1. Introduction

The term “toxin-antitoxin system” usually abbreviated as “TA system” comprises a functional element consisting of a biologically active protein molecule and a corresponding inhibitor, whose nature and inhibitory mechanism depend on the system’s class affiliation. Components of such systems are encoded within polycistronic operons, often with partially overlapping open reading frames. These systems are wide spread among bacteria as well as archaea[1,2,3,4] and evolved to carry out diverse functions. However, their common feature is an enzymatic activity detrimental for the cell metabolism. Such toxic activity has been demonstrated to switch bacterial cells over to a dormant state, leading to cell death during prolonged exposure. In most cases various stress stimuli are responsible for TA system activation.

The signaling pathway in such instances is often related to other stress-induced response pathways. Moreover, it is well documented that in some cases the activity of TA systems stabilizes mobile genetic elements, therefore comprising an important mechanism of plasmids maintenance.

The biological activity of a toxin comprising a component of a TA system is usually (but not always) that of an endoribonuclease. The classification of TA systems is based on the mechanism of inhibition of the toxin as well as on operon auto regulatory functions. Initially two classes of TA systems were identified[5], but subsequent discoveries extended the classification to three classes[6]. Class I includes systems in which the antitoxin is an antisense RNA forming duplexes with the toxin mRNA. This leads to inhibition of translation in a process known as RNA interference. Examples of such systems are chromosomally located operons found in Escherichia coli namely tisAB[7] and symER[8], as well as plasmid loci parB[9] of E. coli and par of Enterococcus faecalis[10][11] and a homologous plasmid operon of Staphylococcus aureus[12]. Class II encompasses a wide range of TA systems; Antitoxins of this class are proteins. The biological activities exhibited by the toxins include transcription inhibition by targeting gyrase function and interference with translation through an mRNA interferase activity, which may or may not rely on ribosome binding. The endoribonucleolytic activity of mRNA interferases is often sequence specific gives a short overview of the class II TA systems and their characteristics. Class III comprises a single member only. This system is encoded in the toxIN operon of Erwinia carotonovora, a plant pathogen. In this case inhibition of ToxN toxin activity is driven by RNA molecules directly interacting with the toxin molecules[13][14]. On the basis of different TA Modules, bacteria have developed multiple complex mechanisms ensuring an adequate response to environmental changes. In this context, bacterial cell division and growth are subject to strict control to ensure metabolic balance and cell survival.

X. nematophila is a motile gram-negative bacteria belonging to the family Enterobacteriaceae[15]. It forms symbiotic association in the gut of a soil nematode of family Steinernematidae[16]. The X. nematophila isolated, studied so far have been obtained from nematodes harvested from soil samples. Free-living forms of the bacterium have not yet been isolated from soil or water sources, which suggest that the symbiotic association may be essential for the survival of the bacteria in the environment. The bacteria in turn, are essential for effective killing of the insect host and are required by the nematode to complete its life cycle [17][18]. As the bacteria enter the stationary phase of their growth cycle, they secrete several extracellular products, which include lipase (s), phospholipase (s), protease (s), and several broad spectrum antibiotics[19][20]. These products are believed to be secreted in the insects hemolymph when the bacteria enter stationary phase conditions.

Since TA modules are involved in the survival of bacterium under stress conditions. Therefore, it could be possible that similar kind of survival mechanism may exist in X. nematophila which protects bacterium during its stress full life cycle inside insect hemolymph or in insect carcass (due to exponential growth of bacterium which leads to nutrient deprivation). In our earlier study we have identified three putative TA modules includes relB, relE and mazF[21] from the genome of X. nematophila. In this study we have identified three additional putative TA modules such as hip-AB, ccd-BA and yoe-yef-BM. These operons located in the genome of X. nematophila, molecular weights as well as isoelectric point of each toxin and antitoxin proteins have been identified with the help of Expasy server (www.expasy.org) which suggests that all three novel identified TA modules in this study belongs to Type II TA category. Extensive promoter analysis were done with BPROM (www.sofberry.com) to dissect the various transcription factors which control the transcription of such novel identified putative TA module in X. nematophila.

