PubMed Health⌕ Search

Biomedical subjects

T J Foster

Publications and source records attributed to T J Foster.

At least 91 records · Page 5Linked to original sources
PubMed1988

Molecular cloning and genetic analysis of the determinant for gamma-lysin, a two-component toxin of Staphylococcus aureus.

The gamma-lysin determinant of Staphylococcus aureus strain Smith 5R has been cloned in phage lambda and plasmid vectors in Escherichia coli. Genetic evidence is presented which demonstrates that gamma-lysin requires the co-operative action of two polypeptides expressed by the closely linked hlgA and hlgB genes. Recombinants expressed haemolytic activity in agarose medium but not in agar, a known property of gamma-lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of gamma-lysin, but not by anti-alpha-, anti-beta- or anti-delta-lysin serum. Subcloning and transposon Tn5 mutagenesis identified a 3.5 kb region which was necessary for gamma-lysin expression in E. coli. Two genes (hlgA and hlgB) were mapped and their polypeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of gamma-lysin purified from S. aureus culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of hlgA) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the hlgB gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the S. aureus culture supernate containing component B of gamma-lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the hlgA and hlgB genes are closely linked in the chromosome of several strains of S. aureus.

Bacterial Proteins↗
PubMed1988

Genetic analysis of gentamicin resistance in methicillin- and gentamicin-resistant strains of Staphylococcus aureus isolated in Dublin hospitals.

Methicillin- and gentamicin-resistant strains of Staphylococcus aureus isolated in Dublin hospitals have been classified into groups I, II, and III based on resistance to antimicrobial agents and plasmid profiles. Each group expresses a characteristic level of resistance to gentamicin, tobramycin, and sisomicin. Enzyme assays showed that resistant strains expressed 2"-aminoglycoside phosphotransferase-6'-aminoglycoside transferase activity by a determinant which is known to be chromosomally located. The gentamicin resistance (Gmr) determinants were transferred from group I, II, or III strains by transduction into a laboratory strain where each expressed the same low level of resistance. This finding suggests that high-level resistance in some clinical strains is due to a second, unlinked resistance mechanism. No evidence was obtained by hybridization experiments that clinical isolates or spontaneous mutants expressing high-level Gmr carried more than one copy of the Gmr determinant, thus eliminating the possibility that a gene dosage effect was responsible for high-level resistance. Hybridization experiments with transductants and wild strains suggested that the Gmr determinant was located at homologous sites in wild strains from different groups, although restriction site differences were observed in flanking sequences. Electron microscope analysis of a cloned Gmr determinant and genetic evidence suggested that a Dublin clinical isolate harbored a transposon very similar to Tn4001.

Anti-Bacterial Agents↗
PubMed1987

Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358.

The broad-spectrum mercurial-resistance plasmid pDU1358 was analyzed by cloning the resistance determinants and preparing a physical and genetic map of a 45-kilobase (kb) region of the plasmid that contains two separate mercurial-resistance operons that mapped about 20 kb apart. One encoded narrow-spectrum mercurial resistance to Hg2+ and a few organomercurials; the other specified broad-spectrum resistance to phenylmercury and additional organomercurials. Each determinant governed mercurial transport functions. Southern DNA X DNA hybridization experiments using gene-specific probes from the plasmid R100 mer operon indicated close homology with the R100 determinant. The 2153 base pairs of the promoter-distal part of the broad-spectrum Hg2+-resistance operon of pDU1358 were sequenced. This region included the 3'-terminal part of the merA gene, merD, unidentified reading frame URF1, and a part of URF2 homologous to previously sequenced determinants of plasmid R100. Between the merA and merD genes, an open reading frame encoding a 212 amino acid polypeptide was identified as the merB gene that determines the enzyme organomercurial lyase that cleaves the C--Hg bond of phenylmercury.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of the epidermolytic toxin A gene of Staphylococcus aureus.

The nucleotide sequence of the eta gene, which codes for the epidermolytic toxin serotype A of Staphylococcus aureus TC16, is reported. The coding sequence of 840 nucleotides specifies a protein which, when secreted, has a predicted molecular weight of 26,950. The sequence of eta and the deduced amino acid sequence of the toxin have been compared with those of epidermolytic toxin serotype B. The coding sequences have 52% identical residues, and the polypeptides have 40% identical residues. Amino acid residues have been conserved in the areas of the proteins which correspond to major hydrophobic domains, whereas the regions likely to specify antigenic determinants occur in hydrophilic sequences that have diverged. The level of expression of epidermolytic toxin A in S. aureus 8325-4 was shown to be dependent on the integrity of a regulatory gene called agr.

