scholarly journals Molecular Analysis of Phr Peptide Processing in Bacillus subtilis

2003 ◽  
Vol 185 (16) ◽  
pp. 4861-4871 ◽  
Author(s):  
Sophie Stephenson ◽  
Christian Mueller ◽  
Min Jiang ◽  
Marta Perego
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Signal Peptidase ◽  
Type I ◽  
Peptidase Activity ◽  
Amino Terminal ◽  
Processing Event ◽  
Signal Peptidases

ABSTRACT In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F∼P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

scholarly journals Broadening the Spectrum of β-Lactam Antibiotics through Inhibition of Signal Peptidase Type I

2012 ◽  
Vol 56 (9) ◽  
pp. 4662-4670 ◽  
Author(s):  
Alex G. Therien ◽  
Joann L. Huber ◽  
Kenneth E. Wilson ◽  
Patrick Beaulieu ◽  
Alexandre Caron ◽  
...  
Keyword(s):  
Treatment Options ◽  
Signal Peptidase ◽  
Type I ◽  
New Agents ◽  
Content Type ◽  
Cyclic Depsipeptide ◽  
Serious Infections ◽  
Synthetic Derivative

ABSTRACTThe resistance of methicillin-resistantStaphylococcus aureus(MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem bothin vitroandin vivowith potent efficacy. Thein vitroactivity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

Evaluation of the type I signal peptidase as antibacterial target for biofilm-associated infections of Staphylococcus epidermidis

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3719-3729 ◽  
Author(s):  
Katrijn Bockstael ◽  
Nick Geukens ◽  
Lieve Van Mellaert ◽  
Piet Herdewijn ◽  
Jozef Anné ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Functional Activity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Antimicrobial Treatment ◽  
Signal Peptidase ◽  
Temperature Sensitive ◽  
Type I

The development of antibacterial resistance is inevitable and is a major concern in hospitals and communities. Moreover, biofilm-grown bacteria are less sensitive to antimicrobial treatment. In this respect, the Gram-positive Staphylococcus epidermidis is an important source of nosocomial biofilm-associated infections. In the search for new antibacterial therapies, the type I signal peptidase (SPase I) serves as a potential target for development of antibacterials with a novel mode of action. This enzyme cleaves off the signal peptide from secreted proteins, making it essential for protein secretion, and hence for bacterial cell viability. S. epidermidis encodes three putative SPases I (denoted Sip1, Sip2 and Sip3), of which Sip1 lacks the catalytic lysine. In this report, we investigated the active S. epidermidis SPases I in more detail. Sip2 and Sip3 were found to complement a temperature-sensitive Escherichia coli lepB mutant, demonstrating their in vivo functional activity. In vitro functional activity of purified Sip2 and Sip3 proteins and inhibition of their activity by the SPase I inhibitor arylomycin A2 were further illustrated using a fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we demonstrated that SPase I not only is an attractive target for development of novel antibacterials against free-living bacteria, but also is a feasible target for biofilm-associated infections.

scholarly journals Mechanism of Transcription Activation at the comG Promoter by the Competence Transcription Factor ComK of Bacillus subtilis

2004 ◽  
Vol 186 (4) ◽  
pp. 1120-1128 ◽  
Author(s):  
K. A. Susanna ◽  
A. F. van der Werff ◽  
C. D. den Hengst ◽  
B. Calles ◽  
M. Salas ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Complex Signal ◽  
Type I ◽  
Binding Characteristics ◽  
Α Subunits

ABSTRACT The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type α-subunits and with C-terminal deletion mutants of the α-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.

scholarly journals Regulators of the Bacillus subtilis cydABCD Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD

2007 ◽  
Vol 189 (9) ◽  
pp. 3348-3358 ◽  
Author(s):  
Ankita Puri-Taneja ◽  
Matthew Schau ◽  
Yinghua Chen ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Negative Regulator ◽  
Regulatory Proteins ◽  
Regulation Of Respiration ◽  
Regulator Protein ◽  
Promoter Fusion

ABSTRACT The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions −58 and −107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3′-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions −4 to −33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.

scholarly journals Signal Peptidase Is Necessary and Sufficient for Site 1 Cleavage of RsiV inBacillus subtilisin Response to Lysozyme

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Ana N. Castro ◽  
Lincoln T. Lewerke ◽  
Jessica L. Hastie ◽  
Craig D. Ellermeier
Keyword(s):  
Transmembrane Domain ◽  
Signal Peptidase ◽  
Peptidase Activity ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Proteolytic Cascade ◽  
Σ Factor ◽  
Necessary And Sufficient

ABSTRACTExtracytoplasmic function (ECF) σ factors are a diverse family of alternative σ factors that allow bacteria to sense and respond to changes in the environment. σVis an ECF σ factor found primarily in low-GC Gram-positive bacteria and is required for lysozyme resistance in several opportunistic pathogens. In the absence of lysozyme, σVis inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded via the process of regulated intramembrane proteolysis (RIP). RIP is initiated by cleavage of RsiV at site 1, which allows the intramembrane protease RasP to cleave RsiV within the transmembrane domain at site 2 and leads to activation of σV. Previous work suggested that RsiV is cleaved by signal peptidase at site 1. Here we demonstratein vitrothat signal peptidase is sufficient for cleavage of RsiV only in the presence of lysozyme and provide evidence that multipleBacillus subtilissignal peptidases can cleave RsiVin vitro. This cleavage is dependent upon the concentration of lysozyme, consistent with previous work that showed that binding to RsiV was required for σVactivation. We also show that signal peptidase activity is required for site 1 cleavage of RsiVin vivo. Thus, we demonstrate that signal peptidase is the site 1 protease for RsiV.IMPORTANCEExtracytoplasmic function (ECF) σ factors are a diverse family of alternative σ factors that respond to extracellular signals. The ECF σ factor σVis present in many low-GC Gram-positive bacteria and induces resistance to lysozyme, a component of the innate immune system. The anti-σ factor RsiV inhibits σVactivity in the absence of lysozyme. Lysozyme binds RsiV, which initiates a proteolytic cascade leading to destruction of RsiV and activation of σV. This proteolytic cascade is initiated by signal peptidase, a component of the general secretory system. We show that signal peptidase is necessary and sufficient for cleavage of RsiV at site 1 in the presence of lysozyme. This report describes a role for signal peptidase in controlling gene expression.

