A conserved signal-peptidase antagonist modulates membrane homeostasis of actinobacterial sortase critical for surface morphogenesis.

Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for a transpeptidase that anchors many extracellular proteins to the Gram-positive bacterial cell wall. Here, we uncover the hitherto unknown function of this class of conserved proteins, which we name SafA, as a topological modulator of sortase in the oral Actinobacterium Actinomyces oris. Genetic deletion of safA induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the safA mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions-the phenotypes that are mirrored by srtA depletion. The pleiotropic defect of the safA mutant is rescued by ectopic expression of safA from not only A. oris, but also Corynebacterium diphtheriae or Corynebacterium matruchotii. Importantly, the SrtA N terminus harbors a tripartite-domain feature typical of a bacterial signal peptide, including a cleavage motif AXA, mutations in which prevent SrtA cleavage mediated by the signal peptidase LepB2. Bacterial two-hybrid analysis demonstrates that SafA and SrtA directly interact. This interaction involves a conserved motif FPW within the exoplasmic face of SafA, since mutations of this motif abrogate SafA-SrtA interaction and induce SrtA cleavage and excretion as observed in the safA mutant. Evidently, SafA is a membrane-imbedded antagonist of signal peptidase that safeguards and maintains membrane homeostasis of the housekeeping sortase SrtA, a central player of cell surface assembly.

The bacterial copper resistance protein CopG contains a cysteine-bridged tetranuclear copper cluster.

CopG is an uncharacterized protein ubiquitous in Gram-negative bacteria whose gene frequently occurs in clusters of copper resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system.

New Paradigms of Pilus Assembly Mechanisms in Gram-Positive Actinobacteria.

Adhesive pili in Gram-positive bacteria represent a variety of extracellular multiprotein polymers that mediate bacterial colonization of specific host tissues and associated pathogenesis. Pili are assembled in two distinct but coupled steps, an orderly crosslinking of pilin monomers and subsequent anchoring of the polymer to peptidoglycan, catalyzed by two transpeptidase enzymes - the pilus-specific sortase and the housekeeping sortase. Here, we review this biphasic assembly mechanism based on studies of two prototypical models, the heterotrimeric pili in Corynebacterium diphtheriae and the heterodimeric pili in Actinomyces oris, highlighting some newly emerged basic paradigms. The disparate mechanisms of protein ligation mediated by the pilus-specific sortase and the spatial positioning of adhesive pili on the cell surface modulated by the housekeeping sortase are among the notable highlights.