2. Methods

2.1. Identification and Genetic Organization of Putative TA Modules

Complete genome sequence of X. nematophila ATCC 19061 was available in NCBI server (www.ncbi.nlm.nih.gov) under RefSeq database with accession number NC_014228.1. The entire genome sequence of X. nematophila was analyzed bioinformatically for the presence of hip-AB, ccd-BA, and yoeB-yef M homolog’s. Operons coding these TA modules were downloaded and analyzed with ORF (Open Reading Frame) finder Software from NCBI server.

2.2. Protein-Protein Blast

Protein databank was searched by protein-protein blast performed with putative toxin-antitoxins encoded by the various operon from the genome of X. nematophila using NCBI server (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.3. Phylogenetic Analysis

Different annotated protein sequences were multiple aligned and phylogenetic trees were constructed by using CLC Genomics Workbench (version 4.9) software.

2.4. Physiochemical Properties

All the physio-chemical properties of putative toxin- antitoxin proteins have been determined by Expasy server (www.expasy.org).

2.5. Promoter Analysis

Identification of putative pro- moters associated with ORFs was determined by software BPROMO(www.softberry.com).

2.6. Genomic DNA Isolation

X. nematophila culture was inoculated from glycerol stock in 50 ml and grown overnight at 28℃, 200 rpm. Overnight grown culture was pelleted down by centrifugation at 5000 rpm for 10 minutes at 4℃. The pellet was resuspended in 4 ml TE buffer pH8 (10 mM Tris HCl, 1mM EDTA) and 0.5 ml of 10% SDS was added. 30 μl of proteinase K (20mg/ml) was added to the resuspended culture and incubated at 37℃ for one hour. After complete lysis of the cells, 1 ml of 5 M NaCl was added and mixed gently. 750 μl CTAB NaCl mixture was added to the lysate and incubated at 65℃ for 20 minutes. Later equal volume of chloroform: Isoamyl alcohol mix (approax 7.5ml) was added and mixed gently. It was centrifuged at 12,000 rpm at 4℃ for 30 minutes. To the aqueous phase containing genomic DNA, 12.5 μl of RNase (2mg/ml) was added. The supernatant was incubated at 37℃ for one and half hours; equal volume of phenol: chloroform:isoamyl alcohol mixture was added and mixed properly. The tubes were centrifuged at 12,000 rpm, for 30 minutes at 4℃. The supernatant was again extracted with equal volume of phenol:chloroform:isoamyl alcohol mixture. The supernatant (aqueous phase) was collected in korex tube and 0.6 volume of isopropanol was added and mixed properly. korex tube was centrifuged at 10,000 rpm for 20 minutes at 4℃. The pellet was washed with 70 % ethanol and kept for drying in room temperature. Finally the pellet was dissolved in 0.5 ml autoclaved water and run on 0.8% agarose gel.

2.7. Amplification of Putative HipAB, ccdAB and yoeB-yefM Operon by Polymerase Chain reaction (PCR) from Genome

The DNA sequence encoding putative hipA toxin and its antitoxin hipB respectively were obtained by PCR amplification using genomic DNA as template (Denaturation at 94ºC for 40 sec, annealing at 60ºC for 40 sec and extension at 68ºC for 1 min 30 sec, total 30 cycles) for cloning with primer pair JSR25 and JSR26. Sequence encoding putative ccdB toxin and its antitoxin ccdA respectively were obtained by PCR amplification using genomic DNA as template (Denaturation at 94ºC for 40 sec, annealing at 60ºC for 40 sec and extension at 68ºC for 50 sec, total 30 cycles) for cloning with primer pair JSR30 and JSR31. Sequence encoding putative yoeB toxin and its antitoxin yefM respectively were obtained by PCR amplification using genomic DNA as template (Denaturation at 94ºC for 40 sec, annealing at 60ºC for 40 sec and extension at 68ºC for 50 sec, total 30 cycles) for cloning with primer pair JSR20 and JSR21.

