Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris

Abstract

Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial vitamin K epoxide reductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of the A. oris membranespanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent ␤-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of the Mycobacterium tuberculosis VKOR analog in the A. oris VKOR deletion (ΔVKOR) mutant rescued its defects, in contrast to the expression of M. tuberculosis VKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA in Escherichia coli. Altogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the class Actinobacteria. IMPORTANCE It has recently been shown in the high-GC Gram-positive bacteria (or Actinobacteria) Actinomyces oris and Corynebacterium diphtheriae that oxidative folding of nascent polypeptides transported by the Sec machinery is catalyzed by a membrane-anchored oxidoreductase named MdbA. In A. oris, reoxidation of MdbA requires a bacterial VKOR-like protein, and yet, how VKOR mediates MdbA reoxidation is unknown. We show here that the A. oris membrane-spanning protein VKOR employs two pairs of exoplasmic cysteine residues, including the canonical CXXC thioredoxinlike motif, to oxidize MdbA via a disulfide relay mechanism. This mechanism of disulfide relay is essential for pilus assembly, polymicrobial interactions, and bio- Editor Olaf Schneewind, The University of Chicago

Structure and Mechanism of LcpA, a Phosphotransferase That Mediates Glycosylation of a Gram-Positive Bacterial Cell Wall-Anchored Protein

“…We previously reported that disulfide bond formation in A. oris requires the activity of a membrane-bound thiol-disulfide oxidoreductase named MdbA (20), and reactivation of MdbA involves another oxidoreductase called VKOR (22, 23). Because mdbA is an essential gene, whereas a mutant devoid of vkor is viable although it exhibits severe defects in oxidative protein folding (20), we examined if LcpA stability is affected in the vkor mutant.…”

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC

“…Transmission electron microscopy was performed according to a previously published protocol ( 48 ). Briefly, fusobacteria grown in TSPC broth were harvested by centrifugation and suspended in 0.1 M NaCl.…”

Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii

“…The primer pair pCmMdbA-HindIII-A/ pCmMdbA-B (Table S2) was used to amplify the C. diphtheriae mdbA promoter region from the genomic DNA of strain NCTC 13129, whereas the primer pair pCmMdbA-C/pCmMdbA-BamHI-D was used to amplify the C. matruchotii mdbA coding sequence from the genomic DNA of strain ATCC 14266. Overlapping PCR was employed to link the promoter region to the coding sequence using both PCR products as the templates and primers pCmMdbA-HindIII-A and pCmMdbA-BamHI-D according to a Penicillin-binding protein; transglycosylase 2 published protocol (18). The generated PCR product was gel purified and digested with HindIII-HF and BamHI-HF (New England BioLabs) and subsequently ligated into pCGL0243 (40).…”