Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase.

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase.

In Gram-positive bacteria, the biphasic assembly of pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by anchoring of the resulting polymers to the cell wall mediated by the housekeeping sortase. Uniquely, in Actinomyces oris , the SrtC2 sortase not only polymerizes FimA pilins to assemble type 2 fimbriae, but it can also act as the anchoring sortase, which joins both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural comparisons of SrtC2 and SrtC1 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is unaffected by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of the tip adhesin CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions that require CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

Ribonuclease J-Mediated mRNA Turnover Modulates Cell Shape, Metabolism and Virulence in Corynebacterium diphtheriae.

Controlled RNA degradation is a crucial process in bacterial cell biology for maintaining proper transcriptome homeostasis and adaptation to changing environments. mRNA turnover in many Gram-positive bacteria involves a specialized ribonuclease called RNase J (RnJ). To date, however, nothing is known about this process in the diphtheria-causative pathogen Corynebacterium diphtheriae, nor is known the identity of this ribonuclease in this organism. Here, we report that C. diphtheriae DIP1463 encodes a predicted RnJ homolog, comprised of a conserved N-terminal ß-lactamase domain, followed by ß-CASP and C-terminal domains. A recombinant protein encompassing the ß-lactamase domain alone displays 5'-exoribonuclease activity, which is abolished by alanine-substitution of the conserved catalytic residues His186 and His188. Intriguingly, deletion of DIP1463/rnj in C. diphtheriae reduces bacterial growth and generates cell shape abnormality with markedly augmented cell width. Comparative RNA-seq analysis revealed that RnJ controls a large regulon encoding many factors predicted to be involved in biosynthesis, regulation, transport, and iron acquisition. One upregulated gene in the ∆rnj mutant is ftsH, coding for a membrane protease (FtsH) involved in cell division, whose overexpression in the wild-type strain also caused cell-width augmentation. Critically, the ∆rnj mutant is severely attenuated in virulence in a Caenorhabditis elegans model of infection, while the FtsH-overexpressing and toxin-less strains exhibit full virulence as the wild-type strain. Evidently, RNase J is a key ribonuclease in C. diphtheriae that post-transcriptionally influences the expression of numerous factors vital to corynebacterial cell physiology and virulence. Our findings have significant implications for basic biological processes and mechanisms of corynebacterial pathogenesis.