Table 1. List of primers used in this study Primer Sequences JSR25 5’CGC GGA TCC ATG AAG ATC ACA TCA CCA CAA AC 3’ JSR26 5’CCC AAG CTT TTA CCC TGC ACA AAG CCT GCC 3’ JSR30 5’CGC GGA TCC ATG ACA CGT GTG CTT ACA CG 3’ JSR31 5’CCC AAG CTT TCA AAA CCC GTC AAA GAG TAA ATC 3’ JSR20 5’CGC GGA TCC ATG AAT GTT ATG AGC TAT TCA GC 3’ JSR21 5’ CCC AAG CTT TTA GTA GTG ATA GCG ACA TTG 3’

3. Result and Discussion

3.1. Genetic Organization of hip-AB, ccd-BA, and yoeB-yefM Homolog’s

X. nematophila genome was analyzed bioinformatically and in this study we have identified three loci corresponding to three different TA modules. HipA toxin homolog encoded by 1299 bp and its antitoxin homolog HipB encoded by 251 bp was located in an operon designated as "XNC1_operon 0810" which lies between 4076692-4077990 base pair of X. nematophila genome in complementary orientation as shown in Figure1 (a).

In the second identified putative TA module, putative CcdB toxin encoded by 305 bp whereas its antitoxin homolog CcdA encoded by 278bp. Both genes were located in locus designated as operon"XNC1 _operon0014" which lies between 82145-83430 base pair as shown Figure1 (b).

Third identified putative TA module is homolog of yoeB-yefM TA module. Putative YoeB toxin encoded by 254bp whereas its putative antitoxin YefM homolog encoded by 254 bp which is located in operon designated as operon "XNC1_operon0711”. This operon lies between 3607111-3607616 base pair in the genome of X. nemtaophila as shown in Figure 1(c).

3.2. Analysis by ORF Finder (Open Reading Frame Finder) and Identification of Protein Families

All the corresponding protein sequences encoded by different TA modules were deduced by ORF finder. First putative HipA toxin compose of 432 amino acids and its putative HipB antitoxin composed of 83 amino acids. The Protein-protein blast with HipA protein showed a conserved domain belongs to couple_ hip-A super family, hipA_N super family, and hipA_C super family as shown in Figure 2 (a). Blast results showed its similarity with hip-A domain containing protein of Enterobacter sp.638 with 69% identity (accession no. YP_001176760.1) Similarly, protein-protein blast with HipB protein showed a conserved domain belongs to HTX_XRE super family as shown in Figure 2 (b). The blast results showed its 59% identity with XRE super family transcriptional regulators of Enterobacter sp.638 (accession no. YP_00117676.1).

Putative CcdA antitoxin composed of 92 amino acids and its putative toxin CcdB composed of 113 amino acids. Protein-protein blast with putative CcdA protein showed conserved domain belongs to CcdA super family as shown in Figure 3 (a). It showed 68% identity with CcdA protein from Photorhabdus luminescens subsp. laumondii TTO1 (Accession no. NP_ 929535.1). Protein-Protein blast with putative CcdB protein showed a conserved domain belongs to CcdB super family as shown in Figure 3 (b). Blast result with putative CcdB showed that CcdB toxin protein from X. nematophila was 67% identical with CcdB cytotoxic from Photorhabdus luminescens subsp. laumondii TTO1 (Accession no. NP _929536.1)

Putative YoeB toxin composed of 84 amino acids and its putative YefM antitoxin composed of 84 amino acids. Protein-protein blast with putative YefM antitoxin protein showed a conserved domain belongs to PhdYefM_antitoxin super family as shown in Figure 4 (a). Blast results showed its similarity with yefM antitoxin protein from Prevent host death protein from Delta proteobacterium MLMS-1. (Accession no. ZP_01288746.1) with 77% identity. Blast results showed that putative YoeB toxin has a conserved domain at N-terminal which belongs to plasmid stability super family as shown in Figure 4 (b). Blast results showed that putative YoeB toxin showed 81% identity with hypothetical protein from Legionella drancourtii LLAP12 LDG_6480 (Accession no. ZP_ 09620091.1). Although putative toxin was encoded by genome but belong to plasmid stability family, this could be possible only due to the horizontal gene transfer commonly present in the prokaryotic system.

3.3. Physiochemical Properties

The physiochemical properties of putative toxin-antitoxin proteins have been determined by Expasy server (www.expasy.org) which showed that HipA toxin had pI of 6.57 with molecular weight of 48,446 Da whereas its putative antitoxin HipB had pI 10.16 with molecular weight 9480 Da. CcdA antitoxin had pI of 8.89 with molecular weight of 10,546 Da where as its putative antitoxin CcdB had pI 5.09 with molecular weight of 11,543 Da. YoeB toxin had pI of 8.76 with molecular weight of 10,246 Da whereas its putative antitoxin YefM had pI 5.25 with molecular weight of 9512 Da.

