Functional Characterization of Escherichia coli GlpG and Additional Rhomboid Proteins Using an aarA Mutant of Providencia stuartii

Abstract

The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and required for the production of an extracellular signaling molecule that regulates cellular functions including peptidoglycan acetylation, methionine transport, and cysteine biosynthesis. Additional aarA-dependent phenotypes include (i) loss of an extracellular yellow pigment, (ii) inability to grow on MacConkey agar, and (iii) abnormal cell division. Since these phenotypes are easily assayed, the P. stuartii aarA mutant serves as a useful host system to investigate rhomboid function. The Escherichia coli GlpG protein was shown to be functionally similar to AarA and rescued the above aarA-dependent phenotypes in P. stuartii. GlpG proteins containing single alanine substitutions at the highly conserved catalytic triad of asparagine (N154A), serine (S201A), or histidine (H254A) residues were nonfunctional. The P. stuartii aarA mutant was also used as a biosensor to demonstrate that proteins from a variety of diverse sources exhibited rhomboid activity. In an effort to further investigate the role of a rhomboid protein in cell physiology, a glpG mutant of E. coli was constructed. In phenotype microarray experiments, the glpG mutant exhibited a slight increase in resistance to the ␤-lactam antibiotic cefotaxime.

Core principles of intramembrane proteolysis: comparison of rhomboid and site-2 family proteases

“…The issue of triad versus dyad has been a point of debate ever since the original observation that mutation of a transmembrane asparagine, proposed to be the third catalytic residue, abolished the activity of Drosophila rhomboid-1, while the asparagine mutant had mild effects in human RHBDL2 [12]. Similar discrepancies have been observed for bacterial rhomboid enzymes in vivo [31,45,46] and in vitro [32,47]. Therefore, the structures unequivocally show that no other residue is hydrogen-bonded to the histidine, and the candidate asparagine on TM2 lies on the opposite side of the serine such that it could not bond with the histidine.…”

Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase

“…Prov. stuartii aarA mutants exhibit a prominent chaining phenotype (7,20) and are similar in this respect to E. coli tat mutants (22). A Prov.…”

“…mirabilis as a multicopy suppressor of an aarA mutation was not unique. In a search for E. coli genes that restored aarA mutant phenotypes, we previously reported identification of the rhomboid GlpG (20). However, additional E. coli genes that restored pigment production and extracellular signal were tatA and tatE (data not shown).…”

“…Prov. stuartii aarA mutations are pleiotropic, and the resulting phenotypes include loss of an extracellular signaling molecule, abnormal cell division (cell chaining), inability to grow on MacConkey agar, and loss of a diffusible yellow pigment (6,7,20). In a search for rhomboid-like proteins in the related organism Prot.…”

Expressed sequence tags of Aspergillus fumigatus: Extension of catalogue and their evaluation as putative drug targets and/or diagnostic markers

“…Two of the opportunistic fungal pathogens from different class of fungi, C. albicans and C. neoformans were found to have Homologues of few of these putative drug targets have already been known as virulence factors in other organisms; adelylate kinase has been implicated in virulence of Pseudomonas aeruginosa (13), whereas , succinyl-CoA synthetase has been shown to be important for biofilm formation in bacteria (14). Rhomboid factors are serine proteases (15) and few of the serine proteases have already been known to play a role in virulence of A. fumigatus by digesting the host tissue and also by acting as allergen (16). Autoimmune diseases have been known to contribute to the extensive tissue damage observed in allergic bronchopulmonary aspergillosis (ABPA) patients (17).…”