scholarly journals Transcriptional Activation by Bacillus subtilis ResD: Tandem Binding to Target Elements and Phosphorylation-Dependent and -Independent Transcriptional Activation

2004 ◽  
Vol 186 (7) ◽  
pp. 2028-2037 ◽  
Author(s):  
Hao Geng ◽  
Shunji Nakano ◽  
Michiko M. Nakano
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
Gene Activation ◽  
Phosphorylation Site ◽  
Oxygen Limitation ◽  
Nitrate Respiration ◽  
Expression Of Genes

ABSTRACT The expression of genes involved in nitrate respiration in Bacillus subtilis is regulated by the ResD-ResE two-component signal transduction system. The membrane-bound ResE sensor kinase perceives a redox-related signal(s) and phosphorylates the cognate response regulator ResD, which enables interaction of ResD with ResD-dependent promoters to activate transcription. Hydroxyl radical footprinting analysis revealed that ResD tandemly binds to the −41 to −83 region of hmp and the −46 to −92 region of nasD. In vitro runoff transcription experiments showed that ResD is necessary and sufficient to activate transcription of the ResDE regulon. Although phosphorylation of ResD by ResE kinase greatly stimulated transcription, unphosphorylated ResD, as well as ResD with a phosphorylation site (Asp57) mutation, was able to activate transcription at a low level. The D57A mutant was shown to retain the activity in vivo to induce transcription of the ResDE regulon in response to oxygen limitation, suggesting that ResD itself, in addition to its activation through phosphorylation-mediated conformation change, senses oxygen limitation via an unknown mechanism leading to anaerobic gene activation.

scholarly journals A Truncated Soluble Bacillus Signal Peptidase Produced in Escherichia coli Is Subject to Self-Cleavage at Its Active Site

2000 ◽  
Vol 182 (20) ◽  
pp. 5765-5770 ◽  
Author(s):  
M. L. van Roosmalen ◽  
J. D. H. Jongbloed ◽  
A. Kuipers ◽  
G. Venema ◽  
S. Bron ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptidase ◽  
Soluble Form ◽  
Peptidase Activity ◽  
Mutant Proteins ◽  
E Coli ◽  
Amino Terminal ◽  
Complete Inactivation ◽  
High Level

ABSTRACT Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm ofEscherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the −1, −3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.

scholarly journals Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins.

1994 ◽  
Vol 127 (2) ◽  
pp. 343-355 ◽  
Author(s):  
M M Falk ◽  
N M Kumar ◽  
N B Gilula
Keyword(s):  
Plasma Membrane ◽  
Gap Junction ◽  
Proteolytic Processing ◽  
Signal Peptidase ◽  
Membrane Insertion ◽  
Competent Cell ◽  
In Vitro Synthesis ◽  
Amino Terminal

Connexins, the proteins that form gap junction channels, are polytopic plasma membrane (PM) proteins that traverse the plasma membrane bilayer four times. The insertion of five different connexins into the membrane of the ER was studied by synthesizing connexins in translation-competent cell lysates supplemented with pancreatic ER-derived microsomes, and by expressing connexins in vivo in several eucaryotic cell types. In addition, the subcellular distribution of the connexins was determined. In vitro-synthesis in the presence of microsomes resulted in the signal recognition particle-dependent membrane insertion of the connexins. The membrane insertion of all connexins was accompanied by an efficient proteolytic processing that was dependent on the microsome concentration. Endogenous unprocessed connexins were detectable in the microsomes used, indicating that the pancreatic microsomes serve as a competent recipient in vivo for unprocessed full length connexins. Although oriented with their amino terminus in the cytoplasm, the analysis of the cleavage reaction indicated that an unprecedented processing by signal peptidase resulted in the removal of an amino-terminal portion of the connexins. Variable amounts of similar connexin cleavage products were also identified in the ER membranes of connexin overexpressing cells. The amount generated correlated with the level of protein expression. These results demonstrate that the connexins contain a cryptic signal peptidase cleavage site that can be processed by this enzyme in vitro and in vivo in association with their membrane insertion. Consequently, a specific factor or condition must be required to prevent this aberrant processing of connexins under normal conditions in the cell.

scholarly journals Cloning and Characterization of Archaeal Type I Signal Peptidase from Methanococcus voltae

2003 ◽  
Vol 185 (20) ◽  
pp. 5936-5942 ◽  
Author(s):  
Sandy Y. M. Ng ◽  
Ken F. Jarrell
Keyword(s):  
Gene Product ◽  
Signal Peptidase ◽  
Type I ◽  
Peptidase Activity ◽  
E Coli ◽  
Methanococcus Voltae ◽  
Signal Peptidase I ◽  
Archaeal Gene ◽  
Enzyme Source

ABSTRACT Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.

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