Figure 1. Genetic organization of TA homolog in the genome of X. nematophila. (a) hipAB TA homolog (b) ccdBA TA homolog (c) yoeB-yefM homolog

Figure 2. (a). Conserved domain of putative HipA toxin protein. (b). Conserved domain of putative HipB antitoxin protein

Figure 3. (a). Conserved domain of putative CcdA antitoxin protein. (b) Conserved domain of putative CcdB toxin protein

Figure 4. (a). Conserved domain of YefM antitoxin protein (b) Conserved domain of YoeB toxin protein

3.4. Phylogenetic Analysis of HipAB, CcdBA, and YoeB-YefM

Phylogenetic analysis of HipA toxin from X. nemtophila revealed that it forms a distinct branch of toxin from other bacteria as shown in Figure 5. Toxins from other bacteria such as Salmonella enterica subsp. enterica serovar Montevideo str. SARB30 (EHL 47545.1) and Aeromonas hydrophila (YP_0029 95621.1), which are closed to the X. nematophila and located at proximal site in the phylogenetic tree. Whereas toxins from Photobacterium leiognathi subsp mandapamensis svers.1.1. (ZP_08311955.1),Photobacterium angustum S14 (ZP_01233605.1), formed a separate cluster. Rahnella sp. Y9602 (YP_00421 5426.1), Serratia sp. AS12 (YP_0044 98968.1), forms the third cluster located at distal from X. nematophila. Phylogenetic analysis of HipB antitoxin from X. nemtophila revealed that it forms very close related toxin with Enterobacter sp. 638 (YP_001176761.1), and both formed a unique cluster separated from the other two clusters formed by toxins from other bacteria as shown in Figure 6.

Phylogenetic analysis of the CcdB toxin revealed that there were three distinct clusters formed by toxins from different bacteria however, toxin from X. nematophila ATCC 19061is very close Photorhabdus luminescens subsp. laumondii TTO1 as shown in Figure 7. Second cluster of toxin has formed from Pectobacterium carotovorum subsp. Brasiliensis PBR1692 (ZP_03827872.1), Citrobacter sp.30_2 (ZP_04558763.1). Third cluster of toxin has formed Klebsiella oxytoca KCTC 1686 (AEX_029 92.1), Escherichia fergusonii B253 (EGC_06526.1). In between second cluster and X. nematophila ATCC 19061, the antitoxin of Yersinia enterocolitica (YP_002643112.1) has separated out evolutionary. CcdA antitoxins from other bacteria such as Yersinia enterocolitica (YP_002643111.1), was separated in very beginning from the putative antitoxin from X. nematophila ATCC 19061 as shown in Figure 8. Similarly another antitoxin from other bacteria such as Citrobacter sp. 30_2 (ZP_04558764.1) separated apart later.

Figure 5. Phylogenetic analysis of Toxin hip-A protein. Phylogenetic tree constructed based on amino acid sequence of toxin from other bacteria sp. Sequences were aligned by using CLC Genomics workbench (VERSION 4.9) software. Gene accession number for the various protein were as follows: X.nematophila ATCC 19061 (YP_003714330.1), Enterobacter sp. 638 (YP_001176760.1), Salmonella enterica subsp. enterica serovar Montevideo str. SARB30 (EHL47545.1) Aeromonas hydrophila (YP_002995621.1) Tolumonas auensis DSM 9187 (YP_002894289.1) Rahnella sp. Y9602 (YP_004215426.1) Serratia sp.AS12(YP_004498968.1), Photobacterium leiognathisubsp. Mandapamensis svers.1.1. (ZP_08311955.1) Photobacterium angustumS14 (ZP_01233605.1), Acaryochloris sp. CCMEE 5410 (ZP_09251245.1)

