N., Gottesman, S., Gerdes, K. and van Melderen, L. (2004)

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