A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis

Kobayashi, Tetsuo; Zhu, Yingfang; Nicholls, N; Lampen, J. O.

doi:10.1128/jb.169.9.3873-3878.1987

Cited by 79 publications

(65 citation statements)

References 39 publications

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“…The heavier fragment was observed only in the absence of arylomycin; while the amount of the smaller fragment decreased with added inhibitor, it was still present at the highest arylomycin concentration examined. A canonical N-terminal signal peptide was not detected with two of the identified proteins, LtaS (SERP0379), which is involved in lipoteichoic acid synthesis (36), and BlaR1 (SERP1460), which is a component of a two-component response regulator involved in the sensing of ␤-lactam antibiotics (30). However, LtaS is predicted by sequence analysis to have five N-terminal transmembrane helices and a C-terminal, extracellular domain (which has been annotated as a sulfatase [UniProt accession number Q5HR16]).…”

Section: Vol 193 2011 Type I Spase and Protein Secretion In S Epidmentioning

confidence: 99%

Type I Signal Peptidase and Protein Secretion inStaphylococcus epidermidis

et al. 2011

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Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the ␤-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationaryphase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.

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Section: Vol 193 2011 Type I Spase and Protein Secretion In S Epidmentioning

confidence: 99%

Type I Signal Peptidase and Protein Secretion inStaphylococcus epidermidis

et al. 2011

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“…In the absence of ␤-lactams antibiotics, the BlaI repressor prevents the transcription of the blaP gene (1)(2)(3). Two additional genes, blaR1 and blaR2, are also involved in the induction of the ␤-lactamase synthesis (4,5). The blaP, blaI, and blaR1 genes are clustered in a divergeon (bla divergeon) in which blaI and blaR1 form an operon (Fig.…”

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confidence: 99%

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

Filée

Vreuls

Herman

et al. 2003

Journal of Biological Chemistry

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In the absence of penicillin, the ␤-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 M by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the ␤-lactamase locus (blaP-blaIblaR1) were lower than (1.9 M) or in the same range as (75 M) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K dOP1 ‫؍‬ 1.7 ؎ 0.5 10 The blaP gene encodes the class A ␤-lactamase of Bacillus licheniformis 749/I. In the absence of ␤-lactams antibiotics, the BlaI repressor prevents the transcription of the blaP gene (1-3). Two additional genes, blaR1 and blaR2, are also involved in the induction of the ␤-lactamase synthesis (4, 5). The blaP, blaI, and blaR1 genes are clustered in a divergeon (bla divergeon) in which blaI and blaR1 form an operon (Fig. 1). The blaR1 gene encodes a transmembrane protein that acts as penicillin receptor (6 -8), and blaR2 is not identified yet and is not linked to the bla divergeon. The BlaR2 protein is essential for the inactivation of BlaI. In Staphylococcus aureus, the blaZ and mecA genes, encoding respectively a ␤-lactamase and the low affinity penicillinbinding protein 2Ј, are regulated by similar elements (9, 10).The blaI gene encodes a 128-residue protein characterized by two functional and separate domains (11). The DNA-binding domain, a helix-turn-helix recognition motif, is located in the N-terminal region, and the dimerization domain is in the Cterminal region. After deletion of the C-terminal domain, the repressor becomes unable to dimerize and to form a stable complex with its operators. DNase footprinting experiments and filter binding reactions revealed the presence of three regulatory regions that are recognized specifically by BlaI (11). These operators present a 23-bp-long dyad symmetry and show the deduced 5Ј-AAAGTATTACATATGTAACNTTT-3Ј consensus sequence (Fig. 1).In the presence of ␤-lactam antibiotics, the C-terminal domain of BlaR1 is acylated (6, 7). This event triggers the activation of the putative cytoplasmic metalloprotease motif of BlaR1 by self-proteolysis (8, 12). In S. aureus, the ␤-lactamase induction correlates with BlaI proteolysis between residues Asn 101 and Phe 102 , and it is postulated that the activated form of BlaR1 proteolyses BlaI (12-...

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“…The portion of the gene encoding the mature PBP4a was cloned in phase with the signal sequence of the Bacillus licheniformis 749i ␤-lactamase under the control of the B. licheniformis blaP promoter (15,18). The resulting vector was first cloned in E. coli TG1, isolated, and used to transform B. subtilis 1A751, which was grown in LB medium for 16 h at 37°C.…”

Section: Resultsmentioning

confidence: 99%

Purification and Characterization of PBP4a, a New Low-Molecular-Weight Penicillin-Binding Protein from Bacillus subtilis

et al. 2001

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Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781-788, 1992), which is rapidly inactivated by many ␤-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k 2 /K) for the acylation of the essential serine by benzylpenicillin is 300,000 M ؊1 s ؊1 for the Actinomadura sp. strain R39 peptidase, 1,400 M ؊1 s ؊1 for B. subtilis PBP4a, and 7,000 M ؊1 s ؊1 for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k 2 /K ‫؍‬ 46,000 M ؊1 s ؊1 ). PBP4a is also much more thermostable than the R39 enzyme.The Bacillus subtilis genome (16) contains a gene that encodes a putative 491-residue protein with sequence similarities to low-molecular-weight penicillin-binding proteins (PBPs). Successively referred to as pbp (25) and dacC (19), this gene was overexpressed in Escherichia coli by Pedersen et al. (19), who showed that dacC does indeed encode a membrane-bound PBP migrating between PBP4 and PBP5 of B. subtilis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein is now referred to as PBP4a. The phenotypic effect of dacC mutations and the role of PBP4a in the synthesis of the B. subtilis spore peptidoglycan and its influence on the properties of the spore were investigated, but no physiological role or enzymatic activity could be assigned to the protein (20).As already reported for Actinomadura sp. strain R39 PBP4 (in short, the R39 DD-peptidase) (14), E. coli PBP4 (17), and a Haemophilus influenzae putative PBP (4), the B. subtilis PBP4a possesses a large insert (175 residues) between the first and second active-site-defining motifs, and it can therefore be classified as a class C low-molecular-weight PBP (14). Importantly, although preliminary X-ray data for E. coli PBP4 are available (24), no three-dimensional structure of a protein belonging to this class of proteins has been presently established.In this work, we describe the overexpression of PBP4a in E. coli, its purification, and its kinetic characterization as an in vitro DD-carboxypeptidase. Values for the parameter of inactivation (k 2 /K, the second-order rate constant characterizing the acylation reaction) by some penicillins and cephalosporins were also determined and compared to those for the R39 DD-peptidase, to which PBP4a has the highest degree of sequence similarity (46% identity and 61% similarity) (8, 14). MATERIALS AND METHODSRecombinant DNA techniques, bacterial strains, plasmids, and growth conditions. B. subtilis 168 1A1 (Genetic Stock Center) was grown at 37°C in LuriaBertani (LB) medium, and its genomic DNA was extracted with the Qiagen genomic tip 20/G kit. (Westburg, ...

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A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis

Cited by 79 publications

References 39 publications

Type I Signal Peptidase and Protein Secretion inStaphylococcus epidermidis

Type I Signal Peptidase and Protein Secretion inStaphylococcus epidermidis

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

Purification and Characterization of PBP4a, a New Low-Molecular-Weight Penicillin-Binding Protein from Bacillus subtilis

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