Sorting Out the Details: The Roles of a Novel GlyGly-CTERM Domain, Rhombosortase, and the Type II Secretion System in Surface Localization of a Vibrio cholerae serine protease, VesB.

Some proteins carry recognition motifs that allow them to be sorted into different compartments of the cell. In Gram-positive bacteria, proteins with the LPXTG sorting motif are localized to the cell surface where they are attached to peptidoglycan by a membrane bound cysteine protease called sortase. The work presented here entails a novel Gram-negative sorting mechanism that includes the rhombosortase protease and the C-terminal recognition motif, GlyGly-CTERM. GlyGly-CTERM domains contain serines and glycines, followed by a stretch of hydrophobic and positively charged residues. The composition of this domain makes it an ideal candidate to span the membrane and serve as a possible substrate for rhombosortase, a rhomboid-like protease. Rhomboid proteases are intramembrane serine proteases that cleave substrates with a single transmembrane domain that contains helix-destabilizing residues. With this information, we postulated that rhombosortase cleaves and promotes cell surface association of GlyGly-CTERM containing proteins similar to sortase. In order to test this hypothesis, the GlyGly-CTERM containing protein Vibrio extracellular serine protease B (VesB) was utilized. VesB contains an N-terminal signal peptide, a protease domain that includes the propeptide and the catalytic triad, and a C-terminal domain with the GlyGly-CTERM extension. VesB is a trypsin-like serine protease that has an apparent preference for arginine and cleaves both peptides and intact proteins. Using activity assays, western blotting, proteomic analysis and fluorescent microscopy, these studies showed that the type II secretion (T2S) system is responsible for outer membrane translocation of VesB, while rhombosortase promotes its surface retention by cleaving the GlyGly-CTERM domain and perhaps further modifying the newly generated C-terminus of VesB. It was demonstrated that native VesB in a rhombosortase mutant and VesB produced without its GlyGly-CTERM domain were inactive and no longer cell-associated. This latter finding suggests that removal of the N-terminal propeptide and thereby activation of VesB occurs on the cell surface. Taken together, rhombosortase cleaves and promotes cell surface retention of the GlyGly-CTERM domain containing protein, VesB, thereby highlighting a novel protein sorting mechanism in Gram-negative bacteria.PhDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108933/1/sgadwal_1.pd

The two faces of ToxR: activator of ompU , co‐regulator of toxT in Vibrio cholerae

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87152/1/j.1365-2958.2011.07681.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/87152/2/MMI_7681_sm_FigureS1_TableS1-2.pd