Characterization of the Sortase Repertoire in Bacillus anthracis

Abstract

LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss), bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins.

Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal

“…Notably, its side chain methyl does not appear to be recognized as it is positioned on the surface of the enzyme in between Leu 103 in helix H2 and Ile 61 in the N-terminal appendage. This surface appears to be large enough to accommodate a range of amino acid side chains, consistent with the documented promiscuity of Ba SrtA for this site within the sorting signal (62). The side chain of the threonine residue in the substrate is buried in the S1 pocket formed by the side chains of Ile 185 in strand ␤7, Ala 124 in strand ␤4, and Leu 103 in helix H2 (Fig.…”

“…The genome of B. anthracis encodes class A, B, and D sortases, known as Ba SrtA, Ba SrtB, and Ba SrtC, respectively (19). Ba SrtA, the focus of this study, anchors seven proteins to the cell wall by joining the threonine of the LPXTG sorting signal at the C terminus of the protein to the amine group of meso-diaminopimelic acid (DAP) within lipid II (20).…”

Solution Structure of the Sortase Required for Efficient Production of Infectious Bacillus anthracis Spores

“…B. anthracis also encodes a housekeeping class A enzyme (BaSrtA) that anchors a different set of proteins to the cell wall. 20,21 Interestingly, BaSrtC and BaSrtA specifically recognize very closely related sorting signals. 16,17 BaSrtA recognizes a canonical LP X TG-type sorting signal present in seven B. anthracis proteins (three of these proteins are involved in collagen adhesion, while the functions of the other proteins are not known).…”

A new structural class of bacterial thioester domains reveals a slipknot topology

“…SaTIE and BaTIE have been experimentally confirmed to contain thioester bonds . BaTIE was among the first surface proteins from B. anthracis described, and has previously been studied as BasC and BA5258 . It has been suggested to function as a collagen‐binding protein .…”