Survey of mycobacterial fluoroquinolone resistance protein conservon (mfp conservon) in Mycobacteriaceae and identification of its promoter activity

Abstract

Mycobacteria develops resistance to fluoroquinolones antibiotics either through acquired mutations in the gyrase genes or intrinsic resistance contributed by cell wall barrier, efflux pumps or pentapeptide repeat proteins (PRPs). MfpA (Mycobacterial fluoroquinolone resistance protein A) is a PRP, that contributes to intrinsic resistance to fluoroquinolones through its co-ordinated function with Gyrase and MfpB (Mycobacterial fluoroquinolone resistance protein B). Together, these proteins maintain the intrinsic resistance to fluoroquinolones by protecting the bacterial cell from topoisomerase poisons. In this study, we surveyed the distribution and arrangement of mfp conservon in Mycobacteriaceae and predicted its promoter elements. Mycobacterium tuberculosis mfp intergenic region was then cloned into pSD5B, a promoterless vector and its promoter activity was measured. BLAST analysis of mfp conservon against RefSeq representative genome database of Mycobacteriaceae produced a total of 202 unique hits. 155 species were found to have full length conservon consisting of all 5 mfp genes, while 15 among them had a second conservon without the mfpA gene. 28 species did not have mfpA gene although they have other four genes in the conservon. 10 species were found to have only mfpA gene, present along with a completely different set of genes around them. Intergenic region of 188 species were taken and analysed for the presence of promoter elements. Promoter prediction showed the presence of a characteristic phoB box TGTCATA in the intergenic region of most species including M. tuberculosis. A 235 bp intergenic region of M. tuberculosis was further cloned in a promoterless vector and it showed the highest promoter activity when the first 158 bases containing phoB box was expressed. In summary, mfpA gene was observed in different forms and the first 158 bases of the intergenic region showed the highest promoter activity.