Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli.
(1/1221)
Enlargement of the stress-bearing murein sacculus of bacteria depends on the coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chromatography and surface plasmon resonance (SPR) studies were performed. The membrane-bound lytic transglycosylase MltA when covalently linked to CNBr-activated Sepharose specifically retained the penicillin-binding proteins (PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escherichia coli. In the presence of periplasmic proteins also PBP1A was specifically bound. At least five different non-PBPs showed specificity for MltA-Sepharose. The amino-terminal amino acid sequence of one of these proteins could be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltA-interacting protein, named MipA, in addition binds to PBP1B, a bifunctional murein transglycosylase/transpeptidase. SPR studies with PBP1B immobilized to ampicillin-coated sensor chips showed an oligomerization of PBP1B that may indicate a dimerization. Simultaneous application of MipA and MltA onto a PBP1B sensor chip surface resulted in the formation of a trimeric complex. The dissociation constant was determined to be about 10(-6) M. The formation of a complex between a murein polymerase (PBP1B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzyme, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli. (+info)
Transcription of the pcbAB, pcbC and penDE genes of Penicillium chrysogenum AS-P-78 is repressed by glucose and the repression is not reversed by alkaline pHs.
(2/1221)
Glucose repressed transcription of the penicillin biosynthesis genes pcbAB, pcbC and penDE when added at inoculation time to cultures of Penicillium chrysogenum AS-P-78 but it had little repressive effect when added at 12 h and no effect when added at 24 or 36 h. A slight increase in the expression of pcbC and penDE (and to a smaller extent of pcbAB) was observed in glucose-grown cultures at pH 6.8, 7.4 and 8.0 as compared with pH 6.2, but alkaline pHs did not override the strong repression exerted by glucose. Transcription of the actin gene used as control was not significantly affected by glucose or alkaline pHs. Repression by glucose of the three penicillin biosynthetic genes was also observed using the lacZ reporter gene coupled to each of the three promoters in monocopy transformants with the constructions integrated at the pyrG locus. Glucose repression of the three genes encoding enzymes of penicillin biosynthesis therefore appears to be exerted by a regulatory mechanism independent from pH regulation. (+info)
Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis.
(3/1221)
Genes required for natural transformation of Haemophilus influenzae Rd were identified by a cassette mutagenesis protocol consisting of the following steps: random insertional mutagenesis, phenotypic screening, sequencing of genome sequence tags from the DNA flanking the insertion in the selected mutants and comparison of genome sequence tags to genomic sequence data. The cassette mutagenesis screen for transformation genes resulted in five distinct mutant classes, two of which have been identified in previous studies. Insertions in the three newly identified loci interrupted genes with predicted protein products homologous to a type IV pilin-like protein biogenesis operon, drug-efflux transporters and a phospholipid-biosynthesis enzyme. The most significant finding of this screen is the requirement for type IV pilin-like proteins in genetic transformation of H. influenzae. These surface structures are utilized for DNA uptake in a number of Gram-positive and Gram-negative bacteria, and appear to be the common component among the systems for DNA binding. (+info)
Penicillin-binding protein-mediated resistance in pneumococci and staphylococci.
(4/1221)
Target alteration underlies resistance to beta-lactam antibiotics in both Staphylococcus species and Streptococcus pneumoniae. The penicillin-binding protein (PBP) targets in penicillin-resistant strains of S. pneumoniae are modified, low-binding-affinity versions of the native PBPs. Multiple PBP targets may be modified by transformation and homologous recombination with DNA from PBP genes of viridans streptococci. The level of resistance is determined by how many and to what extent targets are modified. In contrast, methicillin resistance in staphylococci is due to expression of PBP 2a, a novel, low-affinity PBP for which there is no homologue in methicillin-susceptible strains. PBP 2a is encoded by mecA, a highly conserved gene most likely acquired by a rare transposition from Staphylococcus sciuri or a closely related ancestor. Expression of resistance can be highly variable, but this seems not to be determined by PBP modifications. Several non-PBP factors are required for high-level resistance. (+info)
Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus.
(5/1221)
The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates. (+info)
Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated.
(6/1221)
Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain. (+info)
Disulfide bridges are not involved in penicillin-binding protein 1b dimerization in Escherichia coli.
(7/1221)
PBP1b can be found as a dimer in Escherichia coli. Previous results suggested that dimerization involved the cysteine(s) in an intermolecular disulfide bond. We show that either deletion mutants or a mutant without cysteines is fully active and still binds penicillin and that the latter can also form dimers. (+info)
BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins.
(8/1221)
We describe a new, sensitive, rapid, and nonradioactive method involving the use of the commercially available BOCILLIN FL, a fluorescent penicillin, as a labeling reagent for the detection and study of penicillin-binding proteins (PBPs). This method allowed rapid detection of 30 ng of a purified PBP protein under UV light and of 2 to 4 ng of the protein with the aid of a FluorImager. This method also allowed rapid determination of the PBP profiles of Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae. The PBP profiles obtained are virtually identical to those reported previously with 3H-, 14C-, or 125I-labeled penicillin. Using this method enabled us to determine the 50% inhibitory concentrations of the penicillin-sensitive and -resistant PBP2x proteins of S. pneumoniae for penicillin G, thereby allowing a direct evaluation of their relative affinities for penicillin G. Finally, this method also allowed us to compare relative affinities of a PBP2x protein for different beta-lactam antibiotics with the aid of fluorescence polarization technology and to monitor a PBP2x protein during purification. (+info)