Biomedical subjects

B G Spratt

Publications and source records attributed to B G Spratt.

At least 19 recordsLinked to original sources

PubMed1992

Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species.

The two pathogenic species of Neisseria, N. meningitidis and N. gonorrhoeae, have evolved resistance to penicillin by alterations in chromosomal genes encoding the high molecular weight penicillin-binding proteins, or PBPs. The PBP 2 gene (penA) has been sequenced from over 20 Neisseria isolates, including susceptible and resistant strains of the two pathogenic species, and five human commensal species. The genes from penicillin-susceptible strains of N. meningitidis and N. gonorrhoeae are very uniform, whereas those from penicillin-resistant strains consist of a mosaic of regions resembling those in susceptible strains of the same species, interspersed with regions resembling those in one, or in some cases, two of the commensal species. The mosaic structure is interpreted as having arisen from the horizontal transfer, by genetic transformation, of blocks of DNA, usually of a few hundred base pairs. The commensal species identified as donors in these interspecies recombinational events (N. flavescens and N. cinerea) are intrinsically more resistant to penicillin than typical isolates of the pathogenic species. Transformation has apparently provided N. meningitidis and N. gonorrhoeae with a mechanism by which they can obtain increased resistance to penicillin by replacing their penA genes (or the relevant parts of them) with the penA genes of related species that fortuitously produce forms of PBP 2 that are less susceptible to inhibition by the antibiotic. The ends of the diverged blocks of DNA in the penA genes of different penicillin-resistant strains are located at the same position more often than would be the case if they represent independent crossovers at random points along the gene. Some of these common crossover points may represent common ancestry, but reasons are given for thinking that some may represent independent events occurring at recombinational hotspots.

Base Sequence↗

PubMed1992

Genetic diversity of penicillin-resistant Neisseria meningitidis.

The genetic relatedness of 42 penicillin-resistant Neisseria meningitidis isolates obtained during a 2-year period from a single hospital was studied by multilocus enzyme electrophoresis and by restriction fragment length polymorphism (RFLP) analysis of penicillin-binding protein (PBP) 2 genes. The PBP 2 genes of 7 susceptible strains gave identical RFLP profiles. Sixteen different PBP 2 RFLP profiles were found among the 42 resistant strains, but 4 were found in greater than 1 resistant isolate. Multilocus enzyme electrophoresis revealed a high level of genetic diversity. Four clusters of resistant strains could be distinguished at a genetic distance of 0.75. Resistant strains with the most common PBP 2 RFLP profile were restricted to one of these clusters and may be derived from a common ancestral strain. However, resistant strains with the 3 other common RFLP profiles were distributed in two or more of the clusters.

Adolescent↗

PubMed1992

Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene.

Studies of natural populations of Neisseria meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0-0.6% for fbp, 0-1.3% for argF, and 0-3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli 'housekeeping' genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcal argF gene: two regions of the gene differed by greater than 13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by less than 1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.

Amino Acid Sequence↗

PubMed1992

Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae.

Resistance to third-generation cephalosporins in a clinical isolate of Streptococcus pneumoniae was shown to be due to the production of altered forms of penicillin-binding proteins (PBPs) 2X and 1A. The cloned PBP2X gene from the resistant strain was able to transform a susceptible strain to an intermediate level of resistance. The resulting transformant could be transformed to the full level of resistance of the clinical isolate using the cloned PBP1A gene from the latter strain. Chromosomal DNA from the resistant strain (and from other resistant strains) could readily transform a susceptible strain to the full level of resistance to third-generation cephalosporins (greater than 250-fold for cefotaxime; greater than 100-fold for ceftriaxone) in a single step (transformation frequency of about 10(-5)). The resistant transformants obtained with chromosomal DNA were shown by gene fingerprinting to have gained both the PBP1A and PBP2X genes from the DNA donor.

Bacterial Proteins↗

PubMed1991

Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins.

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.

Acyltransferases↗

PubMed1991

The mutation Lys234His yields a class A beta-lactamase with a novel pH-dependence.

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

Ampicillin↗

PubMed1991

Localized sex in bacteria.

