Identification of penicillin-binding protein 5a of Bacillus megaterium KM as a DD-carboxypeptidase.

Measurement of the stabilities of DD-carboxypeptidase activity and the penicillin-binding activity of proteins 5 and 5a in membranes isolated from vegetative cells and stage-V forespores suggests that the unique sporulation-specific protein 5a may be a penicillin-sensitive DD-carboxypeptidase.     

Carnosine reacts with a glycated protein

Oxidation and glycation induce formation of carbonyl (CO) groups in proteins, a characteristic of cellular aging. The dipeptide carnosine (beta-alanyl-L-histidine) is often found in long-lived mammalian tissues at relatively high concentrations (up to 20 mM). Previous studies show that carnosine reacts with low-molecular-weight aldehydes and ketones. We examine here the ability of carnosine to react with ovalbumin CO groups generated by treatment of the protein with methylglyoxal (MG). Incubation of MG-treated protein with carnosine accelerated a slow decline in CO groups as measured by dinitrophenylhydrazine reactivity. Incubation of [(14)C]-carnosine with MG-treated ovalbumin resulted in a radiolabeled precipitate on addition of trichloroacetic acid (TCA); this was not observed with control, untreated protein. The presence of lysine or N-(alpha)-acetylglycyl-lysine methyl ester caused a decrease in the TCA-precipitable radiolabel. Carnosine also inhibited cross-linking of the MG-treated ovalbumin to lysine and normal, untreated alpha-crystallin. We conclude that carnosine can react with protein CO groups (termed "carnosinylation") and thereby modulate their deleterious interaction with other polypeptides. It is proposed that, should similar reactions occur intracellularly, then carnosine's known "anti-aging" actions might, at least partially, be explained by the dipeptide facilitating the inactivation/removal of deleterious proteins bearing carbonyl groups.     

Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli

We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C beta-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acid of PBP 2 was identified as His633.     

Identification of an epitope of type 1 streptococcal M protein that is shared with a 43-kDa protein of human myocardium and renal glomeruli.

The localization of opsonic and tissue-cross-reactive epitopes within the amino terminus of type 1 streptococcal M protein was investigated by using murine mAb raised against synthetic peptides of type 1 M protein. Two mAb (IIIA2 and IIIB8) reacted with epitopes located within amino acid residues 1-12 of type 1 M protein. These antibodies opsonized type 1 streptococci and did not cross-react with human kidney and heart tissue. Another mAb (IC7) reacted with mesangial cells of renal glomeruli and human myocardium. The cross-reactive epitope of mAb IC7 was localized to position 13-19, indicating that it is not the same epitope as the previously described vimentin-cross-reactive epitope at position 23-26 of type 1 M protein. In Western blots of mesangial cell and myocardial proteins, mAb IC7 cross-reacted with a 43-kDa protein. Neither vimentin nor actin inhibited the binding of mAb IC7 to the cross-reactive protein, as determined by Western blot or immunofluorescence inhibition tests. These results provide evidence that type 1 M protein contains at least one autoimmune epitope shared with both human glomeruli and myocardium.     

Synthesis of a 125I-radiolabeled penicillin for penicillin-binding proteins studies

Radioiodination of penicillin X (p-hydroxybenzylpenicillin) with 125INa, using the chloramine-T method, is simple and almost quantitative. The product thus obtained can be used without further purification for the penicillin-binding proteins (PBPs) assay. The chemical structure of 125I-penicillin X is very close to that of penicillin G, and the affinity of the two compounds for the PBPs are very similar. 125I-penicillin X can then advantageously replace [14C]penicillin G in these experiments, as its specific radioactivity is 2000 Ci/mol, in our preparations, instead of about 50 Ci/mol; thus, the experimental procedure is simplified and the autoradiography exposure time is reduced to 12-48 h.     

