Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.7/September-2014/17.pdf Veterinary World, EISSN: 2231-0916 712 RESEARCH ARTI CLE Open Access Cloning and sequencing of protein L-isoaspartyl O -methyl transferase of Salmonella Typhimurium isolated from poultry S. K. Dixit 1 , D. P. Hota 2 , M. Kumawat 2 , T. K. Goswami 1 and M. Mahawar 2 1. Immunology Section, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India; 2. Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India. Corresponding author: S. K. Dixit, e-mail: sunildixit1987@gmail.com, DPH: durgaprasad.hota04@gmail.com, MK: 0711mworld@gmail.com, TKG: goswami.tapas@gmail.com, MM: manishbiochemistry@gmail.com Received: 16-05-2014, Revised: 28-07-2014, Accepted: 04-08-2014 , Published online: 25-09-2014 doi: 10.14202/vetworld.2014.712-716. How to cite this article: Dixit SK, Hota DP, Kumawat M, Goswami TK, Mahawar M (2014) Cloning and sequencing of protein L-isoaspartyl O-methyl transferase of Salmonella Typhimurium isolated from poultry, Veterinary World 7 (9): 712-716. Abstract Aim: To clone the Salmonella Typhimurium protein L-isoaspartyl O-methyl transferase (PIMT) enzyme and to analyze the sequence with PIMT gene of other pathogenic serovars of Salmonella. Materials and Methods: Salmonella Typhimurium strain E-2375 was procured from the National Salmonella Center, IVRI. The genomic DNA was isolated from Salmonella Typhimurium. Polymerase chain reaction (PCR) was carried out to amplify PIMT gene using the designed primers. The PCR product was cloned into pET28c plasmid vector and transformed into Escherichia coli DH5α cells. The plasmid was isolated from E. coli and was sequenced. The sequence was analyzed and submitted in Genbank. Results: The PCR product revealed a distinct amplicon of 627 bp. The clone was confirmed by PCR. Sequencing data revealed 100% homology between PIMT sequences from Salmonella Typhimurium strain E-2375 (used in the current study) and PIMT sequences of standard reported strain (Salmonella Typhimurium str. LT2) in NCBI data base. This submitted sequence in Genbank having accession no. KJ575536. Conclusions: PIMT gene of Salmonella is highly conserved in most of the pathogenic Salmonella serovars. The PIMT clone can be used to isolate PIMT protein. This PIMT protein will be helpful to identify isoaspartate containing proteins thus can help in study Salmonella virulence. Keywords: cloning, sequencing, Salmonella Typhimurium protein L-isoaspartyl O-methyl transferase, virulence. Introduction Salmonella enterica serovar Typhimurium (shortly Salmonella Typhimurium) is a Gram-negative, flagellated and facultative intracellular anaerobe grouped under the family enterobactericeae is widely known for its highly zoonotic importance [1]. This pathogenic bacterium infects wide range of animal host causing severe morbidity and mortality in ani- mals and human [2,3]. Poultry birds mainly act as an asymptomatic reservoir host and its contaminated meat and eggs are the source of infection causing severe gastroenteritis in human and typhoid like disease in rodents [4]. Growing resistance to antibiotics [5], endemicity of infection and their career state encour- ages understanding the several aspects of its physiol- ogy and metabolism for novel vaccine development and its effective control. There are several virulence factor associated with the severity of infection [6]. The virulence associated enzyme mediated protein repairing modification mechanism associated with Salmonella for the confrontation of a hostile environ- ment inside the host immune system. The phagocytic cells damage various macromolecules such as DNA, RNA and proteins by producing a battery of antimi- crobials such as reactive oxygen species and reactive nitrogen species [7]. The proteins are the major target for this oxidative damage due to their abundance and oxidation leads to covalent modification of the amino acid residues and conformational changes in protein structure [8]. This protein modification brought about by various protein repairing enzymes may be present in Salmonella Typhimurium rescue from the oxidative damage and assist for intraphagosomal survival and its replication. Out of the several protein repairing enzymes reported, protein L-isoaspartyl O-methyl transferase (PIMT) have been found to be effective for all the domains of the life, and this enzymes extensively studied in Escherichia coli [9] and Pyrococcus [10] and Vibrio cholera [11]. The way PIMT works are by repairing the damaged proteins like L-isoaspartyl resi- dues to normal L-aspartyl in proteins by methyl trans- ferase activities [12]. The housekeeping role of this enzyme catalyzes the methyl esterification by transfer of methyl group from the S-adenosylmethionine to the α-carboxyl group of L-isoaspartyl and β-carboxyl group of D-aspartyl residues via succinimide inter- mediate formation, which is hydrolyzed to a mixture of L-aspartyl and L-isoaspartyl residues in the ratio of 1:3 and subsequently restore their biological func- tions [13]. Copyright: The authors. This article is an open access article licensed under the terms of the Creative Commons Attributin License (http:// creative commons.org/licenses/by/2.0) which permits unrestricted use, distribution and reproduction in any medium, provided the work is properly cited.