Rhomboid homologs in mycobacteria: insights from phylogeny and genomic analysis

<p>Abstract</p> <p>Background</p> <p>Rhomboids are ubiquitous proteins with diverse functions in all life kingdoms, and are emerging as important factors in the biology of some pathogenic apicomplexa and <it>Providencia stuartii</it>. Although prokaryotic genomes contain one rhomboid, actinobacteria can have two or more copies whose sequences have not been analyzed for the presence putative rhomboid catalytic signatures. We report detailed phylogenetic and genomic analyses devoted to prokaryotic rhomboids of an important genus, <it>Mycobacterium</it>.</p> <p>Results</p> <p>Many mycobacterial genomes contained two phylogenetically distinct active rhomboids orthologous to Rv0110 (rhomboid protease 1) and Rv1337 (rhomboid protease 2) of <it>Mycobacterium tuberculosis </it>H37Rv, which were acquired independently. There was a genome-wide conservation and organization of the orthologs of Rv1337 arranged in proximity with glutamate racemase (<it>mur1</it>), while the orthologs of Rv0110 appeared evolutionary unstable and were lost in <it>Mycobacterium leprae </it>and the <it>Mycobacterium avium </it>complex. The orthologs of Rv0110 clustered with eukaryotic rhomboids and contained eukaryotic motifs, suggesting a possible common lineage. A novel nonsense mutation at the Trp73 codon split the rhomboid of <it>Mycobacterium avium </it>subsp. <it>Paratuberculosis </it>into two hypothetical proteins (MAP2425c and MAP2426c) that are identical to MAV_1554 of <it>Mycobacterium avium</it>. Mycobacterial rhomboids contain putative rhomboid catalytic signatures, with the protease active site stabilized by Phenylalanine. The topology and transmembrane helices of the Rv0110 orthologs were similar to those of eukaryotic secretase rhomboids, while those of Rv1337 orthologs were unique. Transcription assays indicated that both mycobacterial rhomboids are possibly expressed.</p> <p>Conclusions</p> <p>Mycobacterial rhomboids are active rhomboid proteases with different evolutionary history. The Rv0110 (rhomboid protease 1) orthologs represent prokaryotic rhomboids whose progenitor may be the ancestors of eukaryotic rhomboids. The Rv1337 (rhomboid protease 2) orthologs appear more stable and are conserved nearly in all mycobacteria, possibly alluding to their importance in mycobacteria. MAP2425c and MAP2426c provide the first evidence for a split homologous rhomboid, contrasting whole orthologs of genetically related species. Although valuable insights to the roles of rhomboids are provided, the data herein only lays a foundation for future investigations for the roles of rhomboids in mycobacteria.</p

Rhomboids of Mycobacteria: Characterization Using an <em>aarA</em> Mutant of <em>Providencia stuartii</em> and Gene Deletion in <em>Mycobacterium smegmatis</em>

<div><h3>Background</h3><p>Rhomboids are ubiquitous proteins with unknown roles in mycobacteria. However, bioinformatics suggested putative roles in DNA replication pathways and metabolite transport. Here, mycobacterial rhomboid-encoding genes were characterized; first, using the <em>Providencia stuartii</em> null-rhomboid mutant and then deleted from <em>Mycobacterium smegmatis</em> for additional insight in mycobacteria.</p> <h3>Methodology/Principal Findings</h3><p>Using in silico analysis we identified in <em>M. tuberculosis</em> genome the genes encoding two putative rhomboid proteins; Rv0110 (referred to as “rhomboid protease 1”) and Rv1337 (“rhomboid protease 2”). Genes encoding orthologs of these proteins are widely represented in all mycobacterial species. When transformed into <em>P. stuartii</em> null-rhomboid mutant (Δ<em>aarA</em>), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of <em>P. stuartii</em>). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in <em>M. smegmatis</em>, the ΔMSMEG_4904 single mutant (which lost the gene encoding MSMEG_4904, orthologous to Rv1337, “rhomboid protease 2”) formed the least biofilms and was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. However, the ΔMSMEG_5036 single mutant (which lost the gene encoding MSMEG_5036, orthologous to Rv0110, “rhomboid protease 1”) was not as susceptible. Surprisingly, the double rhomboid mutant ΔMSMEG_4904–ΔMSMEG_5036 (which lost genes encoding both homologs) was also not as susceptible suggesting compensatory effects following deletion of both rhomboid-encoding genes. Indeed, transforming the double mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ΔMSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin).</p> <h3>Conclusions/Significance</h3><p>Mycobacterial rhomboid-encoding genes exhibit differences in complementing <em>aarA</em> whereby it's only genes encoding “rhomboid protease 2” orthologs that fully restore AarA activity. Additionally, gene deletion data suggests inhibition of DNA gyrase by MSMEG_4904; however, the ameliorated effect in the double mutant suggests occurrence of compensatory mechanisms following deletion of genes encoding both rhomboids.</p> </div

