Transcribed intergenic regions exhibit a lower frequency of nucleotide polymorphism than the untranscribed intergenic regions in the genomes of Escherichia coli and Salmonella enterica.

The temporary exposure of single-stranded regions in the genome during the process of replication and transcription makes the region vulnerable to cytosine deamination resulting in a higher rate of C→T transition. Intraoperon intergenic regions undergo transcription along with adjacent co-transcribed genes in an operon, whereas interoperon intergenic regions are usually devoid of transcription. Hence these two types of intergenic regions (IGRs) can be compared to find out the contribution of replication-associated mutations (RAM) and transcription-associated mutations (TrAM) towards bringing variation in genomes. In our work, we performed a polymorphism spectra comparison between intraoperon IGRs and interoperon IGRs in genomes of two well-known closely related bacteria such as Escherichia coli and Salmonella enterica. In general, the size of intraoperon IGRs was smaller than that of interoperon IGRs in E. coli and S. enterica. Interestingly, the polymorphism frequency at intraoperon IGRs was 2.5-fold lesser than that in the interoperon IGRs in E. coli genome. Similarly, the polymorphism frequency at intraoperon IGRs was 2.8-fold lesser than that in the inter-operon IGRs in S. enterica genome. Therefore, the intraoperon IGRs were often observed to be more conserved. In the case of interoperon IGRs, the T→C transition frequency was a minimum of two times more frequent than T→A transversion frequency whereas in the case of intraoperon IGRs, T→C transition frequency was similar to that of T→A transversion frequency. The polymorphism was purine-biased and keto-biased more in intraoperon IGRs than the inter-operon IGRs. In E. coli, the transition/transversion ratio was observed as 1.639 and 1.338 in inter-operon and in intraoperon IGRs, respectively. In S. enterica, the transition/transversion ratio was observed as 2.134 and 2.780 in inter-operon and in intraoperon IGRs, respectively. The observation in this study indicates that transcribable IGRs might not always have higher polymorphism frequency than nontranscribable IGRs. The lower polymorphism frequency at intraoperon IGRs might be attributed to different events such as the transcription-coupled DNA repair, sequences facilitating translation initiation and avoidance of Rho-dependent transcription termination.

Incorporation of transition to transversion ratio and nonsense mutations, improves the estimation of the number of synonymous and non-synonymous sites in codons.

A common approach to estimate the strength and direction of selection acting on protein coding sequences is to calculate the dN/dS ratio. The method to calculate dN/dS has been widely used by many researchers and many critical reviews have been made on its application after the proposition by Nei and Gojobori in 1986. However, the method is still evolving considering the non-uniform substitution rates and pretermination codons. In our study of SNPs in 586 genes across 156 Escherichia coli strains, synonymous polymorphism in 2-fold degenerate codons were higher in comparison to that in 4-fold degenerate codons, which could be attributed to the difference between transition (Ti) and transversion (Tv) substitution rates where the average rate of a transition is four times more than that of a transversion in general. We considered both the Ti/Tv ratio, and nonsense mutation in pretermination codons, to improve estimates of synonymous (S) and non-synonymous (NS) sites. The accuracy of estimating dN/dS has been improved by considering the Ti/Tv ratio and nonsense substitutions in pretermination codons. We showed that applying the modified approach based on Ti/Tv ratio and pretermination codons results in higher values of dN/dS in 29 common genes of equal reading-frames between E. coli and Salmonella enterica. This study emphasizes the robustness of amino acid composition with varying codon degeneracy, as well as the pretermination codons when calculating dN/dS values.

Stem Region of tRNA Genes Favors Transition Substitution Towards Keto Bases in Bacteria.

Transversion and transition mutations have variable effects on the stability of RNA secondary structure considering that the former destabilizes the double helix geometry to a greater extent by introducing purine:purine (R:R) or pyrimidine:pyrimidine (Y:Y) base pairs. Therefore, transversion frequency is likely to be lower than that of transition in the secondary structure regions of RNA genes. Here, we performed an analysis of transition and transversion frequencies in tRNA genes defined well with secondary structure and compared with the intergenic regions in five bacterial species namely Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Staphylococcus aureus and Streptococcus pneumoniae using a large genome sequence data set. In general, the transversion frequency was observed to be lower than that of transition in both tRNA genes and intergenic regions. The transition to transversion ratio was observed to be greater in tRNA genes than that in the intergenic regions in all the five bacteria that we studied. Interestingly, the intraspecies base substitution analysis in tRNA genes revealed that non-compensatory substitutions were more frequent than compensatory substitutions in the stem region. Further, transition to transversion ratio in the loop region was observed to be significantly lesser than that among the non-compensatory substitutions in the stem region. This indicated that the transversion is more deleterious than transition in the stem regions. In addition, substitutions from amino bases (A/C) to keto bases (G/T) were also observed to be more than the reverse substitutions in the stem region. Substitution from amino bases to keto bases are likely to facilitate the stable G:U pairing unlike the reverse substitution that facilitates the unstable A:C pairing in the stem region of tRNA. This work provides additional support that the secondary structure of tRNA molecule is what drives the different substitutions in its gene sequence.