Figure 6. Phylogenetic analysis of Antitoxin hip-B protein. Phylogenetic tree constructed based on amino acid sequence of antitoxin from other bacterial sp. Sequences were aligned by using CLC Genomics workbench (version 4.9) software. Gene accession number for the various protein were as follows X. nematophila ATCC (YP_003714331.1) , Aeromonas hydrophila (YP_002995620) Aeromonas salmonicida subsp. salmonicida A449 (YP_001144159.1 ), Enterobacter sp. 638 (YP_001176761.1), Photobacterium sp. SKA34 ( ZP_01160296.1), Photobacterium angustums14 (ZP_01233604), Serratia proteamaculans 568 (YP_001476801.1) Rahnella sp.Y9602 (YP_004215427), Photorhadus asymbiotica subsp. asymbiotica ATCC43949 (YP_003039273), Escherichia coli o127:h6 str.e2348/69 (YP_002329162)

Figure 7. Phylogenetic analysis of Toxin ccd-B protein. Phylogenetic tree constructed based on amino acid sequence of toxin from other bacterial sp. Sequence were aligned by using CLC Genomics workbench (version 4.9) software. Gene accession number for the various protein were as follows X. nematophila ATCC 19061 (YP_003710432.1), Photorhabdus luminescens subsp. laumondii TTO1 (NP_929536.1), Salmonella enterica subsp.enterica serovar Paratyphi B str. SPB7 (YP_001591751.1), Yersinia enterocolitica (YP_002643112.1), Dickeya dadantii 3937 (YP_003881572.1), Pectobacterium carotovorum subsp. brasiliensis PBR1692 (ZP_03827872.1), Klebsiella oxytoca KCTC1686(AEX_02992.1), Citrobacter sp. 30_2 (ZP_04558763.1), Providencia rettgeri DSM 1131 (ZP_06124214.1), Escherichia fergusonii B253 (EGC_06526.1)

Figure 8. Phylogenetic analysis of ccd-A Antitoxin protein. Phylogenetic tree constructed based on amino acid sequence of antitoxin from other bacterial sp. Sequence were aligned by using CLC Genomics workbench (version 4.9) software. Gene accession number for the various protein were as follows X. nematophila ATCC 19061(YP_003710431.1), Photorhabdus luminescens subsp. laumondii TTO1(NP_929535), Salmonella enterica subsp. enterica serovar Derby (AAV53396.1), Citrobacter sp.30_2(ZP_04558764.1), Providencia rettgeri DSM 1131(ZP_06124213.1), Dickeya dadantii 3937(YP_003881571.1) Yersinia enterocolitica (YP_002643111.1), Klebsiella oxytoca KCTC 1686 (AEX02991.1),Serratia proteamaculans 568 (YP_001477786.1), Pantoea vagans C9-1(YP_003932332)

Figure 9. Phylogenetic analysis of Yoe-B Toxin protein. Phylogenetic tree constructed based on amino acid sequence of toxin from other bacterial sp. Sequence were aligned by using CLC Genomics workbench (version 4.9) software. Gene accession number for the various protein were as follows X. nematophila ATCC 19061 (YP_003713875.1), Legionella drancourtii LLAP12(ZP_09620091) Delta proteobacterium MLMS-1(ZP_01288747.1), Francisella philomiragia subsp. philomiragia ATCC 25017 (YP_003097662.1) Aggregatibacter actinomycetemcomitans RhAA1 (EHK89628.1) ,Cyanothece sp. ATCC 51142 (YP_001805675.1) Acinetobacter sp. P8-3-8 (ZP_09142029.1),Crocosphaera watsonii WH 8501 (ZP_00516315.1), Pantoea sp. aB (ZP_07380623.1),Halomonas sp. HAL1 (ZP_08959993.1)

Phylogenetic analysis of the YoeB toxin from X. nematophila ATCC 19061 revealed that toxin of Legionella drancourtii LLAP12 (ZP_096200 91) was the most closed to it as shown in Figure 9. The toxin from another bacterium such as Delta proteobacterium MLMS-1(ZP_01288747.1) form a cluster with toxin of Legionella drancourtii LLAP12 (ZP_09620091).There was another cluster of toxin from bacteria such as Halomonas sp. HAL1 (ZP_08959993.1) and Pantoea sp. A B (ZP_073 80623.1) located at distal from X. nematophila. The toxin from Cyanothece sp. ATCC 51142 (YP_0018 05675.1) and Crocosphaera watsonii WH 8501 (ZP_00516 315.1) was separated from the beginning. Analysis of the YefM antitoxin from X. nematophila ATCC 19061 revealed that it form distinct clusters formed by antitoxins from other bacteria as shown in Figure 10.

