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RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.

RNase HII removes RNA from RNA/DNA hybrids, such as single ribonucleotides and RNA primers generated during DNA synthesis. Both, RNase HII substrates and RNase HII deficiency have been associated with genome instability in several organisms, and genome instability is a major force leading to the acquisition of drug resistance in bacteria. Understanding the mechanisms that underlie this phenomenon is one of the challenges in identifying efficient methods to combat bacterial pathogens. The aim of the present study was set to investigate the role of rnhB, presumably encoding RNase HII, in maintaining genome stability in the M. tuberculosis model organism Mycobacterium smegmatis. We performed gene replacement through homologous recombination to obtain mutant strains of Mycobacterium smegmatis lacking the rnhB gene. The mutants did not present an altered phenotype, according to the growth rate in liquid culture or susceptibility to hydroxyurea, and did not show an increase in the spontaneous mutation rate, determined using the Luria-Delbrück fluctuation test for streptomycin resistance in bacteria. The mutants also did not present an increase in the level of RNase HII substrates, measured as the level of alkaline degradation of chromosomal DNA or determined through immunodetection. We conclude that proteins other than RnhB proteins efficiently remove RNase HII substrates in M. smegmatis. These results highlight differences in the basic biology between Mycobacteria and eukaryotes and between different species of bacteria.