Cloning , Sequencing and Phylogenetic Analysis of stn gene of Salmonella Typhimurium

Salmonella Typhimurium is an important facultative bacillus pathogen having broad host range and high physiological adoptability. Several genes contribute in virulence and determine pathogenicity of this isolate. Stn is an important virulent gene which code for Salmonella toxin, increases the level of c-AMP in the host, and ultimately results into diarrhoea and vomiting. In present study stn gene was cloned, sequenced and on basis of sequence information of stn gene, phylogenetic relation was deduced between different serovars of Salmonella Typhimurium. Genomic DNA was isolated from field isolate of Salmonella Typhimurium (isolate No-A201) and stn gene was amplified using gene specific primer and cloned in pJET vector by the positive selection system. Amplification of stn gene yielded a product of approximately 750 bp. Subsequently gene was sequenced and a complete ORF of 750 bp was obtained. The sequence was submitted to NCBI Genbank and allotted the Accession No KF032246 was allocated. Sequence was further used for bioinformatics analysis of Stn protein, which exhibited two major domains and one amino acid substitution at 609 residue. On phylogenetic analysis, S.Typhimurium exhibited 99% similarity with Salmonella enterica subsp. enterica serovar Newport. Our findings indicate that stn is an important toxin gene, which is conserved among many serovars of Salmonella.

Gastrointestinal diseases of infectious origin usually arise upon ingestion of contaminated foods or water and can have a wide number of etiological agents, known as enteric pathogens.Among them, the genus Salmonella is of particular clinical relevance in both developed and developing countries, where this pathogen is one of the most common causes of food-borne illness and is a major cause of diarrheal diseases, respectively (Kozak et al.,2013;Vojdani et al.,2008;Ansari et al., 2012 ;Kabir et al.,2012;Kariuki et al,. 2006).Several outbreaks attributed to different Salmonella serovars are reported each year, highlighting the frequency of S. enterica serovar Typhimurium and S. enterica serovar Enteritidis among the most common causal agents (Kozak et al.,2013;Vojdani et al.,2008;Ansari et al., 2012 ;Kabir et al.,2012;Kariuki et al,. 2006) Salmonella enterica serovar Typhimurium is a Gram negative, facultative bacillus and a leading cause of human gastroenteritis, which is often associated with non typhoid symptoms such as diarrhoea and abdominal pain ( McClelland et al.,2001;Everest et al.,1999).In poultry, S. Typhimurium has been isolated from broilers, breeders, and commercial egg-laying flocks (Chima et al., 1998;Jamshidi et al., 2009).The backyard poultry practices are very common in India so there is always a possibility of transferring of Salmonella Typhimurium from poultry to human populations where it can cause serious health problems (Moreno et al., 2000;Swe et al., 2008;Saxena et al., 2006).Therefore, Salmonella control in poultry would contribute a significant advantage in improving public health.Hence, there is an emergent need for devising various strategies to develop vaccine against poultry salmonellosis for reducing the infection caused by this organism.
Salmonella enterotoxin (Stn) is a putative virulence factor and causative agent of diarrhoea (Chopra et al., 1994;.Chopra et al.,1999).It has been shown that the stn gene is specifically distributed only in Salmonella spp.irrespective of their serotypes (Dinjus et al., 1997;Makino et al., 1999;Moore et al., 2007).Biological activities of Stn are important to Salmonella virulence, especially acute gastroenteritis.It has shown an enterotoxic activity in a murine ileal loop model (Chopra et al., 1999).Therefore, Stn is a Salmonella virulence factor and is responsible for the enterotoxicity of Salmonella.It is possible that Stn will play a pivotal role in special functions of Salmonella.To explore more information about Stn protein in the present study stn gene of Salmonella Typhimurium was cloned, sequenced and phylogenetic analysis was done by using bioinformatics approach.

