Identification of a fmtA-like gene that has similarity to other PBPs and beta-lactamases in Staphylococcus aureus

We identified a gene from Staphylococcus aureus, flp (fmtA-like protein), encoding a protein of 489 amino acid residues with a molecular mass of 56.4 kDa. The deduced amino acid sequence shows similarity to previously characterized penicillin binding proteins (PBPs) and FmtA of S. aureus (one of the factors which affect methicillin resistance). FLP protein has three motifs, which are conserved in PBPs and beta-lactamases, suggesting that it might be associated with cell wall synthesis. Recombinant FLP protein, however, lacks penicillin binding activity, and the inactivation of flp in two methicillin-resistant S. aureus strains did not cause a reduction in the methicillin resistance.     

Impaired processing of FLP and NLP peptides in carboxypeptidase E (EGL-21)-deficient Caenorhabditis elegans as analyzed by mass spectrometry

Biologically active peptides are synthesized from inactive pre-proproteins or peptide precursors by the sequential actions of processing enzymes. Proprotein convertases cleave the precursor at pairs of basic amino acids, which are then removed from the carboxyl terminus of the generated fragments by a specific carboxypeptidase. Caenorhabditis elegans strains lacking proprotein convertase EGL-3 display a severely impaired neuropeptide profile (Husson et al. 2006, J. Neurochem.98, 1999-2012). In the present study, we examined the role of the C. elegans carboxypeptidase E orthologue EGL-21 in the processing of peptide precursors. More than 100 carboxy-terminally extended neuropeptides were detected in egl-21 mutant strains. These findings suggest that EGL-21 is a major carboxypeptidase involved in the processing of FMRFamide-like peptide (FLP) precursors and neuropeptide-like protein (NLP) precursors. The impaired peptide profile of egl-3 and egl-21 mutants is reflected in some similar phenotypes. They both share a severe widening of the intestinal lumen, locomotion defects, and retention of embryos. In addition, egl-3 animals have decreased intestinal fat content. Taken together, these results suggest that EGL-3 and EGL-21 are key enzymes for the proper processing of neuropeptides that control egg-laying, locomotion, fat storage and the nutritional status.     

PilM/N/O/P Proteins Form an Inner Membrane Complex That Affects the Stability of the Pseudomonas aeruginosa Type IV Pilus Secretin

The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. With the exception of the outer membrane secretin, a multimer of PilQ subunits, the specific functions of the products encoded by this gene cluster are poorly characterized. Orthologous proteins in the related bacterial type II secretion system have been shown to interact to form an inner membrane complex required for protein secretion. In this study, we provide evidence that the PilM/N/O/P proteins form a functionally equivalent type IVa pilus complex. Using Pseudomonas aeruginosa as model organism, we found that all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane. Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. Furthermore, complementation studies revealed that the stoichiometry of the components is important for the correct formation of a stable complex in vivo. We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa, as PilQ stability is negatively affected in its absence. Finally, we show that, in the absence of the pilin subunit, the levels of membrane-bound components of the inner membrane complex are negatively regulated by the PilR/S two-component system, suggesting a role for PilR/S in sensing the piliation status of the cell.