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B G Spratt

Publications and source records attributed to B G Spratt.

At least 91 records · Page 5Linked to original sources
PubMed1990

Mapping of conformational epitopes of monoclonal antibodies against Escherichia coli penicillin-binding protein 1B (PBP 1B) by means of hybrid protein analysis: implications for the tertiary structure of PBP 1B.

We have analyzed the location of the epitope areas of the four monoclonal antibody groups against penicillin-binding protein 1B (PBP 1B; T. den Blaauwen, F. B. Wientjes, A. H. J. Kolk, B. G. Spratt, and N. Nanninga, J. Bacteriol. 171:1393-1401). They could be specified by studying monoclonal antibody binding patterns to amino- and carboxy-terminal truncated PBP 1B molecules. Monoclonal antibodies against conformational epitopes, with the exception of one epitope area, did not recognize PBP 1B molecules that had not been translocated across the membrane. Apparently, translocation is required for PBP 1B to fully obtain its native conformation.

Antibodies, Monoclonal↗
PubMed1989

Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae.

Resistance to penicillin in clinical isolates of Streptococcus pneumoniae has occurred by the development of altered penicillin-binding proteins (PBPs) that have greatly decreased affinity for the antibiotic. We have investigated the origins of penicillin-resistant strains by comparing the sequences of the transpeptidase domain of PBP2B from 6 penicillin-sensitive and 14 penicillin-resistant strains. In addition we have sequenced part of the amylomaltase gene from 2 of the sensitive and 6 of the resistant strains. The sequences of the amylomaltase gene of all of the strains and of the PBP2B gene of the penicillin-sensitive strain show that S. pneumoniae is genetically very uniform. In contrast the PBP2B genes of the penicillin-resistant strains show approximately equal to 14% sequence divergence from those of the penicillin-sensitive strains and the development of penicillin resistance has involved the replacement, presumably by transformation, of the original PBP2B gene by a homologous gene from an unknown source. This genetic event has occurred on at least two occasions, involving different sources, to produce the two classes of altered PBP2B genes found in penicillin-resistant strains of S. pneumoniae. There is considerable variation among the PBP2B genes of the resistant strains that may have arisen by secondary transformation events accompanied by mismatch repair subsequent to their original introductions into S. pneumoniae.

Aminoacyltransferases↗
PubMed1989

Recruitment of a penicillin-binding protein gene from Neisseria flavescens during the emergence of penicillin resistance in Neisseria meningitidis.

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria meningitidis produce altered forms of penicillin-binding protein 2 that have decreased affinity for penicillin. The sequence of the penicillin-binding protein 2 gene (penA) from a penicillin-resistant strain of N. meningitidis was compared to the sequence of the same gene from penicillin-sensitive strains and from penicillin-sensitive and penicillin-resistant strains of Neisseria gonorrhoeae. The penA genes from penicillin-sensitive strains of N. gonorrhoeae and N. meningitidis were 98% identical. The gene from the penicillin-resistant strain of N. meningitidis consisted of regions that were almost identical to the corresponding regions in the penicillin-sensitive strains (less than 0.2% divergence) and two regions that were very different from them (approximately 22% divergence). The two blocks of altered sequence have arisen by the replacement of meningococcal sequences with the corresponding regions from the penA gene of Neisseria flavescens and result in an altered form of penicillin-binding protein 2 that contains 44 amino acid substitutions and 1 amino acid insertion compared to penicillin-binding protein 2 of penicillin-sensitive strains of N. meningitidis. A similar introduction of part of the penA gene of N. flavescens, or a very similar commensal Neisseria species, appears to have occurred independently during the development of altered penA genes in non-beta-lactamase-producing penicillin-resistant strains of N. gonorrhoeae.

Acyltransferases↗
PubMed1989

Penicillin-binding protein 2 genes of non-beta-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae.

