Biology subjects

Aswin Sai Narain Seshasayee

Publications and source records attributed to Aswin Sai Narain Seshasayee.

3 recordsLinked to original sources

bioRxiv2016

SNP analysis implicates the role of cytosine methylation in introducing consequential mutations in Vibrio cholerae genomes.

Epigenetic modifications play a key role in gene regulation and in recognition of self DNA in bacteria. In-spite of their positive role in cell survival, modifications like cytosine methylation incur a mutational cost. Cytosine methylation, specifically 5-methylcytosine, is prone to hydrolytic deamination which leads to C [->] T and G [->] A transitions. Here, we first study the abundance of mutagenic cytosine methylation target motifs and show that bacteria like Vibrio cholerae might use motif avoidance as a strategy to minimize the mutational effect of deamination of methylated cytosine. Second by performing SNP analysis on whole genome sequence data from Vibrio cholerae patient isolates we show a) high abundance of cytosine methylation-dependent mutations in the cytosine methylation target motif RCCGGY, b) 95% of these C [->] T and G [->] A transitions in the coding region lead to non-synonymous substitutions and c) many of these transitions are associated with membrane proteins and are implicated in virulence. Thus, our SNP analysis of V. cholerae genomes implicates the role of cytosine methylation in generating genotypic diversity with adaptive potential.

Genomics↗

bioRxiv2016

Modulation of global transcriptional regulatory networks as a strategy for increasing kanamycin resistance of EF-G mutants

Evolve and resequence experiments have provided us a tool to understand bacterial adaptation to antibiotics by the gain of genomic mutations. In our previous work, we used short term evolution to isolate mutants resistant to the ribosome targeting antibiotic kanamycin. We had reported gain of resistance to kanamycin via multiple different point mutations in the translation elongation factor G (EF-G). Furthermore, we had shown that the resistance of EF-G mutants could be increased by second site mutations in the genes rpoD / cpxA / topA / cyaA. Mutations in three of these genes had been discovered in earlier screens for aminoglycoside resistance. In this work we expand on our understanding of these second site mutations. Using genetic tools we asked how mutations in the cell envelope stress sensor kinase (CpxAF218Y) and adenylate cyclase (CyaAN600Y) could alter their activities to result in resistance. We found that the mutation in cpxA most likely results in an active Cpx stress response. Further evolution of an EF-G mutant in a higher concentration of kanamycin than what was used in our previous experiments identified the cpxA locus as a primary target for a significant increase in resistance. The mutation in cyaA results in a loss of catalytic activity and probably results in resistance via altered CRP function. Despite a reduction in cAMP levels, the CyaAN600Y mutant has a transcriptome indicative of increased CRP activity, pointing to an unknown non-catalytic role for CyaA in gene expression. From the transcriptomes of double and single mutants we describe the epistasis between the mutation in EF-G and these second site mutations. We show that the large scale transcriptomic changes in the topoisomerase I (FusAA608E-TopAS180L) mutant likely result from increased negative supercoiling in the cell. Finally, genes with known roles in aminoglycoside resistance were present among the mis-regulated genes in the mutants.

Genomics↗

bioRxiv2016

Regulation of global transcription in E. coli by Rsd and 6S RNA

In Escherichia coli, the sigma factor {sigma}70 directs RNA polymerase to transcribe growth-related genes, while {sigma}38 directs transcription of stress response genes during stationary phase. Two molecules hypothesized to regulate RNA polymerase are the protein Rsd, which binds to {sigma}70, and the non-coding 6S RNA which binds to the RNA polymerase- {sigma}70 holoenzyme. Despite multiple studies, the functions of Rsd and 6S RNA remain controversial. Here we use RNA-Seq in five phases of growth to elucidate their function on a genome-wide scale. We show for the first time that Rsd and 6S RNA facilitate {sigma}38 activity throughout bacterial growth, while 6S RNA also regulates widely different genes depending upon growth phase. We discover novel interactions between 6S RNA and Rsd and show widespread expression changes in a strain lacking both regulators. Finally, we present a mathematical model of transcription which highlights the crosstalk between Rsd and 6S RNA as a crucial factor in controlling sigma factor competition and global gene expression.

Genomics↗