A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria

Abstract

Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a comparative analysis of 72 sequenced microbial genomes. We show that sortase enzymes can be partitioned into five distinct subfamilies based upon their primary sequences and that most of their substrates can be predicted by making a few conservative assumptions. Most bacteria encode sortases from two or more subfamilies, which are predicted to function nonredundantly in sorting proteins to the cell surface. Only ϳ20% of sortase-related proteins are most closely related to the well-characterized Staphylococcus aureus SrtA protein, but nonetheless, these proteins are responsible for anchoring the majority of surface proteins in gram-positive bacteria. In contrast, most sortase-like proteins are predicted to play a more specialized role, with each anchoring far fewer proteins that contain unusual sequence motifs. The functional sortase-substrate linkage predictions are available online (http://www.doe-mbi.ucla.edu/Services/Sortase/) in a searchable database.Pathogenic bacteria display an array of surface proteins to adhere to a site of infection, invade host cells, and evade the immune response. Many surface proteins are covalently attached to the cell wall by membrane-associated transpeptidases, called sortases (reviewed in references 18, 45, 48, and 53). The archetype sortase is the SrtA protein from Staphylococcus aureus, which anchors proteins that contain a C-terminal cell wall sorting signal (CWS) consisting of an LPXTG motif, followed by a hydrophobic domain and a tail of mostly positively charged residues (see Fig. 1A). An N-terminal secretion signal enables the precursor surface protein to be translocated across the membrane, where SrtA cleaves it in between the threonine and glycine residues of the LPXTG motif (47). SrtA then catalyzes the formation of an amide link between the carboxyl-group of the threonine and the cell wall precursor lipid II (57, 61), which is subsequently incorporated into the peptidoglycan via the transglycosylation and transpeptidation reactions of bacterial cell wall synthesis (66). An analysis of bacterial genomes indicates that this anchoring mechanism is conserved in gram-positive bacteria, since nearly all species encode SrtA homologs and proteins bearing a CWS (34, 55). Sortases may be excellent targets for new antimicrobial agents, since pathogens deficient in these enzymes exhibit reduced virulence (11,12,23,35,43,46).A large number of proteins are related to SrtA, but their functions have yet to be determined (55). Consistent with playing a role in surface protein chemistry, all SrtA homologs contain appropriately positioned active site residues (SrtA residues H120 and C184) (32)...

Cell evolution and Earth history: stasis and revolution

“…Replacing DNA gyrase by histones had marked coevolutionary repercussions on DNA-handling enzymes, which were substantially modified during the neomuran revolution; archaebacteria alone invented reverse gyrase by fusing two eubacterial genes, a hyperthermophilic adaptation, showing they and hyperthermophily must be more recent (Cavalier-Smith 2002a Cell evolution and Earth history T. Cavalier-Smith 973 posibacteria before the N-terminal signal sequence is cleaved. Most posibacteria have sortases of 2-4 subfamilies with different substrate specificities (Comfort & Clubb 2004). Sortases are a synapomorphy for posibacteria (comprising subphyla Endobacteria and Actinobacteria), strongly supporting their monophyly (Cavalier-Smith 2006a).…”

“…The lipid tails of posibacterial lipoproteins are in the CM outer leaflet; their protein part is covalently attached to the thick murein wall by sortase enzymes that recognize cell wall sorting signals near the C-terminus of its precursor, after its export to the periplasmic space by SRP (Comfort & Clubb 2004). Prior to this, sortases cleave the hydrophobic C-terminal sequence that temporarily anchors it to the CM.…”

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

“…Based on their primary sequences members of the sortase superfamily can be partitioned into at least six subfamilies, called class A to F enzymes (6,17,18). The genome of B. anthracis encodes class A, B, and D sortases, known as Ba SrtA, Ba SrtB, and Ba SrtC, respectively (19).…”

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

“…1), allowing us to hypothesize on the role of these two conserved motifs in sortase specificity and recognition. This expands on the work by Comfort and Clubb that identified prevalent LPXTG motif for each sortase isoform (50). To verify our hypothesis, we established a L. lactis model encoding a minimal pilus cassette and a series of relevant mutated derivatives.…”