Amino Acid Sequence↗
PubMed1987

The genetics and biochemistry of mercury resistance.

The ability of bacteria to detoxify mercurial compounds by reduction and volatilization is conferred by mer genes, which are usually plasmid located. The narrow spectrum (Hg2+ detoxifying) Tn501 and R100 determinants have been subjected to molecular genetic and DNA sequence analysis. Biochemical studies on the flavoprotein mercuric reductase have elucidated the mechanism of reduction of Hg2+ to Hg0. The mer genes have been mapped and sequenced and their protein products studied in minicells. Based on the deduced amino acid sequences, these proteins have been assigned a role in a mechanistic scheme for mercury flux in resistant bacteria. The mer genes are inducible, with regulatory control being exerted at the transcriptional level both positively and negatively. Attention is now focusing on broad-spectrum resistance involving detoxification of organomercurials by an additional enzyme, organomercurial lyase. Lyase genes have recently been cloned and sequencing studies are in progress.

Bacteria↗
PubMed1986

Nucleotide sequence of the R26 chloramphenicol resistance determinant and identification of its gene product.

The cml gene of plasmid R26 is carried on a 1.9-kb HindIII fragment and specifies low-level, inducible resistance to chloramphenicol (Cm). In this paper we report the identification of its product as an approx. 31 kDa protein in minicell experiments, and the determination of the nucleotide sequence of cml, which indicates that the gene product is a relatively hydrophobic protein of Mr 33,800. The protein has no detectable homology to other characterised chloramphenicol-resistance (CmR) proteins, nor any to the membrane-associated tetracycline-resistance (TcR) proteins. The presumptive ribosome-binding site (RBS) of cml mRNA is within a region showing potential for secondary structure.

Amino Acid Sequence↗
PubMed1986

Polypeptides specified by the mercuric resistance (mer) operon of plasmid R100.

Overlapping deletion mutations were constructed in chimaeric plasmids carrying the mer operon of plasmid R100. Polypeptides specified by the mutant plasmids in Escherichia coli minicells correlated with the mer genes as follows: merT, 17- and 16-kDa polypeptides; merP, 9.8- and 9.5-kDa polypeptides; merC, a 14-kDa polypeptide; merA, 65- and 62-kDa polypeptides. The products of the merR and merD genes were not identified. The revised nomenclature of the mer genes is explained.

Bacterial Proteins↗
PubMed1986

Inactivation of the alpha-haemolysin gene of Staphylococcus aureus 8325-4 by site-directed mutagenesis and studies on the expression of its haemolysins.

S. aureus strain 8325-4 was shown to produce alpha-, beta-, delta- and gamma-haemolysins by haemolytic assays and immunoblotting. Hybridization experiments indicated that a single copy of the alpha-haemolysin gene (hla) resides in the chromosome. Site-directed mutagenesis was used to inactivate the hla gene. This gene, which had previously been cloned in E. coli, was inactivated in vitro by inserting a fragment carrying an erythromycin resistance marker. Shuttle plasmids were constructed and transformed into 8325-4 and non-haemolytic recombinants enriched by a plasmid incompatibility technique. A previously isolated Tn551 insertion defective in alpha-haemolysin was not located in hla. It had pleiotropic defects in expression of alpha-, beta- and delta-haemolysins. Expression of alpha-haemolysin from a plasmid-located hla gene was very low. In contrast, hla-erm mutants were deficient only in alpha-haemolysin and allowed high level expression of the plasmid-borne hla gene. The Tn551 insertion is probably located in a gene encoding a positive regulatory element required for expression of several exoproteins. An hla-erm mutant was less virulent than the otherwise isogenic 8325-4 hla+ strain in a mouse peritonitis model, confirming that alpha-haemolysin is an important virulence factor.

Bacterial Toxins↗
PubMed1986

Molecular cloning and expression of the epidermolytic toxin A gene of Staphylococcus aureus.