A novel beta-lactamase activity from a penicillin-binding protein of Treponema pallidum and why syphilis is still treatable with penicillin

Treponema pallidum, the causative agent of syphilis, is sensitive to penicillins. Yet, an abundant membrane-bound protein of this organism, Tp47, turns over penicillins. It is shown herein that the turnover process is a hydrolytic reaction that results in the corresponding penicilloates, products that have their beta-lactam bonds hydrolyzed. This is the reaction of beta-lactamases, bona fide resistance enzymes to beta-lactam antibiotics. Remarkably, the x-ray structure of Tp47 bears no resemblance to any other beta-lactamases or the related penicillin-binding proteins. Furthermore, evidence is presented that the reaction of Tp47 takes place in the absence of the zinc ion and does not involve intermediary acyl enzyme species. Hence, the beta-lactamase activity of Tp47 is the fifth known mechanism for turnover of beta-lactam antibiotics. Tp47 also exhibits a penicillin binding reaction, in the process of which the enzyme is covalently modified in the active site. The two reactions take place in two different active sites, and the events of the beta-lactamase activity are over 2,000-fold more rapid than the penicillin binding reaction. The level of beta-lactamase activity is high and is held back only by a strong product-inhibition component to the catalytic process. If natural selection would result in a mutant variant of Tp47 that overcomes product inhibition for the beta-lactamase activity, a novel bona fide resistance to penicillins will emerge in Treponema, which will be a disconcerting clinical development. The physiological functions of Tp47 are not known, but it is likely that this is at least a bifunctional enzyme involved in the processing of the Treponema peptidoglycan as a substrate.     

The bacterial outer membrane protein that reacts with anti-HLA-B27 antibodies is the OmpA protein

A bacterial outer membrane protein of 35-kDa Mr has been reported to react with several anti-HLA-B27 mAb. Here, we demonstrated that this protein showed the heat-modifiability of the OmpA protein during SDS-PAGE. Further, the protein was not detected in mutants of Escherichia coli in which the expression of the OmpA protein has been suppressed. The protein would be reexpressed when one of the mutants was transformed with an expression vector carrying the OmpA gene. Finally, the identity of the reactive protein to OmpA protein was verified by homology in amino acid sequences. An NH2-terminal fragment of this protein was generated by tryptic digestion. Inasmuch as this was unreactive with the anti-HLA-B27 antibody, we concluded that the carboxyl-terminus contributed directly or indirectly to the reactive domain.     

Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.     

Purification and characterization of the penicillin-binding protein that is the lethal target of penicillin in Bacillus megaterium and Bacillus licheniformis. Protein exchange and complex stability.

The penicillin-binding protein that is thought to be the lethal target of penicillin in Bacillus megaterium (protein 1) has been purified to greater than 95% homogeneity. The membrane-bound penicillin-binding proteins were solubilized with a non-ionic detergent and partially separated from each other by ion-exchange chromatography on DEAE-Sepharose CL-6B. Protein 1 was subsequently purified by covalent affinity chromatography on ampicillin-affinose. Bacillus licheniformis contains an equivalent penicillin-binding protein (protein 1) that can be more readily purified to virtual homogeneity in a one-step procedure. It was separated from the other penicillin-binding proteins by utilizing the observation that in this organism, this particular protein is the only one whose covalent complex with benzylpenicillin subsequently breaks down. Membranes were treated with saturating concentrations of benzylpenicillin followed by the removal of free penicillin and further incubation to allow the complex between benzylpenicillin and protein 1 to break down. The penicillin-binding proteins were then solubilized and applied to a column of ampicillin-affinose to which only protein 1 was bound as the other penicillin-binding proteins still had benzylpenicillin bound to them. Pure protein 1 was eluted from the affinity resin with hydroxylamine. The interaction of benzylpenicillin with purified protein 1 has been studied by separating unbound antibiotic from the benzylpenicillin . protein complex by paper electrophoresis. Benzylpenicillin reacts with the protein rapidly to form a covalent complex and the fully saturated complex has a molar ratio of bound [14C] benzylpenicillin: protein of 0.7:1. The complex breaks down, obeying first-order kinetics, with a half-life of 16 min at 35 degrees C, a value identical to that obtained with the membrane-bound protein. The concentration of benzylpenicillin that results in the formation of 50% of the maximum amount of benzylpenicillin . protein complex is that at which the molar amount of benzylpenicillin present is equal to 50% of the molar amount of penicillin-binding protein, rather than being a measure of any of the kinetic parameters of the binding reaction. This observation may be significant in the interpretation of previous results where the amounts of penicillins needed to kill cells or to inhibit penicillin-sensitive reactions have been expressed as concentrations. The possible importance of the breakdown of beta-lactam . protein complexes in the clinical use of these antibiotics is discussed.     

