Abstract
Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes.
Highlights
Ety of extractable lipids [1, 2]
To exclude that this was an antibiotic-specific effect, we examined whether rv1698 had a similar effect on the efficacy of chloramphenicol against M. smegmatis
Expression of both mspA and rv1698 significantly increased the susceptibility of the ⌬mspA mutant MN01 to chloramphenicol. These results indicated that Rv1698 increased the outer membrane permeability of the M. smegmatis ⌬mspA mutant
Summary
Bacterial Strains and Growth Conditions—All of the bacterial strains used in this study are listed in supplemental Table S1. The broken cells were harvested by centrifugation and resuspended in lysis buffer followed by sonication as described above. Purification of Rv1698His from M. bovis BCG—M. bovis BCG carrying the plasmid pML911 was grown in Middlebrook 7H9 liquid medium supplemented with 10% oleic acid albumin dextrose complex, 0.05% Tween 80, and hygromycin. The samples were boiled for 20 min to allow cell lysis and centrifuged again to remove insoluble debris, and 50 l of the protein extracts were separated on a 10% polyacrylamide gel and analyzed in Western blots using standard protocols [22]. These values were taken as the widths of the molecules and used for a minimal estimate of the pore size of Rv1698
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