Mycobacterium tuberculosis is one of the most deadly human pathogens. The major mechanism for the adaptations of M. tuberculosis is nucleotide substitution. Previous studies have relied on the nonsynonymous-to-synonymous substitution rate (d(N)/d(S)) ratio as a measurement of selective constraint based on the assumed selective neutrality of synonymous substitutions. However, this assumption has been shown to be untrue in many cases. In this study, we used the substitution rate in intergenic regions (d(i)) of the M. tuberculosis genome as the neutral reference, and conducted a genome-wide profiling for d(i), d(S), and the rate of insertions/deletions (indel rate) as compared with the genome of M. canettii using a 50 Kb sliding window. We demonstrate significant variations in all of the three evolutionary measurements across the M. tuberculosis genome, even for regions in close vicinity. Furthermore, we identified a total of 233 genes with their d(S) deviating significantly from d(i) within the same window. Interestingly, d(S) also varies significantly in some of the windows, indicating drastic changes in mutation rate and/or selection pressure within relatively short distances in the M. tuberculosis genome. Importantly, our results indicate that selection on synonymous substitutions is common in the M. tuberculosis genome. Therefore, the d(N)/d(S) ratio test must be applied carefully for measuring selection pressure on M. tuberculosis genes.
