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

Abstract

BackgroundRhomboids 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 Providencia stuartii null-rhomboid mutant and then deleted from Mycobacterium smegmatis for additional insight in mycobacteria.Methodology/Principal FindingsUsing in silico analysis we identified in M. tuberculosis 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 P. stuartii null-rhomboid mutant (ΔaarA), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of P. stuartii). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in M. smegmatis, 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).Conclusions/SignificanceMycobacterial rhomboid-encoding genes exhibit differences in complementing aarA 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.

A Rhomboid Protease Gene Deletion Affects a Novel Oligosaccharide N-Linked to the S-layer Glycoprotein of Haloferax volcanii

“…2C). Similarly, an increased sensitivity to novobiocin has also been reported for mycobacterial Rho null mutants (9). In addition, cell motility was slightly reduced in the mutant strain (Fig.…”

“…Processing of TatA activates the translocation process, allowing the export of an unknown quorumsensing signal (8). In Mycobacteria, Rho null mutants display impaired biofilm formation and increased antibiotic sensitivity (9). Rho homologs are widely represented in archaeal genomes, and the predicted proteins contain the typical multispanning transmembrane domains (TMDs) and the amino acid residues involved in catalysis (Ser-His dyad), suggesting that they are probably functional enzymes (10).…”

Structure-aware M. tuberculosis functional annotation uncloaks resistance, metabolic, and virulence genes

“…This gene had been annotated as a putative secreted protein, which does not indicate its involvement in drug resistance and biofilm formation. Also among our unique mannotations is Rv1337 as a rhomboid protease 74 , a far more informative annotation than that from TubercuList (possible integral membrane protein), PATRIC, RefSeq, or Mtb Network Portal (S7 Table). Also absent from these databases is the involvement of Rv1337 in ciprofloxacin and novobiocin resistance and in biofilm formation 74 , which we added as functional notes.…”

“…Also among our unique mannotations is Rv1337 as a rhomboid protease 74 , a far more informative annotation than that from TubercuList (possible integral membrane protein), PATRIC, RefSeq, or Mtb Network Portal (S7 Table). Also absent from these databases is the involvement of Rv1337 in ciprofloxacin and novobiocin resistance and in biofilm formation 74 , which we added as functional notes. Another mannotated gene, Rv3005c, encodes an oxidoreductase responsive to oxidative stress 75 .…”

Archaeal membrane-associated proteases: insights on Haloferax volcanii and other haloarchaea

“…In bacteria, rhomboid null mutants show phenotypes that may be related to defective cell envelope and/or cell-surface structures. In Bacillus subtilis , a mutant strain in the rhomboid homolog YqgP displayed a slight decrease in glucose uptake and a defect in cell division leading to the formation of filamentous cells (Mesak et al, 2004); Mycobacterium smegmatis rhomboid mutants showed reduced capacity for biofilm formation and increased sensitivity to antibiotics (Kateete et al, 2012). So far only TatA, a protein component of the Tat translocon in the pathogenic bacterium Providencia stuartii, has been experimentally confirmed as a rhomboid substrate (Stevenson et al, 2007).…”