Nucleotide Sequence Determination of A Gene Encoding Sortase D from Rossellomorea aquimaris MKSC 6.2
Dewi Chairunnissa Wiyadi, Fernita Puspasari, Ihsanawati

Institut Teknologi Bandung

Abstract

Surface proteins located on the cell walls of Gram-positive bacteria play an important role in the physiology and pathogenesis of these microbes. Sortase is a type of cysteine protease that helps the assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria. There are six classes of sortase, which has a different biological function, one of them is sortase D, involved in the spore formation and pili assembly. Sortase D in Bacillus anthracis is important for protein binding to the cell wall to facilitate sporulation, while sortase D in Bacillus cereus and other Gram-positive bacteria plays a role in pili assembly through the transpeptidase reaction mechanism. Current research on sortase D is still limited. Therefore, the aim of this study is to determine the nucleotide sequence of the gene encoding sortase D from the marine bacterium Rossellomorea aquimaris MKSC 6.2 (RaqsrtD). The study began with the design of degenerate primers based on information of the nucleotide sequence coding for class D sortases from various R. aquimaris strains in Genbank. Degenerate Polymerase Chain Reaction (degPCR) was performed to amplify the RaqsrtD gene fragment from the R. aquimaris MKSC 6.2 chromosome and it was inserted into pGEM-T Easy cloning vector. Escherichia coli TOP10F competent cells were transformed with this recombinant plasmid. The final step was determining the nucleotide sequence of the recombinant plasmid using the dideoxy Sanger method. Degenerate PCR produced a 260 bp gene fragment. The results of the transformation of E. coli TOP10F cells with pGEM-T-Easy-RaqsrtD260 produced 4 colonies that gave positive result in colony PCR analysis. Analysis of the nucleotide sequence of these 4 colonies using blastx on the NCBI website, showed that the size of RaqsrtD gene fragment is 263 bp and has similar nucleotide sequences (98.84%) to class D sortase from Bacillus sp. Marseille-Q1617 and Rossellomorea aquimaris CH159a_5T and CH87b_3T strains with percent identity 88.37%. These results underlied the preparation of primers to amplify the whole RaqsrtD gene (approx. 560 bp). Determination of the inserted gene sequence on the recombinant plasmid pGEM-T-Easy-RaqsrtD provided information that the size of the RaqsrtD gene that encode mature protein is 540 bp. The nucleotide sequence of RaqsrtD has percent identity of 99.47% with the nucleotide sequence coding sortase D from Bacillus sp. Marseille-Q1617. The study about determination of the nucleotide sequence of D sortase allows further characterization of this enzyme to develop drug target in preventing Gram-positive bacteria infection.

Keywords: Cysteine protease, Sortase D, Rossellomorea aquimaris MKSC 6.2, degenerate PCR, nucleotide analysis

Topic: Biokimia Medis