Electrophoretic studies suggest that bacterial populations consist of a number of independent clones and that genetic recombination is rare in nature. But DNA sequencing reveals that individual bacterial genes have a mosaic structure that could have arisen only by recombination. How can these observations be reconciled and what is their relevance to prokaryotic evolution?

Bacteria↗

PubMed1991

Intercontinental spread of a multiresistant clone of serotype 23F Streptococcus pneumoniae.

Isolates of serotype 23F Streptococcus pneumoniae with high levels of resistance of penicillin have been commonly recovered in Spain for more than a decade. Recently penicillin-resistant serotype 23F S. pneumoniae strains were also isolated from children attending a day-care center in Cleveland. A number of Spanish and Cleveland isolates were compared by electrophoretic analysis of penicillin-binding protein (PBP) profiles and DNA restriction endonuclease cleavage profiles of the PBP 2X and 2B genes amplified with the polymerase chain reaction and by multilocus enzyme electrophoresis. All strains were identical by these criteria. The findings demonstrate that the Spanish and Cleveland isolates are clonally related and suggest that this antibiotic resistant clone of serotype 23F S. pneumoniae has spread intercontinentally from Spain to the United States.

Bacterial Proteins↗

PubMed1991

Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae.

Penicillin resistance in pneumococci is due to the appearance of high molecular-weight penicillin-binding proteins (PBPs) that have reduced affinity for the antibiotic. We have compared the PBX 2x genes (pbpX) of one penicillin-susceptible and five penicillin-resistant clinical isolates of Streptococcus pneumoniae isolated from various parts of the world. All of the resistant isolates contained a low-affinity form of PBP 2x. The 2 kb region of the two penicillin-susceptible isolates differed at only eight nucleotide sites (0.4%) and resulted in one single amino acid difference in PBP 2x. In contrast, the sequences of the PBP 2x genes from the resistant isolates differed overall from those of the susceptible isolates at between 7 and 18% of nucleotide sites and resulted in between 27 and 86 amino acid substitutions in PBP 2x. The altered PBP 2x genes consisted of regions that were similar to those of susceptible strains (less than 3% diverged), alternating with regions that were very different (18-23% diverged). The presence of highly diverged regions within the PBP 2x genes of the resistant isolates contrasts with the uniformity of the sequences of the amylomaltase genes from the same isolates, and with the uniformity of the PBP 2x genes in the two susceptible isolates. It suggests that the altered PBP 2x genes have arisen by localized interspecies recombinational events involving the PBP 2x genes of closely related streptococci, as has been suggested to occur for altered PBP 2b genes (Dowson et al., 1989b). The PBP 2x genes from the resistant isolates could transform the susceptible strain R6 to increased levels of resistance to beta-lactam antibiotics, indicating that the altered forms of PBP 2x in the resistant isolates contribute to their resistance to penicillin.

Amino Acid Sequence↗

PubMed1991

Horizontal transfer of multiple penicillin-binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae.

Multiply antibiotic-resistant serotype 23F isolates of Streptococcus pneumoniae are prevalent in Spain and have also been recovered recently in the United Kingdom and the United States. Analysis of populations of these isolates by multilocus enzyme electrophoresis, and restriction endonuclease cleavage electrophoretic profiling of penicillin-binding protein (PBP) genes, has demonstrated that these isolates are a single clone (Muñoz et al., 1991). Here we report studies of non-serotype 23F penicillin-resistant pneumococci isolated in Spain and the United Kingdom. One of the isolates expressed serotype 19 capsule but was otherwise indistinguishable from the serotype 23F clone on the basis of multilocus enzyme electrophoresis, antibiotic resistance profiling, and restriction endonuclease patterns of genes encoding PBP1A, PBP2B and PBP2X, a result which suggests that horizontal transfer of capsular biosynthesis genes had occurred. These same techniques revealed that six other resistant isolates, all expressing serotype 9 polysaccharide capsule, represent a clone. Interestingly, the chromosomal lineage of this clone is not closely related to the 23F clone; however, the serotype 9 and 23F clones harbour apparently identical PBP1A, -2B and -2X genes. To explain these data, we favour the interpretation that horizontal gene transfer in natural populations has distributed genes encoding altered forms of PBP1A, -2B and -2X to distinct evolutionary lineages of S. pneumoniae.