The gene coding for serotype A of epidermolytic (exfoliative) toxin has been cloned from Staphylococcus aureus in Escherichia coli phage lambda and plasmid vectors. The coding sequence for eta was localised by subcloning and transposon Tn5 mutagenesis experiments. The eta gene was probably expressed from its natural promoter in E. coli. The protein synthesised in E. coli was located predominantly in the periplasm. It was immunochemically indistinguishable from the toxin purified from S. aureus culture supernatants and had the same molecular weight. Furthermore, subcutaneous injection of this material caused epidermal splitting (the Nikolsky reaction) showing that it was biologically active. An eta shuttle plasmid was transformed into protoplasts of S. aureus. The level of expression of toxin in strain 8325-4 was shown to be dependent on the integrity of the agr gene which is known to be required for the expression of several exoproteins.

Bacterial Toxins↗
PubMed1985

Susceptibility to antimicrobial agents and analysis of plasmids in gentamicin- and methicillin-resistant Staphylococcus aureus from Dublin hospitals.

Methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) strains isolated from Dublin Hospitals were classified into two groups (phenotypes). Phenotype-I strains expressed high level resistance to gentamicin and were susceptible to fusidic acid; strains resistant to tetracycline harboured a 3 X 10(6)-mol. wt plasmid. Strains in phenotype II usually expressed low level resistance to gentamicin, were resistant to fusidic acid and often harboured a (22-24) X 10(6)-mol. wt plasmid that specified resistance to ethidium bromide, tetracycline, kanamycin, neomycin and trimethoprim, or to combinations of these markers. A few phenotype-II strains expressed higher levels of resistance to gentamicin and other aminoglycosides. All MGRSA strains carried a 21 X 10(6)-mol. wt plasmid conferring resistance to penicillin, ethidium bromide, cadmium and mercury. Gentamicin resistance was invariably chromosomal and all strains carried chromosomal resistance to methicillin, erythromycin, streptomycin and spectinomycin. Several methicillin-resistant S. aureus (MRSA) strains isolated before the emergence of gentamicin resistance harboured a 21 X 10(6)-mol. wt penicillinase plasmid with the same restriction endonuclease profile as that from some MGRSA strains. Some MRSA strains carried other plasmids related to those found in MGRSA strains.

Anti-Bacterial Agents↗
PubMed1985

Molecular analysis of multiple-resistance plasmids transferred from gram-negative bacteria isolated in a urological unit.

Forty-one isolates of multiply resistant gram-negative bacteria causing infection in a urological unit of a Dublin hospital were collected during a 6-month period. Twenty-one isolates transferred multiple resistance to an Escherichia coli K-12 recipient in liquid matings. Serratia marcescens, Proteus morganii, Proteus vulgaris, and E. coli isolates harbored similar 120-megadalton IncC plasmids, whereas Enterobacter cloacae strains transferred a 160-megadalton plasmid of a different Inc group. Southern hybridization experiments were performed with purified fragments cloned from one IncC plasmid as probes. They were hybridized to plasmid sequences in total cellular DNA extracts, showing that the IncC plasmids were very closely related. This suggests that the same plasmid has transferred to different bacterial species in the hospital environment.

Bacteriuria↗
PubMed1985

Expression of the cloned toxic shock syndrome toxin 1 gene (tst) in vivo with a rabbit uterine model.

Toxic shock syndrome (TSS) toxin 1 (TSST1) is produced by strains of Staphylococcus aureus associated with TSS. Purified TSST1 induces in rabbits a shock-like illness with many features similar to TSS in humans. These symptoms were also induced by TSST1-producing bacteria in diffusion chambers implanted in the rabbit uterus. Naturally occurring TSST1+ strains and a TSST1- strain harboring a pE194-derived plasmid carrying the cloned TSST1 determinant tst gave the same symptoms. TSST1- strains and a TSST1- strain carrying a pE194-tst plasmid with a deletion of the tst gene had no effect in rabbits. The results with the plasmid-carrying TSST1+ and TSST1- strains, which were isogenic apart from tst, show that the toxin is responsible for the illness in rabbits and suggest that it is a major factor in the pathogenesis of TSS.

Animals↗
PubMed1985

Posttranscriptional regulation of the inducible nonenzymatic chloramphenicol resistance determinant of IncP plasmid R26.