Identification of Treponema pallidum penicillin-binding proteins.

Penicillin-binding proteins of 180, 89, 80, 68, 61, 41, and 38 kilodaltons were identified in Treponema pallidum (Nichols) by their covalent binding of [35S]benzylpenicillin. Penicillin-binding proteins are localized in the plasma membranes of many bacterial species and may serve as useful markers for determining plasma membrane intactness in T. pallidum fractionation studies.     

Identification of a mouse cDNA fragment whose expressed polypeptide reacts with anti-recA antibodies.

We have previously reported the in vivo detection of a mouse nuclear protein that cross-reacts with antibodies raised against E coli recA protein. Here, we characterize monospecific anti-recA antibodies, their use for the immunological screening of a cDNA expression library and the isolation of a mouse cDNA fragment which codes for a polypeptide recognized by anti-recA antibodies. The cDNA fragment is 601 nucleotide long and was called KIN17(601). It contains an open reading frame coding for a 200 amino acid polypeptide. In kin17(200) polypeptide, there are amino acids identical to those that form one of the major antigenic determinants of recA protein. Kin17(200) polypeptide also displays a significant similarity with the helix 1 motif of several homeoproteins.     

Major peptidoglycan transglycosylase activity in Streptococcus pneumoniae that is not a penicillin-binding protein

Abstract In this communication, results support the conclusion that succinate can repress hexose-catabolizing enzymes in Rhizobium sp. 32H1 in a manner similar to catabolite repression, such as that seen in Pseudomonas aeruginosa [9,27]. Enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways were only detected in cells cultured on hexoses and were not present in succinate-grown cells. Initial enzymes of fructose and mannitol metabolism were present in cells gorwn on fructose and mannitol, respectively. NADP-linked 6-phosphogluconate dehydrogenase was not detected in cell-free extracts regardless of the carbon source.     

Identification and cloning of the gene encoding penicillin-binding protein 7 of Escherichia coli.

Penicillin-binding protein (PBP) 7 of Escherichia coli is a poorly characterized member of the family of enzymes that synthesize and modify the bacterial cell wall. The approximate chromosomal position of the gene encoding this protein was determined by measuring the expression of PBPs during lytic infection of E. coli by each of the 476 miniset members of the Kohara lambda phage genomic library. Phages lambda 363 and lambda 364, encompassing the region from 47.7 to 48 min of the chromosome, overproduced PBP 7. One open reading frame, yohB, was present on both these phages and directed the expression of PBPs 7 and 8. The predicted amino acid sequence of PBP 7 contains the consensus motifs associated with other PBPs and has a potential site near the carboxyl terminus where proteolysis by the OmpT protein could occur, creating an appropriately sized PBP 8. The PBP 7 gene (renamed pbpG) was interrupted by insertion of a kanamycin resistance gene cassette and was moved to the chromosome of E. coli. No obvious growth defects were observed, suggesting that PBP 7 is not essential for growth under normal laboratory conditions.     

Changes in peptidoglycan composition and penicillin-binding proteins in slowly growing Escherichia coli.

The composition of peptidoglycan of chemostat-grown cultures of Escherichia coli was investigated as a function of growth rate. As the generation time was lengthened from 0.8 to 13.8 h, there was a decrease in the major monomer (disaccharide tetrapeptide) and dimer (bis-disaccharide tetrapeptide), while disaccharide tripeptide moieties increased to greater than 50% of the total wall. The average chain length became much shorter; lipoprotein density tripled, and the number of unusual diaminopimelyl-diaminopimelic acid crossbridges increased fivefold. As cells grew more slowly, amounts of penicillin-binding proteins (PBPs) 1a-1b complex and 4 decreased, while amounts of PBPs 3 and the 5-6 complex increased. We propose that the chemical composition of E. coli cell walls changes with growth rate in a manner consistent with alterations in the activities of PBPs and cell shape.     

