RNase HI Is Essential for Survival of Mycobacterium smegmatis

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.The study was supported by POIG.01.01.02-10-107/09 project implemented under Innovative Economy Operational Programme, years 2007–2013 "Studies of the molecular mechanisms at the interface the human organism - the pathogen - environmental factors" and by grant of Polish National Center of Science 2011/01/N/NZ6/04186 “Identification of a novel mechanism of initiation of DNA replication in Mycobacterium smegmatis”

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses

Institutional Review Board Statement: The experimental procedures were approved and conducted according to guidelines of the appropriate Polish Local Ethics Commission for Experiments on Animals No. 9 in Lodz (Agreement 9/ŁB87/2018). Acknowledgments: We thank Jeremy Rock and Sarah Fortune for providing us with the pLJR965 vector and detailed instructions for the generation of Cas9-regulated strains in M. tuberculosis. The authors thank the mass spectrometry service at the Institute of Biochemistry and Biophysics PAS in Warsaw for MS analysis. The MS analysis equipment used for the analysis was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project cosponsored by the European Regional Development Fund and Innovative Economy, the National Cohesion Strategy of Poland.Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.A.B. was supported by grant “OPUS” from the National Science Centre, Poland, UMO2015/19/B/NZ6/02978. P.P. was supported by grant “OPUS” from the National Science Centre, Poland, UMO-2019/33/B/NZ1/02770

The deletion of rnhB in Mycobacterium smegmatis does not affect the level of RNase HII substrates or influence genome stability.

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

Growth rates in the presence of HU.

<p>A) Growth rates based on the optical density of the cultures. B) Growth rates based on the number of colony forming units (white: <i>M. smegmatis</i> mc² 155; gray: ∆<i>rnhB</i> mutants).</p

Susceptibility of the ∆<i>rnhB</i> mutant and <i>M. smegmatis</i> mc² 155 strains to HU.

<p>A) Logarithmic and B) stationary phase cultures were serially diluted and plated onto medium containing different concentrations of HU. We observed that 20 mM HU inhibited the growth of the analyzed strains, but we did not observe differences in susceptibility between the strains.</p

List of the <i>M. smegmatis</i> strains used in this study.

<p>List of the <i>M. smegmatis</i> strains used in this study.</p

Primers used in this study.

<p>Primers used in this study.</p

Southern blot analyses confirming the generation of ∆rnhA/∆4305/∆rnhBattB::rnhA and ∆rnhA/∆4305/∆rnhBattB::4305 strains.

<p>We used the double mutant strains ∆rnhA/∆rnhB and ∆4305/∆rnhB to obtain the triple conditional mutants ∆rnhA/∆4305/∆rnhBattB::rnhA and ∆rnhA/∆4305/∆rnhBattB::4305. The intermediate steps of the gene replacement procedure are denoted SCO.</p

Sequence comparisons of the RNase HII domains in the RnhB proteins of <i>E. coli</i> K12_MG1655, <i>M. smegmatis</i> mc² 155 and <i>M. tuberculosis</i> H37Rv.

<p>Sequence comparisons of the RNase HII domains in the RnhB proteins of <i>E. coli</i> K12_MG1655, <i>M. smegmatis</i> mc² 155 and <i>M. tuberculosis</i> H37Rv.</p