The rpsL gene and streptomycin resistance in Streptococcus gordonii and Streptococcus pyogenes Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/tx31qm91x

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  • Streptomycin resistance in both gram-positive and gram-negative bacteria is usually caused by a single mutation in the rpsL gene. The rpsL gene encodes the S12 protein of the ribosomal complex. The rpsL genes of various bacteria have consensus regions in their sequences. Primers were designed from these consensus pockets and a fragment of the rpsL gene was sequenced from S. gordonii using PCR based methodologies. Using the Multiplex Restriction Sequence PCR(mRS PCR), which used the known primer at one end and a restriction site primer on the other, a gene walk was conducted. In streptomycin resistant strains of S. gordonii, namely GP204, SP204 and SP635, the AAA coding for Lys56 was mutated to ACA, coding for Thr56. The lysine to threonine transition, causing resistance to streptomycin was identical to that expected from the literature. The streptomycin resistance gene of S. pyogenes was mapped using similar techniques. Streptomycin resistant strains S43 ATCC, 543/192/4 and S43/192/30R were studied. In streptomycin resistant S43 ATCC and S43/192/30R strains, the lysine 56 changed to isoleucine and threonine respectively. Surprisingly, the 192/4 had two mutations, in each of the two hotspots in the rpsL gene where mutations due to streptomycin resistance occur. It had the amino acid 56, lysine, mutated to arginine and lysine 101 changed to asparagine. To check if this mutation was stable in the host animal, S43/192/4 P8 (S43/192/4 passaged eight times in mice) was sequenced and the sequence was identical to the streptomycin resistant 192/4. Hence, the lys101 mutation was stable and unlike the ancillary mutations in E.coli and S. typhimurium, which are compensated by new mutations. The pathogenesis of S. pyogenes depends in part on the ability of the pathogen to adhere to the epithelial cells of the throat and the quantity of M protein. Pathogenesis studies done on mice revealed the avirulence of S43/192/4smR strain. To elucidate the reason for this avirulence, the adherence properties and the production of M protein of the two strains S43/192/4smR and S43/192/30R were tested. Qualitative immunoblot analysis of the M protein of 192/4 and 30R revealed no significant difference. Competition ELISA was conducted to quantitate the M protein, and this also did not show any significant difference in the M protein levels. The adherence of 30R and 192/4 was measured on human pharyngeal epithelial cell line. The adherence properties of S43/192/4 SmR, was no different from other strains in this experiment. Electron microscopy, using immunogold to highlight the M protein on the cell surface showed no differences.
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