Investigation of the Molecular Basis for Transcriptional Regulation of Tn916 and Macrolide Resistance in Bacillus subtilis

Antibiotic resistance (AR) is one of the most serious threats to modern healthcare today. To understand how resistance spreads, we need to investigate the genetic basis of transferable AR. Conjugative transposons (CTns) have acquired the vast majority of resistance genes we currently know about which makes them one of the major vectors involved in their spread. This study aims to investigate how Tn916 and Tn916-like elements maintain their stability following insertion into a bacterial genome. We identified putative rho-independent terminators upstream of the conjugation genes of Tn2010, Tn5397, Tn6000, Tn6002, Tn6003, Tn6087 and Tn916 and hypothesised that their role is to prevent transcriptional readthrough into the conjugation genes upon integration into a new insertion site. To verify this experimentally, the terminator was cloned in between the tet(M) promoter and a gusA reporter in pHCMC05. We demonstrated the level of β-glucuronidase enzyme activity decreased, confirming termination activity. We have for the first time, identified and verified a group of conserved terminators in the conjugation region of the Tn916-like family of CTns. Further data supports our hypothesis that the terminator efficiency is modulated upon excision and circularisation of Tn916, which is the exact time when Tn916 would require expression of its conjugation genes. A fundamental understanding of the current antibiotic resistance mechanisms employed by bacteria is also essential to minimise the emergence of resistance and to devise effective resistance-control strategies. Another aim of this study is to investigate the molecular mechanism underlying macrolide resistance in Bacillus subtilis. Macrolide-resistant B. subtilis were generated as part of the project and analysis revealed a new genetic mutation to be responsible for the macrolide resistance phenotype. Comparative genome analysis revealed 21 bp and 54 bp duplication in the rplV of these mutants in comparison to the wild type strain. The rplV encodes the large ribosomal subunit protein, L22. Alteration in L22 has led to a predicted alteration in the C-terminal loop of the protein, predicted to change the shape of the exit tunnel within the ribosome. Ectopic expression of the rplV mutants containing the 21 bp and 54 bp duplication in B. subtilis BS34A confers resistance to macrolides. This is the first observation of macrolide resistance due to 54 bp duplication in the B. subtilis rplV gene