A common approach to estimate the strength and direction of selection acting on protein codingsequences 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 two-fold degenerate codons were higher in comparison to that in four-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 Escherichia 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.

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Ralstonia solanacearum causes a lethal bacterial wilt disease in many plants by colonizing the vascular tissues of the hosts. Upon inoculation of tomato seedlings through either leaf or root, the wilting symptoms occur first at the apical region and then proceed downward along the shoot. The systemic order of the disease initiation and progression in the host, independent of the site of pathogen inoculation, is yet to be investigated. To understand the disease progression more clearly, we have carried out a systematic study of the pathogen localization by GUS staining of inoculated tomato seedlings, at 24‐hour intervals from 0 days post‐inoculation (dpi) to 5 dpi. In both inoculation methods, pathogen colonization was observed at 1 dpi at the apical meristem as well as the cotyledon leaves, where the disease initiates. As the disease progressed, colonization by the pathogen towards the lower region of the shoot was observed. Disease consistency and pathogenicity magnitude were observed to be higher using the leaf inoculation method than the root inoculation method. Several R. solanacearum transposon‐induced mutants that were reduced in virulence by root inoculation but virulent by leaf inoculation were obtained. Using GUS staining, it was observed that these mutants were unable to localize in the shoot region when inoculated in the root. Our study indicates that the apical meristem and the cotyledon leaves are the first regions to be colonized in inoculated tomato seedlings, which might explain the disease initiation from this region.

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Transition to transversion ratio (ti/tv) is important in the study of molecular evolution, which provides the understanding of mutational bias in an organism. Since, synonymous polymorphism for two-fold degenerate codons depends only on transitions, two-fold degenerate codons and four-fold degenerate codons are different regarding (Ti/Tv) ratios both for synonymous as well as non-synonymous mutations. Therefore, various coding sequences in any organism having difference in degenerate codon composition is likely to affects the ti/tv ratio. In addition to the above, compositional differences among the coding sequences regarding pretermination codon is also likely to influence the ti/tv ratio because purifying selection is stronger on non-synonymous substitutions leading to termination codons. In this study we have improve the method of finding the ratio between transition to transversion, synonymous transition to synonymous transversion and non-synonymous transition to non-synonymous transversion and also developed a computer program to calculate the values. Our analyses in 2516 coding sequences in the E. coli genome suggested a strong correlation between codon degeneracy with synonymous transition to transversion ratio, also we have observed a moderate correlation between pretermination codons and non-synonymous transition to transversion. The percentage change between the conventional and improved method was the most striking part of our observation.

The previous findings suggest that replication and transcription are two major reasons behind the different substitution patterns of mutations in genomic DNA. In the current work, we have compared the adjacent co-transcribed gene pairs regarding synonymous polymorphism in five different operons in Escherichia coli. It is interesting that the co-transcribed genes were different from each other regarding the polymorphism spectra. The transition to transversion ratio between gene pairs were different due to their compositional differences regarding two-fold degenerate codon and four-fold degenerate codons. Further, the polymorphism spectra difference between the gene pairs was more prominent in four-fold and six-fold degenerate codons than in the two-fold degenerate codons. In case of rpoB and rpoC, the major difference was found at UCC, GUA, CCG, GCU, GGC and CGC codons. Similarly, in case of the other four pairs of co-transcribed genes, the difference was more prominent in the higher degenerate codons than the two-fold degenerate codons. It may be that the restriction of two-fold degenerate codons to transition substitutions only regarding synonymous polymorphism is making these codons different from the higher degeneracy codons in this study.