The rate and role of pseudogenes of the complex.

Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and pseudogenes. Using NCBI's PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and strains and combined transcriptomics and proteomics of H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of and close evaluation of mutational lesions driving pseudogenization suggest that few predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC's evolution and genetic diversity.