The secG deletion mutation of Escherichia coli is suppressed by expression of a novel regulatory gene of Bacillus subtilis

AbstractSecG, a membrane component of E. coli protein translocase, stimulates the translocation of proteins across the cell membrane through the cycle of topology inversion, which is coupled to the membrane-insertion and deinsertion cycle of SecA [Nishiyama et al. (1996) Cell 85, 71–81]. A gene of B. subtilis able to suppress the cold-sensitive phenotype of the secG deletion mutant of E. coli was cloned and found to encode a novel regulatory protein, ScgR. Similarity search revealed homology with known proteins such as GlnR of B. subtilis. Plasmid-encoded ScgR stimulated protein translocation in the deletion mutant. ScgR increased the proportion of cardiolipin at the expense of phosphatidylglycerol, but did not affect the composition of other lipid components of the cell, suggesting that the increased cardiolipin level compensates for the SecG function and thereby stimulates protein translocation

Transcriptome analysis of the responses of Staphylococcus aureus to antimicrobial peptides and characterization of the roles of vraDE and vraSR in antimicrobial resistance

<p>Abstract</p> <p>Background</p> <p>Understanding how pathogens respond to antimicrobial peptides, and how this compares to currently available antibiotics, is crucial for optimizing antimicrobial therapy. <it>Staphylococcus aureus </it>has several known resistance mechanisms against human cationic antimicrobial peptides (CAMPs). Gene expression changes in <it>S. aureus </it>strain Newman exposed to linear CAMPs were analyzed by DNA microarray. Three antimicrobial peptides were used in the analysis, two are derived from frog, temporin L and dermaseptin K4-S4(1-16), and the ovispirin-1 is obtained from sheep.</p> <p>Results</p> <p>The peptides induced the VraSR cell-wall regulon and several other genes that are also up-regulated in cells treated with vancomycin and other cell wall-active antibiotics. In addition to this similarity, three genes/operons were particularly strongly induced by the peptides: <it>vraDE</it>, SA0205 and SAS016, encoding an ABC transporter, a putative membrane-bound lysostaphin-like peptidase and a small functionally unknown protein, respectively. Ovispirin-1 and dermaseptin K4-S4(1-16), which disrupt lipid bilayers by the carpet mechanism, appeared to be strong inducers of the <it>vraDE </it>operon. We show that high level induction by ovispirin-1 is dependent on the amide modification of the peptide C-terminus. This suggests that the amide group has a crucial role in the activation of the Aps (GraRS) sensory system, the regulator of <it>vraDE</it>. In contrast, temporin L, which disrupts lipid bilayers by forming pores, revealed a weaker inducer of <it>vraDE </it>despite the C-terminal amide modification. Sensitivity testing with CAMPs and other antimicrobials suggested that VraDE is a transporter dedicated to resist bacitracin. We also showed that SA0205 belongs to the VraSR regulon. Furthermore, VraSR was shown to be important for resistance against a wide range of cell wall-active antibiotics and other antimicrobial agents including the amide-modified ovispirin-1, bacitracin, teicoplanin, cefotaxime and 10 other β-lactam antibiotics, chlorpromazine, thioridazine and EGTA.</p> <p>Conclusion</p> <p>Defense against different CAMPs involves not only general signaling pathways but also CAMP-specific ones. These results suggest that CAMPs or a mixture of CAMPs could constitute a potential additive to standard antibiotic treatment.</p

Inactivation of the Ecs ABC Transporter of Staphylococcus aureus Attenuates Virulence by Altering Composition and Function of Bacterial Wall

Peer reviewe

ClpXP Protease Regulates the Signal Peptide Cleavage of Secretory Preproteins in Bacillus subtilis with a Mechanism Distinct from That of the Ecs ABC Transporter

Identification and characterization of a suppressor mutation, sup-15, which partially restored secretion in the protein secretion-deficient Bacillus subtilis ecsA26 mutant, led us to discover a novel function of Clp protease. Inactivation of ClpP improved the processing of the precursor of AmyQ α-amylase exposed on the outer surface of the cytoplasmic membrane. A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex. In the absence of ClpXP, the transcription of the sipS, sipT, sipV, and lsp signal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation of ecs mutations. Specific overproduction of SipT enhanced the secretion. These findings extend the regulatory roles of ClpXP to protein secretion. ClpXP also influenced the processing of the lipoprotein PrsA. A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested. In contrast, Ecs did not affect transcription of the sip genes, pointing to a different mechanism of secretion regulation

Characterization of Infecting Strains and Superantigen-Neutralizing Antibodies in Staphylococcus aureus Bacteremia▿

Staphylococcus aureus superantigens (SAgs) are highly potent T cell mitogens. Antibodies against non-enterotoxin gene cluster (non-egc) SAgs are common in healthy adults, whereas neutralizing antibodies against egc SAgs are rare. We investigated the infecting S. aureus strains and the anti-SAg antibody response during S. aureus bacteremia (SAB). This prospective clinical study (www.clinicaltrials.gov, NCT00548002) included 43 injection drug users (IDUs) and 44 group-matched nonaddicts with SAB. spa genotypes and SAg gene patterns (multiplex PCR) of the S. aureus isolates were determined. The neutralizing capacities of sera obtained at the acute phase and the convalescent phase of SAB were tested against the SAg cocktail of the respective infecting strain and a panel of recombinant SAgs. The lineages CC59 and CC30 were more prevalent among bacteremia strains from IDUs than among strains from nonaddicts. SAg gene patterns in isolates from IDUs and nonaddicts were similar. At the acute phase of bacteremia, IDUs had more neutralizing antibodies against non-egc SAgs than did nonaddicts. Antibody titers frequently increased during infection. In contrast, there were no neutralizing antibodies against egc SAgs at disease onset and such antibodies were not induced by SAB. SAB triggers an antibody response only against non-egc SAgs. Preimmunization in IDU patients is probably due to previous exposure to the infecting strain

Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in <i>Bacillus subtilis</i>

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains