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

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

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

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

Southern blot analyses confirming the deletions in the single and double RNase H mutants of <i>M. smegmatis</i>.

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

Growth rate and morphology of the ∆<i>rnhB</i> mutant and <i>M. smegmatis</i> mc² 155 strains.

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

RNA/DNA hybrid level.

<p>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.</p

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

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