Selective Pressure by Rifampicin Modulates Mutation Rates and Evolutionary Trajectories of Mycobacterial Genomes.

Resistance to the frontline antibiotic rifampicin constitutes a challenge to the treatment and control of tuberculosis. Here, we analyzed the mutational landscape of Mycobacterium smegmatis during long-term evolution with increasing concentrations of rifampicin, using a mutation accumulation assay combined with whole-genome sequencing. Antibiotic treatment enhanced the acquisition of mutations, doubling the genome-wide mutation rate of the wild-type cells. While antibiotic exposure led to extinction of almost all wild-type lines, the hypermutable phenotype of the ΔnucS mutant strain (noncanonical mismatch repair deficient) provided an efficient response to the antibiotic, leading to high rates of survival. This adaptative advantage resulted in the emergence of higher levels of rifampicin resistance, an accelerated acquisition of drug resistance mutations in rpoB (ß RNA polymerase), and a wider diversity of evolutionary pathways that led to drug resistance. Finally, this approach revealed a subset of adaptive genes under positive selection with rifampicin that could be associated with the development of antibiotic resistance. IMPORTANCE Rifampicin is the most important first-line antibiotic against mycobacterial infections, including tuberculosis, one of the top causes of death worldwide. Acquisition of rifampicin resistance constitutes a major global public health problem that makes the control of the disease challenging. Here, we performed an experimental evolution assay under antibiotic selection to analyze the response and adaptation of mycobacteria, leading to the acquisition of rifampicin resistance. This approach explored the total number of mutations that arose in the mycobacterial genomes under long-term rifampicin exposure, using whole-genome sequencing. Our results revealed the effect of rifampicin at a genomic level, identifying different mechanisms and multiple pathways leading to rifampicin resistance in mycobacteria. Moreover, this study detected that an increase in the rate of mutations led to enhanced levels of drug resistance and survival. In summary, all of these results could be useful to understand and prevent the emergence of drug-resistant isolates in mycobacterial infections.

Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies.

The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution.

A non-canonical mismatch repair pathway in prokaryotes.

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS-MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS-MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.

Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome.

Ser/Thr/Tyr kinases, which together comprise a major class of regulatory proteins in eukaryotes, were not believed to play an important role in prokaryotes until recently. However, our analysis of 626 prokaryotic genomes reveals that eukaryotic-like protein kinases (ELKs) are found in nearly two-thirds of the sequenced strains. We have identified 2697 ELKs, most of which are encoded by multicellular strains of the phyla Proteobacteria (Myxococcales), Actinobacteria, Cyanobacteria, and Chloroflexi, and 2 Acidobacteria and 1 Planctomycetes. Astonishingly, 7 myxobacterial strains together encode 892 ELKs, with 4 of the strains exhibiting a genomic ELK density similar to that observed in eukaryotes. Most myxobacterial ELKs show a modular organization in which the kinase domain is located at the N terminus. The C-terminal portion of the ELKs is highly diverse and often contains sequences with similarity to characterized domains, most of them involved in signaling mechanisms or in protein-protein interactions. However, many of these architectures are unique to the myxobacteria, an observation that suggests that this group exploits sophisticated and novel signal transduction systems. Phylogenetic reconstruction using the kinase domains revealed many orthologous sequence pairs and a huge number of gene duplications that probably occurred after speciation. Furthermore, studies of the microsynteny in the ELK-encoding regions reveal only low levels of synteny among Myxococcus xanthus, Plesiocystis pacifica, and Sorangium cellulosum. However, extensive similarities between M. xanthus, Stigmatella aurantiaca, and 3 Anaeromyxobacter strains were observed, indicating that they share regulatory pathways involving various ELKs.