Identification of a protein that interacts with the vanilloid receptor

The vanilloid receptor (VR1 or TRPV1) is a capsaicin (CAP)-sensitive non-selective cation channel. Although its channel activity is reportedly modulated through protein-protein interactions, to date very few VR1 interacting proteins have been identified. To address this issue, a yeast two-hybrid screening technique using the C-terminus of rVR1 as bait was employed. Upon interrogation of a mouse brain library, one gene product that interacts with VR1 and is highly homologous to human eferin was found. Its interaction with VR1 was confirmed by GST-pull-down and co-immunoprecipitation. When cotransfected into HEK cells, VR1 and eferin largely colocalize. Furthermore, in rat dorsal root ganglion cells, the rat eferin homologue also colocalizes with rVR1. However, this protein had no significant effect on VR1 channel activity in response to CAP. This was determined by two-electrode recording of oocytes and whole cell recording of HEK cells that were cotransfected with VR1 and human eferin.     

Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding.

All penicillin-binding proteins (PBPs) contain a conserved box of homology in the carboxyl-terminal half of their primary sequence that can be Lys-Thr-Gly, Lys-Ser-Gly, or His-Thr-Gly. Site-saturation mutagenesis was used to address the role of the lysine residue at this position (Lys213) in Escherichia coli PBP 5, a D-alanine carboxypeptidase enzyme. A soluble form of PBP 5 was used to replace Lys213 with 18 other amino acids, and the ability of these mutant proteins to bind [3H]penicillin G was assessed. Only the substitution of lysine with arginine resulted in a protein that was capable of forming a stable covalent complex with antibiotic. The affinity of [14C]penicillin G for the arginine mutant was 1.2-fold higher than for wild-type PBP 5 (4.4 versus 5.1 micrograms/ml for 20 min at 30 degrees C), and both proteins showed identical rates of hydrolysis of the [14C]penicilloyl-bound complex (t1/2 = 9.1 min). Surprisingly, the arginine-substituted protein was unable to catalyze D-alanine carboxypeptidase activity in vitro, which suggests that there is a substantial difference in the geometries of the peptide substrate and penicillin G within the active site of PBP 5.     

A streptococcal penicillin-binding protein is critical for resisting innate airway defenses in the neonatal lung

Group B streptococcus (GBS) is a major cause of neonatal pneumonia. The early interactions between innate airway defenses and this pathogen are likely to be a critical factor in determining the outcome for the host. The surface-localized penicillin-binding protein (PBP)1a, encoded by ponA, is known to be an important virulence trait in a sepsis model of GBS infection that promotes resistance to neutrophil killing and more specifically to neutrophil antimicrobial peptides (AMPs). In this study, we used an aerosolization model to explore the role of PBP1a in evasion of innate immune defenses in the neonatal lung. The ponA mutant strain was cleared more rapidly from the lungs of neonatal rat pups compared with the wild-type strain, which could be linked to a survival defect in the presence of alveolar macrophages (AM). Rat AM were found to secrete beta-defensin and cathelicidin AMP homologues, and the GBS ponA mutant was more susceptible than the wild-type strain to killing by these peptides in vitro. Collectively, our observations suggest that PBP1a-mediated resistance to AM AMPs promotes the survival of GBS in the neonatal lung. Additionally, AM are traditionally thought to clear bacteria through phagocytic uptake; our data indicate that secretion of AMPs may also participate in limiting bacterial replication in the airway.     

Identification of a new mutation in Escherichia coli that suppresses a pbpB (Ts) phenotype in the presence of penicillin-binding protein 1B

Analysis of the functional role of penicillin-binding protein 1B (PBP1B) of Escherichia coli led us to find a new mutation able to suppress thermosensitive growth of the pbpB2158(Ts) mutant strain, which harbors a thermosensitive PBP3 protein only in the presence of a ponB+ background. The mutation, originally isolated in a strain with a high dosage of PBP1B, could also suppress the pbpB(Ts) phenotype when a single copy of the ponB gene was introduced. These results clearly give further support to the implication of PPB1B in the septation process in Escherichia coli.