Oligonucleotides that correspond to regions of the penicillin-binding protein 2 gene (penA) that differ between penicillin-sensitive and penicillin-resistant strains have been used as probes to classify the penA genes in a collection of penicillin-resistant gonococci isolated in Britain. 44/47 of those gonococcal strains that had minimal inhibitory concentrations of greater than or equal to 0.25 microgram benzylpenicillin per ml contained extensively altered penA genes which appeared to be very similar (or identical) to one or other of the two classes of altered penA genes that have been described previously. Since these two classes of altered penA genes are related, it appears that the great majority of the altered penA genes on non-beta-lactamase-producing penicillin-resistant gonococci have a clonal origin. The other three penicillin-resistant strains had altered penA genes that were different to those described previously. A crucial step in the development of the altered forms of PBP2 with decreased affinity for penicillin appears to have been the insertion of an extra codon within the transpeptidase domain of the penA gene. This insertion was found in the penA gene of all gonococci with minimal inhibitory concentrations of greater than 0.016 microgram benzylpenicillin per ml but was not found in any strains with minimal inhibitory concentrations of less than or equal to 0.016 microgram per ml.

Acyltransferases↗
PubMed1989

Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae.

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (greater than 1000-fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin-binding protein 2B from three penicillin-sensitive strains of S. pneumoniae and from a penicillin-resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin-sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin-resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the 'penicillin-resistant' form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin-binding site.

Acyltransferases↗
PubMed1989

A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli.

The feasibility of using a beta-lactamase fusion approach for maximizing the levels of periplasmic or membrane-bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM beta-lactamase was fused after the signal peptide and aminoterminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3* gene of the unmutagenized beta-lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a beta-lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.

Biological Transport↗
PubMed1989

Membrane topology of penicillin-binding protein 3 of Escherichia coli.

The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.

Acyltransferases↗
PubMed1989

Preparation and characterization of monoclonal antibodies against native membrane-bound penicillin-binding protein 1B of Escherichia coli.

We prepared monoclonal antibodies against penicillin-binding protein 1B (PBP 1B) of Escherichia coli to study the membrane topology, spatial organization, and enzyme activities of this protein. The majority of the antibodies derived with PBP 1B as the immunogen reacted against the carboxy terminus. To obtain monoclonal antibodies recognizing other epitopes, we used PBP 1B lacking the immunodominant carboxy-terminal 65 amino acids as the immunogen. Eighteen monoclonal antibodies directed against membrane-bound PBP 1B were isolated and characterized. The epitopes recognized by those monoclonal antibodies were located with various truncated forms of PBP 1B. We could distinguish four different epitope areas located on different parts of the molecule. Interestingly, we could not isolate monoclonal antibodies against the amino terminus, although they were specifically selected for. This is attributed to its predicted extreme hydrophilicity and flexibility, which could make the amino terminus very sensitive to proteolytic degradation. All antibodies reacted against native PBP 1B in a dot-blot immunobinding assay. One monoclonal antibody also recognized PBP 1B in a completely sodium dodecyl sulfate-denatured form. This suggests that all the other monoclonal antibodies recognize conformational epitopes. These properties make the monoclonal antibodies suitable tools for further studies.

Antibodies, Monoclonal↗
PubMed1988

Hybrid penicillin-binding proteins in penicillin-resistant strains of Neisseria gonorrhoeae.

Benzylpenicillin has been used extensively for approximately 40 years in the treatment of gonorrhoea. The intense selective pressures resulting from the continual exposure of Neisseria gonorrhoeae to penicillin have resulted in the emergence of resistant strains that produce altered forms of penicillin-binding proteins (PBPs) with decreased affinity for the antibiotic. A comparison of the sequences of the PBP-2 genes from penicillin-sensitive and penicillin-resistant strains, suggests that penicillin-resistant forms of PBP 2 may have arisen both by amino-acid substitutions and insertions, and by the exchange of a region encoding part of the penicillin-sensitive transpeptidase domain with the homologous region from a closely related species.

Acyltransferases↗
PubMed1988

An amino acid substitution in penicillin-binding protein 3 creates pointed polar caps in Escherichia coli.