Growth inhibition assays.

<p>Depicted are rhomboid mutants (Δ4904, Δ5036 and Δ4904Δ5036) and the wild type (WT) cultured in media with 0.1 µg/ml ciprofloxacin <b>(panel A)</b>; 60 µg/ml novobiocin <b>(panel B)</b>; 100 µg/ml isoniazid <b>(INH, panel C)</b>; and 0.5 µg/ml kanamycin <b>(panel D)</b>. The Δ4904 single mutant (Δ4904) was inhibited by ciprofloxacin (0.1 µg/ml) and novobiocin (60 µg ml⁻¹). In-set are similar data on solid media (7H10) showing that the Δ4904 single mutant (Δ4904) struggles to grow in presence of 0.1 µg ml⁻¹ ciprofloxacin and 60 µg ml⁻¹ novobiocin. Each data point was from an average of four experiments.</p

Figure 2

<p><b>Panels A and B:</b> Wild type <i>M. smegmatis</i> (WT) and rhomboid mutants (Δ4904 single, Δ5036 single and Δ4904Δ5036 double) cultured at 37°C (<b>Panel A</b>) and 42°C (<b>Panel B</b>), showing no difference in growth patterns. <b>Panel C:</b> Colony magnification showing differences in morphology between mutants (Δ4904, Δ5036 and Δ4904Δ5036) and the wild type. <b>Panel D: </b><i>M. smegmatis</i> single rhomboid mutants were inefficient at biofilm formation. The Δ4904 single mutant (Δ4904) formed the least biofilms while the double mutant (Δ4904Δ5036) formed more biofilms than the single mutants. Each data point was from an average of four experiments.</p

Competition and fitness assays.

<p>The Δ4904 single mutant was outcompeted by the wild type. <b>Panels A, B</b> and <b>C</b> depict the number of generations for each cell type; <b>Panels D, E</b> and <b>F</b> depict relative competitive fitness (R). The Δ4904 single mutant had the lowest R while the Δ5036 single mutant, double mutant and wild type had almost similar R values. Each data point was from an average of four experiments.</p

Distribution of “rhomboid protease 1” and “rhomboid protease 2” in selected mycobacterial species/strains.

a<p>Genes encoding “rhomboid protease 1” are missing in <i>M. leprae</i> and the <i>M. avium</i> complex (MAC) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045741#pone.0045741-Kateete1" target="_blank">[14]</a>.</p>b<p>the gene encoding “rhomboid protease 2” in <i>M. ulcerans</i> is a pseudogene (Gene ID number indicated);</p>c<p><i>M. avium subsp. paratuberculosis</i>. Annotation and gene/protein names are as described in KEGG (Kyoto Encyclopedia of Genes and Genomes [<a href="http://www.genome.jp/kegg/" target="_blank">http://www.genome.jp/kegg/</a>]) or from the respective genomes. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045741#pone.0045741.s004" target="_blank">Table S1</a></b> for details.</p