Figure 10. Phylogenetic analysis of Yef-M antitoxin protein. Phylogenetic tree constructed based on amino acid sequence of antitoxin from other bacterial sp. Sequence were aligned by using CLC Genomics workbench (version 4.9) software. Gene accession number for the various protein were as follows X. nematophila ATCC 19061 (YP_003713874 ), Delta proteobacterium MLMS-1 (ZP_01288746.1) Photorhabdus luminescens subsp. laumondii TTO1 (NP_929537), Legionella drancourtii LLAP12 (ZP_09620092.1), Marinomonas sp. MWYL( YP_001339224.1), Aggregatibacter actinomycetemcomitans D11S-1(YP_003256300.1), Acinetobacter sp. P8-3-8 (ZP_09142030.1), Geobacter bemidjiensis Bem (YP_002138737.1), Methylomicrobium alcaliphilum (YP_004915611.1), Rhodobacteraceae bacterium KLH11 (ZP_05125470.1)

3.5. Promoter Analysis

148 base pairs upstream region to a putative hipAB module contained -35 and -10 promoter like elements along with RpoD17 sigma-D (sigma70) binding sequences as shown in Figure 11. The binding sequence of RpoD17 present in upstream region of putative -35 box and some binding sequences of RpoD17 were overlap with it. RpoD17 is the heat shock sigma factor and is transcribed from two promoters PC and PHS. Synthesis of RpoD mRNA from PC is constitutive under both steady-state and heat-shock growth conditions, while that of PHS is transiently induced upon heat-shock[22]. The putative promoter consensus sequences in the -35/-10 region of the RpoD transcriptional initiation site are highly similar to those of E. coli sigma70. Many bacterial species exhibit a general stress response that can be induced by numerous very different stress conditions and, phenotypically renders the cells broadly stress resistant.

Figure 11. 148bp nucleotide sequence upstream region of hip-AB operon showing putative -10 and -35 promoter like elements along with RpoD17 sigma D (sigma 70) binding sequences

152 base pairs upstream region to putative ccdBA TA module contained -35 and -10 promoter like elements, along with the three transcription factor binding site like OxyR, ArgR and ArcA. In ccdBA putative promoter the binding sequences of OxyR and ArgR is overlap with each other, similarly the binding sequences of ArgR and ArcA also overlap with each other. OxyR is the oxidative stress regulator which acts as the transcriptional dual regulator for the expression of antioxidant genes in response to oxidative stress in particular, elevated levels of hydrogen peroxide or stress full chemical environment. The OxyR regulon includes genes involved in peroxide metabolism. In addition, expression of OxyR is positively regulated by cAMP activated Crp protein during exponential growth and negatively regulated by RpoS when cells enter stationary phase[23]. The transcription factor of ArgR transcription factor or protein is a homohexameric protein that, when complexed with arginine, represses arginine biosynthetic genes by binding at sites overlapping their promoters. ArgR complexes with L-arginine represses the transcription of several genes involved in biosynthesis and transport of arginine, transport of histidine and its own synthesis[24] [25][26] and activates genes for arginine catabolism[27]. ArgR is also essential for a site-specific recombination reaction that resolves plasmid ColE1 multimers to monomers and is necessary for plasmid stability[28]. The ArcA is a dual transcriptional regulator for anoxic redox control it acts primarily as a negative transcriptional regulator under anaerobic conditions. ArcA represses operons involved in respiratory metabolism, encoding products such as tricarboxylic acid cycle enzymes, enzymes of the glyoxylate shunt, and enzymes of the pathway for fatty acid degradation[29]. It has been suggested that the most significant role of ArcA is under micro aerobic conditions.

143bp upstream region of yoeB-yefM module was analyzed. It contained putative -35 and -10 promoters like elements. In the upstream of -35 box it has binding region for transcription factors RpoD19 [30], and at the upstream of -10 box it has binding site for another transcription factor called as Tyr. Both the binding sequences of the putative promoter like elements involve in transcription at sigma70 dependent promoters. The general RpoS is a sigma subunit of RNA polymerase, is a very close relative of the vegetative sigma70 RpoD. The homologous of sigma70 are known to activate specific regulons associated with heat-shock or morphological develop- mental stages such as bacterial sporulation or flagellar biogenesis. TyrR can acts as dual character as a repressor and as an activator. This involves in transcription at sigma70 dependent promoters. So, on the basis of all above information it is quite possible that yoeB-yef-M operon might be activated during the heat shock and oxidative stress conditions.