Alleles↗

PubMed1991

Penicillin-resistant isolates of Neisseria lactamica produce altered forms of penicillin-binding protein 2 that arose by interspecies horizontal gene transfer.

Isolates of Neisseria lactamica that have increased resistance to penicillin have emerged in recent years. Resistance to penicillin was shown to be due to the production of altered forms of penicillin-binding protein 2 (PBP 2) that have reduced affinity for the antibiotic. The sequences of the PBP 2 genes (penA) from two penicillin-resistant isolates were almost identical (less than or equal to 1% sequence divergence) to that of a penicillin-susceptible isolate, except in a 175-bp region where the resistant and susceptible isolates differed by 27%. The nucleotide sequences of these divergent regions were identical (or almost identical) to the sequence of the corresponding region of the penA gene of N. flavescens NCTC 8263. Altered forms of PBP 2 with decreased affinity for penicillin in the two penicillin-resistant isolates of N. lactamica appear, therefore, to have arisen by the replacement of part of the N. lactamica penA gene with the corresponding region from the penA gene of N. flavescens.

Amino Acid Sequence↗

PubMed1990

Insertion of an extra amino acid is the main cause of the low affinity of penicillin-binding protein 2 in penicillin-resistant strains of Neisseria gonorrhoeae.

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae (CMRNG strains) produce altered forms of penicillin-binding protein 2 (PBP2) that have decreased affinity for penicillin. A feature of PBP2 from all CMRNG strains is the presence of an additional residue (Asp-345A) that is absent from PBP2 of penicillin-sensitive strains. The role of the additional aspartic acid residue in the decreased affinity of PBP2 is unclear as PBP2 of all previously examined CMRNG strains possess several other amino acid sequence alterations, in addition to the insertion of Asp-345A, compared to PBP2 of penicillin-sensitive strains. Site-directed mutagenesis has been used to insert the Asp-345A codon into the penA gene from a penicillin-sensitive gonococcus. The resulting penA gene expressed an altered form of PBP2 that had a decreased affinity for benzylpenicillin and was able to transform a penicillin-sensitive strain of N. gonorrhoeae to an increased level of resistance to benzylpenicillin. Insertion of amino acids other than aspartic acid did not produce forms of PBP2 that provided increased resistance to penicillin. Removal of the Asp-345A codon from the penA gene of a CMRNG strain reduced its ability to transform a penicillin-sensitive strain to an increased level of penicillin resistance. The reduction in the affinity of PBP2 in CMRNG strains is therefore largely, although not exclusively, due to the insertion of Asp-345A. Clinical isolates that produce altered forms of PBP2 that differ from that of penicillin-sensitive strains only in the insertion of Asp-345A have been identified.

Amino Acid Sequence↗

PubMed1990

Genetic diversity of penicillin-binding protein 2 genes of penicillin-resistant strains of Neisseria meningitidis revealed by fingerprinting of amplified DNA.

A 2-kilobase fragment containing the penicillin-binding protein 2 gene (penA) was amplified by using the polymerase chain reaction with DNA prepared from 35 penicillin-resistant strains of Neisseria meningitidis isolated in England, Ireland, and Spain (MICs, 0.16 to 1.28 micrograms of benzylpenicillin per ml) and from 10 penicillin-susceptible strains (MICs, less than or equal to 0.04 micrograms of benzylpenicillin per ml). The penA genes were digested with HpaII or TaqYI; and the resulting fragments were end-labeled, fractionated on a polyacrylamide gel, and autoradiographed, to produce gene fingerprints. The fingerprints of the penA genes of the 10 penicillin-susceptible strains were identical. In contrast, the fingerprints of the penA genes of all but two of the penicillin-resistant strains differed markedly from those of the penicillin-susceptible strains. The altered penA genes of group B penicillin-resistant strains were very diverse, and 14 different fingerprints were found among the 15 strains that we examined. The penA genes of the 19 resistant group C strains were less diverse, and two major fingerprint patterns predominated.

Bacterial Proteins↗

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↗

PubMed1978