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

Abstract

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.

Figure 1. Sequence comparisons of the RNase HII domains in the RnhB proteins of E. coli K12_MG1655, M. smegmatis mc² 155 and M. tuberculosis H37Rv.

Figure 2. Southern blot analyses confirming the deletions in the single and double RNase H mutants of M. smegmatis.

We used the gene replacement through homologous recombination technique to obtain single and double mutants deficient in rnhB and/or either rnhA or MSMEG4305. The ∆rnhA/∆rnhB mutant was obtained through the introduction of the rnhB gene replacement plasmid into the ∆rnhA-deficient strain. The mutant ∆4305/∆rnhB was obtained through the introduction of the MSMEG4305 gene replacement plasmid into the ∆rnhB strain. The intermediate steps of the gene replacement procedure are denoted SCO.

Figure 3. Southern blot analyses confirming the generation of ∆rnhA/∆4305/∆rnhBattB::rnhA and ∆rnhA/∆4305/∆rnhBattB::4305 strains.

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.

Figure 4. Growth rate and morphology of the ∆rnhB mutant and M. smegmatis mc² 155 strains.

A) Growth rates based on the optical densities of the cultures. B) The cell lengths.

Figure 5. Susceptibility of the ∆rnhB mutant and M. smegmatis mc² 155 strains to HU.

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.

Figure 6. Growth rates in the presence of HU.

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

Figure 7. Alkaline hydrolysis of the genomic DNA.

DNA was isolated from the ∆rnhB mutants and M. smegmatis mc² 155. The strains were grown in 7H9 medium supplemented with OADC. The DNA samples were treated with either NaOH or NaCl as a control. The fragmentation of the samples was visualized on alkaline agarose gels. Lanes 1a) GeneRuler 1-kb DNA Ladder, 2a) M. smegmatis mc² 155 control DNA, 3a) ∆rnhB mutant control DNA, 1b) GeneRuler 1-kb DNA Ladder, 2b) M. smegmatis mc² 155 DNA hydrolyzed with NaOH, and 3b) ∆rnhB mutant DNA hydrolyzed with NaOH. The level of ribonucleotide incorporated in the DNA of both strains was similar, as we did not observe differences in fragmentation of genomic DNA.

Figure 8. RNA/DNA hybrid level.

Comparison of the level of RNA/DNA hybrids in M. smegmatis mc² 155, ∆rnhA/∆4305/∆rnhBattB::rnhA and ∆rnhA/∆4305/∆rnhBattB::4305 strains grown on 7H9 medium in the presence of succinate.