MATERIALS AND METHODS
B a c t e r i a l S t r a i n s .S a l m o n e l l a Typhimurium field isolate (A201) was used in this study which had been characterized by Salmonella specific PCR, biochemical characterization and serotyped at National Salmonella Centre, IVRI Izzatnagar as Salmonella Typhimurium.Escherichia coli DH5á used in cloning experiment was purchased from Bangalore Genei, India and grown in LB broth.Blunt cloning vector pJET 1.2, blunting enzyme, and T4 DNA ligase were procured from Qiagen, USA.The antibiotics (Ampicillin (100 ìg/mL) and Kanamycin (50 ìg/ mL)) used for selection of recombinants were procured from Himedia, India.The cultures were maintained in LB agar slants and their purity was tested by biochemical tests and Salmonella specific PCR.
Cloning of stn gene.Genomic DNA was isolated by CTAB method.Primers were designed for Stn gene of Salmonella Typhimurium using sequence information available on NCBI and using gene tool software.
Stn 1 (Forward) : 5' GGATCC TTG TTA ATC CTG TTG TCT CGC TAT 3' Stn 2 (Reverse) : 5' AAGGTT TTA CTG GCG TTT TTT TGG CAT 3' 50 µl of PCR reaction mixture was set up containing 20 ng of genomic DNA (template), 200 µM dNTPs, 20 picomoles of each primer, 3 unit of Jumpstart TM AccuTaq TM LA (SIGMA) with 5 ul of 10X AccuTaq TM LA PCR buffer.Final volume to 50 ul was made up by using sterilised water.The gene was amplified by PCR using the following program, that is, initial denaturation at 94°C, followed by 30 cycles of denaturation at 94°C, annealing at 51°C, and elongation at 68°C.
PCR product was loaded on 1.5% Agarose gel and the size of the amplicon was measured by comparing with standard molecular weight marker.To remove PCR dimer, PCR product was eluted from agarose gel using Qiagen Mini Elute Gel extraction kit.PCR product was cloned in pJET vector by blunt end cloning.After blunting of PCR product, 1 µl of pJET vector and 1 µl of T4 DNA ligase were added and ligation was carried out at 22ºC for 4 hours.5 ul of ligated product was checked on 1.5 % Agarose gel.Ligated product was transformed into DH5á cells using Calcium chloride method (Sambrook et al., 1989).Recombinants were screened by selecting Ampicillin resistant colonies and analyzing the presence of insert by colony PCR.Plasmids were isolated from selected clones and insert was released by double digestion with Bam H1 and Hind III.The size of insert was measured by comparing with standard molecular weight marker.Selected clones were sent to University of Delhi South Campus for sequencing and obtained sequence was analysed for presence of open reading frame.The sequence was submitted to NCBI.Open reading frame analysis: ORF analysis of stn gene of Salmonella Typhimurium (750 bp) was performed by GENE TOOL software.
Conserved Domain Search: Conserved domain analysis of the protein sequence was performed using CD Search tool of NCBI.
Sequence Similarity and Phylogenetic Analysis: The sequence obtained was subjected to homology search using BLASTn (http://www.ncbi.nlm.nih.gov/).The sequences showing maximum similarity with stn gene was subjected to multiple sequence alignment and a phylogenetic tree was constructed based on the comparative analysis of related sequences using MEGA (Molecular Evolution Genetics Analysis) tool at nucleotide level.Analysis was performed on the default values of the MEGA software and Neighbour-joining statistical method at 1000 bootstrap replication was used for tree construction.

RESULTS AND DISCUSSION
The purity of the culture was checked by biochemical characterization and Salmonellaspecific PCR.The culture was found to be MR+, VP-, Urease-biochemically, which is characteristic of Salmonella Typhimurium.
PCR Amplification and Cloning: The PCR amplification with stn specific primers was conducted with genomic DNA, which resulted in a product of approximate size 750 bp (Figure 1a).The desired product was successfully purified using QIA quick gel extraction kit and cloned in pJET 1.2 blunt cloning vector (Fermentas, USA) and transformed into chemically competent E.coli DH5á cells.Recombinant clones were selected by colony PCR (Figure 1b).The insert was sequenced and complete cds of 750 bp was obtained.The sequence was submitted to NCBI Gene bank and accession no.KF032246 was allocated by NCBI.
GC Content analysis: GC content is found to be variable with different genes but evidence of GC ratio with that of length of the coding region of a gene has shown that the length of the coding sequence is directly proportional to higher G+C content.This has been pointed to the fact that the stop codon has a bias towards A and T nucleotides, and, thus, the shorter the sequence the higher the AT bias and in this case as shown in the graph (Fig

Open reading frame analysis
The sequence when subjected to ORF analysis using GENE TOOL software showed a complete ORF of 750 bp.(Fig 3)

Conserved Domain Search
Nucleotide sequence (750bp) was used to predict the domain region in the sequence.Histidine kinase-like ATPases (25-342bp) and Histidine Kinase A (478-672bp), two domains were found in the stn gene sequence of Salmonella.effective vaccines are not available as in case of Typhoid presently available Vi Polysachharide vaccine has serious limitations such as it cannot be used in pregnant ladies, high cost of production and a debatable immune response in children.(Ochiai et al.,2014) Though to overcome from these problems new adjuvant system has been developed (Tamuly and Saxena 2012) which can produce better immune response and by the use of molecular techniques efforts have been made to differentiate the pathogen at molecular level (Shivachandra et al.,2006;Zheng et al.,2014) to assess the genetic variation among isolates to resolve the problem of vaccination failure.A conserved and immunogenic protein may be a suitable target for various vaccine development against Salmonella (Jha et al., 2015).Stn has been considered as putative virulence factor of Salmonella and causative agent of diarrhoea (Chopra et al., 1999) but in the later stages it was deduced that the mutation in stn gene did not resulted into reduction of virulence of Salmonella (Watson et al., 1998) but it is an interested fact that stn gene is distributed only in Salmonella ssp (Moore et al., 2007) and in our study it has exhibited higher degree of homogeneity among the various serovars of Salmonella.On amino acid sequencing it has shown some similarity with active site of cholera toxin (Dinjus et al., 1997).Therefore, from the findings of the workers and our study we could conclude that though Stn is not an essential protein for virulence of Salmonella but it may be a contributing factor moreover, it seems to be a conserved proteins among the serovars of Salmonella.Therefore like other toxoid vaccine like tetanus (Moreira et al., 2016) it may be a suitable target for the development of toxoid vaccine against salmonellosis.

Fig. 4 .Fig. 5 .
Fig. 4. The sequence was screened for the presence of conserved regions that may exist within a family of gene

Fig. 6 .
Fig. 6.Dendogram for Phylogenetic analysis at nucleotide level of stn gene marked with red square box with other closely related sequences.A is the collapse group of the sequences