The inducible nonenzymatic chloramphenicol resistance (Cmr) determinant of the IncP plasmid R26 was cloned on a 1,900-base-pair restriction endonuclease HindIII fragment. Transposon Tn5 mutagenesis revealed that at least 1,400 base pairs is required for expression of Cmr. There was no increase in the level of Cmr when the copy number of the determinant was raised by cloning in pBR322 or pUB5572. Expression of Cmr by cells carrying a lower-copy-number pUB5572cml+ plasmid was inducible and thus indistinguishable from those with R26 itself. However, pBR322cml+-carrying cells expressed Cmr constitutively, possibly due to the activity of vector promoters or an elevated copy number. Transcriptional and translational cml-lac fusions were constructed. The operon (transcriptional) cml-lac fusion carried by the low-copy-number plasmid pUB5572 caused a low level of constitutive beta-galactosidase activity, which could not be elevated by induction with chloramphenicol and was not affected by a coresident R26cml+ element. In contrast, the gene (translational) cml-lac fusion expressed low-level beta-galactosidase activity, which was elevated fivefold by prior exposure to chloramphenicol. We conclude that the regulation of Cmr occurs posttranscriptionally.

Chloramphenicol↗
PubMed1985

Some mercurial resistance plasmids from different incompatibility groups specify merR regulatory functions that both repress and induce the mer operon of plasmid R100.

Transcription of the mer genes of plasmid R100 is regulated by the product of the merR gene. The merR gene negatively regulates its own expression and also controls the transcription of the merTCA operon both negatively (in the absence of inducer) and positively (in the presence of inducer). We used transcriptional mer-lac fusions of R100-1 in complementation tests to measure the ability of the merR products of different mercury-resistant transposons and plasmids to functionally interact with R100-1. Plasmids from incompatibility groups C, B, S, L, and P, as well as the Pseudomonas transposons Tn501 and Tn3401, regulated the expression of the R100 mer genes in a similar fashion to the R100-1 merR product itself, suggesting that these elements are closely related. Only plasmid R391 (IncJ) did not complement.

DNA, Recombinant↗
PubMed1985

Identification of the merR gene of R100 by using mer-lac gene and operon fusions.

Transcriptional (operon) and translational (gene) fusions between the R100 merR gene and lacZ were constructed in vitro in a pBR322 plasmid carrying the mer genes derived from plasmid R100. The translational fusions were oriented in the opposite direction to and divergently from the merTCAD genes. This shows that the reading frame previously thought to be merR was incorrect. Expression of the gene fusion was repressed in trans by a compatible plasmid carrying the R100 merR+ gene, as was a similarly oriented transcriptional fusion. In contrast, expression of beta-galactosidase by the lac fragment located at the same site but in the opposite orientation was at a lower level and was not repressed by merR+.

DNA Restriction Enzymes↗
PubMed1983

Expression of a cloned Staphylococcus aureus alpha-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus.

A DNA sequence encoding Staphylococcus aureus alpha-hemolysin, which had been previously cloned and mapped in Escherichia coli K-12, was introduced into Bacillus subtilis BD170 and several strains of S. aureus by using plasmid vectors, some of which could replicate in all three organisms. The determinant was cloned on a 3.3-kilobase pair DNA fragment into B. subtilis by using the vector plasmid pXZ105 to form the hybrid plasmid pXZ111. B. subtilis cells harboring pXZ111 produced large zones of alpha-hemolysis after 18 h of growth at 37 degrees C on rabbit blood agar plates, and alpha-hemolysin activity was detected in supernatants prepared from growing cultures of this strain. The alpha-hemolysin was apparently secreted across the B. subtilis cell envelope. Polypeptides of molecular weights 34,000 and 33,000 were precipitated with anti-alpha-hemolysin serum from lysates prepared from BD170 cells harboring pXZ111. A hybrid replicon which could replicate in both E. coli and S. aureus was constructed in E. coli by ligating a HindIII fragment encoding the replication functions and chloramphenicol resistance genes of S. aureus plasmid pCW59 to the pBR322 alpha-hemolysin hybrid plasmid pDU1150. The DNA of this plasmid, pDU1212, was prepared in E. coli and used to transform protoplasts prepared from a non-alpha-hemolytic, nonrestricting strain of S. aureus RN4220. Some of the transformants contained plasmids which had suffered extensive deletions. Some plasmids, however, were transformed intact into RN4220. Such plasmids were subsequently maintained in a stable manner. pDU1212 DNA was prepared from RN4220 and transformed into alpha-hemolytic S. aureus 8325-4 and two mutant derivatives defective in alpha-hemolysin synthesis. All three strains expressed alpha-hemolysin when harboring pDU1212.

Bacillus subtilis↗
PubMed1983

Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes.

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.

Biological Transport↗