The pbpB gene product penicillin-binding protein 3 (PBP3) of Escherichia coli is one of the major targets of beta-lactam antibiotics. At the permissive temperature, the temperature-sensitive pbpBr1 mutant, which was obtained after selection for increased resistance to cephalexin, shows a dramatic change in shape which has never been observed before; the polar caps are pointed. We show that the substitution of amino acid Asn-361 by Ser, previously shown to be responsible for increased cephalexin resistance and for temperature sensitivity, causes the pointed polar caps. However, comparison of the morphological and physiological characteristics of the pbpBr1 mutant with those of other pbpB mutants suggests that the formation of pointed polar caps is not correlated with temperature sensitivity or cephalexin resistance. Partial inactivation of PBP3 by subinhibitory concentrations of cephalexin, furazlocillin, and piperacillin resulted in the formation of slightly pointed polar caps, suggesting that the shape of the polar caps is correlated with PBP3 activity. The large change in the shape of the polar caps was accompanied by a small change in the kinetics of peptidoglycan synthesis and in the local rate of surface synthesis activity along the cell envelope.

Acyltransferases↗
PubMed1987

Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli.

The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.

Bacterial Proteins↗
PubMed1986

Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein.

Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent.

Bacterial Proteins↗
PubMed1986

Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9.

Analogues of the cloning vectors pUC8, pUC9, pEMBL8 +/- and pEMBL9 +/- that have kanamycin resistance (KmR) instead of ampicillin resistance (ApR) as the selectable marker have been developed. HindIII and SmaI sites within the KmR gene have been removed so that all of the cloning sites in the multi-linker region of these plasmids may be used except the AccI site.

Ampicillin↗
PubMed1986

A vector for the construction of translational fusions to TEM beta-lactamase and the analysis of protein export signals and membrane protein topology.

A plasmid vector, pJBS633, that facilitates the construction of translational fusions of genes of interest to the coding region of the mature form of TEM beta-lactamase has been developed. Transformants containing in-frame fusions can be identified by their ability to grow when plated at high inocula on agar containing ampicillin (Ap). The cellular location of the beta-lactamase moiety of the fusion proteins can then be determined since only those that direct the translocation of the beta-lactamase across the cytoplasmic membrane to the periplasm result in the ability of individual cells of Escherichia coli to form isolated colonies in the presence of Ap. Conversely, those fusion proteins in which the beta-lactamase moiety remains cytoplasmic do not protect individual cells against Ap. Transformants expressing the latter class of fusion proteins can, however, be identified when plated at high inocula since, as cells start to lyse, the cytoplasmic beta-lactamase activity is released and provides Ap resistance to the surrounding cells. The vector contains the origin of replication of f1 phage so that single-stranded plasmid DNA can be obtained in the appropriate orientation to allow sequencing across the fusion junction using a universal primer complementary to the start of the coding region of mature TEM beta-lactamase. pJBS633 should be useful as a general vector for the construction of beta-lactamase fusions and, in particular, for the analysis of protein export signals and the determination of the organisation of proteins in the E. coli cytoplasmic membrane.

Bacterial Proteins↗
PubMed1986

Interaction between membrane proteins PBP3 and rodA is required for normal cell shape and division in Escherichia coli.

In Escherichia coli, the products of several genes are required for septation, and the products of several others are required for the maintenance of the rod shape of the cells. We show here that the combination of certain mutations in a division gene (ftsI) with a specific mutation in one of the shape genes (rodA) could produce cells with normal shape and division, although separately these mutations led to a loss of the capacity to divide (ftsI) or to form normal rod-shaped cells (rodA). In contrast, combinations between other mutant alleles of these genes produced double mutants which had lost the capacity both to divide and to form rod-shaped cells. The mutual phenotypic correction observed within particular pairs of mutant genes suggests that the normal morphogenetic cycle of growth and division may require direct interaction between the two membrane proteins which are the products of these genes.

Bacterial Proteins↗