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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.

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.

Bacterial proteinaceous filaments termed pili or fimbriae are nonflagellar, hair‐like structures protruding from the cell surface that are critical for bacterial virulence and fitness. Present in both Gram‐negative and Gram‐positive bacteria, pili are involved in many processes such as conjugation, adherence, twitching motility, biofilm formation and immunomodulation. Considerably diverse and complex, Gram‐negative pili are formed by noncovalent polymerisation of various pilin subunits; many of these pili require chaperones and usher proteins for their assembly. In contrast, fewer pilus systems have been described for the Gram‐positive counterparts; notably well studied are the heterotrimeric or ‐dimeric pili that are covalently assembled by a transpeptidase enzyme called sortase. Furthermore, type IV pili have been identified in several Gram‐positive bacteria, especially in clostridia. Key Concepts Noncovalent pilus polymerisation is a general mechanism of pilus assembly in Gram‐negative bacteria, including the chaperone/usher assembly pathway and type IV pilus pathway, which generates noncovalent pilus polymers. Sortase‐mediated pilus assembly is a general mechanism of pilus assembly in Gram‐positive bacteria that involves a transpeptidase enzyme named sortase, which cleaves pilin precursors at sorting signals between threonine and glycine residues and utilises the side‐chain amino groups of pilin motif sequences to generate covalent links between pilin subunits. The sorting signal consists of an LPXTG motif followed by a hydrophobic domain and a positively charged tail. The pilin motif is an 11 amino acid sequence of WxxxVxVYPKN located near the N ‐terminus of major pilin subunits, in which the electron‐donating lysine residue forms an isopeptide bond with the threonine residue generated from the cleavage of the LPXTG motif of adjacent pilin subunits. Tissue tropism is referred to as the ability of a pathogen to adhere specifically to some particular epithelial cells. Phase variation involves switching of surface antigens such as pili that allows a pathogen to evade the host immune system. Immunomodulation is a process of changing the host's immune system by certain molecules known as immunomodulators including pili that can activate or suppress immune cells. Dental plaque is one of the most complex bacterial biofilms that is formed by sequential colonisation of initial colonisers such as Actinomyces spp. and oral streptococci and late colonisers; this process involves Actinomyces fimbriae. Twitching motility mediated by pili allows translocation between mucosal surfaces, colonisation of host tissues and establishment of biofilms by a pathogen.

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