Figure 12. 152bp nucleotide sequence upstream region of ccd-BA were analyzed. It contained putative -35 and -10 promoter like elements, upstream of -35 box and downstream of -10 box and has oxyR, argR and arcA binding sequences respectively

Figure 13. 143bp nucleotide sequence upstream region of yoeB-yef M were analyzed. It contained putative -35 and -10 promoter like elements, upstream of -35 box and downstream of -10 box and has rpo-D19 and tyr binding sequences respectively

PCR amplification

Genomic DNA was isolated from the X. nematophila as protocol described in method. 585 bp DNA fragment encoding ccdBA module obtained by PCR amplification using genomic DNA as template as shown in lane 1 Figure 14 and 505 bp DNA fragment encoding yoeB-yefM module as shown Figure 14, lane 2. 1550 bp DNA fragment encoding hipAB module obtained by PCR amplification using genomic DNA as template as shown in Figure 14, lane 3.

Figure 14. PCR amplification of ccdBA, yoeB-yefM B and hipAB operon using genomic DNA as template. Lane M, 100bp ladder; lane 1, 585 bp ccdBA operon; lane 2, 505 bp yoeB-yefM operon; and lane 3, 1550 bp hipAB operon

4. Conclusions

TA modules have been associated with bacterial programmed cell death and programmed cell survival or persistence[31][32] under various unfavorable conditions which could be physical, chemical or nutrient depleted conditions. X. nematophila encounters competition with soil microflora in insect cadaver as well as with antibacterial protein from the insect hemolymph in its life cycle. Therefore under these conditions, the role of TA modules in X. nematophila cannot be ruled out. In this study, sequenced and annotated genome of X. nematophila has been studied bioinformatically for the identification of putative TA modules in its genome. Three TA module homolog’s hipBA, ccdAB and yoeB-yefM has been identified. Genomic organization revealed that hipAB module is located in “operon="XNC1_operon 0810”. Protein-protein blast of hipA showed its similarity with hip-A toxin from Enterobacter sp.638 and hipB showed its similarity with XRE super family respectively. Therefore, we can presume that in X. nematophila hipA and hipB will form a putative hip-AB TA module. Difference in their individual pI’s (HipA toxin: pI 6.57 and putative HipB antitoxin: pI 10.16) will tend them to form complex in physiological conditions. From the predicted promoter analysis due to the presence of RpoD17 we inferred that transcription of hipA and putative hipB will occur either under different environmental conditions such as high temperature or stress.

Similarly, second putative ccdBA TA module is located in “XCN_1operon0014”. This TA module encodes for ccdA antitoxin which showed similarity with ccdA antitoxin protein from Photorhabdus luminesense subs laumondii TT 01, where as its putative CcdB toxin showed similarity with CcdB toxin protein from Photorhabdus luminesense subs. laumondii. In this case also difference in individual pI (Putative CcdB toxin: pI 5.09 and CcdA antitoxin: pI 8.89) will tend them to form complex in physiological conditions. From predicted promoter analysis, presence of oxyR, argR, and arcA indicates that transcription of putative ccdBA module will occur either under SOS conditions and might be tightly regulated. Therefore, we can presume that in X. nematophila gene encoding ccdAB will form a novel TA module.

In the third putative yoeB-yefM TA module is located at operon “XCN_1operon0711”. Protein-protein blast with YefM antitoxin protein showed its similarity to the prevent host death protein phd (Antitoxin) from Delta proteobacterium mlms-1 where as YoeB toxin from X. nematophila showed its identity with hypothetical protein from Legionell adrancourtiillap 12 LDG_6480. Therefore, putative YefM antitoxin will form a pair with YoeB toxin. In this case due the differences in individual pI’s (Putative YoeB toxin: pI 8.76 and putative antitoxin YefM pI 5.27) they will form complex in physiological conditions. Promoter predicted revealed the presence of signature sequences for the binding of rpo-D19 and tyr, which indicate transcription form this promoter under heat shock conditions. In silico identification of putative hipBA, ccdBA and yoeB-yefM toxin-antitoxin modules has been confirmed by PCR amplification using genomic DNA as template.