Southern blot confirming deletion of native <i>dnaA</i> gene in ∆<i>rnhA</i>/∆<i>dnaAattB</i>::<i>dnaA</i> mutant strain of <i>M</i>. <i>smegmatis</i>.

<p>We used gene replacement through homologous recombination to obtain mutants deficient in <i>rnhA</i>. Further, we used the same procedure to generate ∆<i>rnhA</i>/<i>dnaA</i>SCO strain where SCO signifies an intermediate step of gene replacement procedure. Next, through complementation procedure, we introduced an additional version of <i>dnaA</i> gene at the <i>attB</i> site of mycobacterial genome. Finally, we removed the native version of <i>dnaA</i>, thereby generating a mutant ∆<i>rnhA</i>/<i>dnaA</i>SCO<i>attB</i>::<i>dnaA</i>. For more information regarding plasmid construction and gene replacement procedure please refer to the text. Schematic representation of analyzed genomic region, including enzymes used for digestion, size of restriction fragments following digestion and the site of hybridization of hybridization probe, is presented in the upper part of the figure. Photographic film presenting results of Southern blot analysis is presented in the lower part of the figure. Bands corresponding to wild type genotype (wt) and mutant genotype (mut) are marked on the right side of the photograph.</p

Western blots presenting quantitative analysis of the RnhA protein in protein extracts of <i>M</i>. <i>smegmatis</i>.

<p>The upper part of the figure presents detection of either LigA or RnhA in protein extracts isolated from mutants used in this study. The lower part of the figure presents quantitative analysis of the amount of RnhA in <i>M</i>. <i>smegmatis</i> mc² 155. Briefly, we detected known concentrations of either recombinant LigA or recombinant RnhA. Further, we compared the intensities of the obtained bands with those obtained from various concentrations of total protein extracts of <i>M</i>. <i>smegmatis</i> mc² 155. We were able to calculate, that RnhA is approximately 133 times less abundant that LigA.</p

SDS-PAGE analysis of the affinity chromatography-purified <i>M</i>. <i>tuberculosis</i> proteins interacting with human IL-8.

<p>(A) Mycobacterial whole-cell extract applied onto the agarose resin (lane 1). (B) Affinity chromatography-purified <i>Mtb</i> proteins binding human IL-8: lane 1-mycobacterial whole-cell lysate, lane 2-flowthrough fraction collected after the washing step, lane 3-eluate fraction 1, lane 4-eluate fraction 2, lane 5-eluate fraction 3. M-protein molecular weight standard.</p

Summary of presumable RNases H of <i>E</i>. <i>coli</i> K12_MG1655, <i>M</i>. <i>tuberculosis</i> H37Rv and <i>M</i>. <i>smegmatis</i> mc² 155.

<p>Summary of presumable RNases H of <i>E</i>. <i>coli</i> K12_MG1655, <i>M</i>. <i>tuberculosis</i> H37Rv and <i>M</i>. <i>smegmatis</i> mc² 155.</p

Primer sequences used for PCR amplification of gene sequences.

<p>Primer sequences used for PCR amplification of gene sequences.</p

Morphology of the cells of RNase H type I deficient mutants.

<p>We harvested cells grown in liquid cultures for 24 hours and analyzed them on Nikon Eclipse TE2000 microscope. The cultures were performed on 7H9 medium with the addition of OADC and Tween80. For the comparison between ∆4305 and <i>M</i>. <i>smegmatis</i> mc² 155 media were additionally supplemented with vitamin B12. We observed no differences in cell lengths between mutant strains and the wild type. (Box- SE, whiskers- 0.95 CI).</p

Comparison of protein sequences of RNases H type I.

<p>Sequence alignment between RNases H type I of <i>E</i>. <i>coli</i> K12_MG1655 (RnhA), <i>M</i>. <i>smegmatis</i> mc² 155 (MSMEG4305 and RnhA) and <i>M</i>. <i>tuberculosis</i> H37Rv (Rv2228c) was performed using MultiAlin and visualized with ESPript 3.0. Highly similar or identical residues between protein sequences are written in bold. Identical residues across all analyzed sequences are shown in white on a black background. Similarities between protein sequences are marked by framing. The span of RNase H domains in each protein sequence, as defined by SMART, is highlighted in yellow.</p

Constitutive stable DNA replication of <i>E</i>. <i>coli</i> ∆<i>rnhA</i> mutants.

<p>An alternative mode of initiation of DNA replication was discovered in <i>E</i>. <i>coli</i> mutants lacking RNase H type I encoded by <i>rnhA</i>. (A,B) The initial strand opening involves RecA dependent hybridization of the RNA transcript to dsDNA, (C) The resulting R-loop is not resolved by RNase HI and therefore RNA persists on DNA strand and serves as a primer for elongation by PolI, (D,E) When the loop opens sufficiently, primosome is loaded on the leading strand. As replication continues, PolI removes persisting RNA transcript and primosome is loaded on the lagging strand.</p

Southern blots confirming deletions in single RNase H type I mutants of <i>M</i>. <i>smegmatis</i>.

<p>We used gene replacement through homologous recombination to obtain mutants deficient in either <i>rnhA</i> or MSMEG4305. Briefly, recombinant plasmids containing genomic regions of either <i>rnhA</i> or MSMEG4305 with large deletions within each gene were introduced into <i>M</i>. <i>smegmatis</i> mc² 155 cells. Following multistep selection we were able to identify clones where the native version of each gene has been replaced with manipulated sequence. Intermediate steps of gene replacement procedure are denoted SCO. For more information regarding plasmid construction and gene replacement procedure please refer to the text. Schematic representation of analyzed genomic regions, including enzymes used for digestion, size of restriction fragments following digestion and the site of hybridization of hybridization probe, is presented in the upper part of the figure. Photographic films presenting results of Southern blot analysis are presented in lower part of the figure. Bands corresponding to wild type genotype (wt) and mutant genotype (mut) are marked on the right side of each photograph.</p

Primers used in this study.

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