RESUMO
The 98th General Meeting of the American Society for Microbiology was held from May 17-21, 1998, in Atlanta, Georgia and was attended by well over 10,000 scientists. The theme of antibiotic resistance dominated the meeting with numerous presentations on resistance mechanisms, new targets and potential antimicrobial agents. Many new insights into the understanding of microbial physiology were provided. Microbial genomics was shown to be revolutionizing the way in which scientists can probe and explore bacteria and fungi.
RESUMO
Clusters of peptidoglycan biosynthesis and cell division genes (DCW genes) were identified and sequenced in two gram-positive cocci, Staphylococcus aureus and Enterococcus faecalis. The results indicated some similarities in organization compared with previously reported bacterial DCW gene clusters, including the presence of penicillin-binding proteins at the left ends and ftsA and ftsZ cell division genes at the right ends of the clusters. However, there were also some important differences, including the absence of several genes, the comparative sizes of the div1B and ftsQ genes, and a wide range of amino acid sequence similarities when the genes of the gram-positive cocci were translated and compared to bacterial homologs.
Assuntos
Proteínas do Citoesqueleto , Enterococcus faecalis/genética , Genes Bacterianos/genética , Hexosiltransferases , Família Multigênica/genética , Peptidoglicano/biossíntese , Peptidil Transferases , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Divisão Celular , Parede Celular/genética , Mapeamento Cromossômico , Clonagem Molecular , Enterococcus faecalis/citologia , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/genética , Fases de Leitura Aberta/genética , Proteínas de Ligação às Penicilinas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Staphylococcus aureus/citologiaRESUMO
The gene required for methicillin resistance in staphylococci, mecA, encodes the low-affinity penicillin-binding protein 2a (PBP2a). Transcriptional regulation of mecA is accomplished in some isolates by mecR1 and mecI, cotranscribed chromosomal genes that encode a putative signal transducer and a transcriptional repressor, respectively. Two Staphylococcus aureus strains that have identical mecR1-mecI nucleotide sequences, BMS1 and N315P, both exhibit low-level, heterotypic expression of methicillin resistance and contain no beta-lactamase coregulatory sequences. mecR1-mecI was amplified from BMS1 by PCR and was shown to be functional on a high-copy-number plasmid when introduced into an S. aureus strain with a deleted mecR1-mecI locus. Cloned mecR1-mecI repressed phenotypic expression of methicillin resistance, mecA transcription and PBP2a production and mediated PBP2a induction in response to certain beta-lactam antibiotics. However, mecR1-mecI had different regulatory activities in its native chromosomal location in N315P compared with those in BMS1. Uninduced mecA transcription was markedly repressed in N315P, and mecI inactivation increased mecA transcription and PBP2a production 5- and 40-fold, respectively. Furthermore, the N315P phenotype changed from low-level, heterotypic resistance with intact mecI to high-level, homotypic resistance in strains with disrupted mecI. In contrast, uninduced BMS1 produced abundant mecA transcript and PBP2a, while the disruption of mecI had no effect on phenotype and little effect on mecA transcription or PBP2a production. Thus, mecI-mediated repression of mecA appears to be dysfunctional in BMS1 because of the presence or absence of additional regulatory cofactors. Furthermore, heterotypic resistance expression in this strain is independent of mecA transcriptional regulation.
Assuntos
Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica , Hexosiltransferases , Resistência a Meticilina/genética , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Peptidil Transferases , Staphylococcus aureus/genética , Transcrição Gênica , Proteínas de Bactérias/genética , Clonagem Molecular , Mutagênese , Proteínas de Ligação às Penicilinas , Reação em Cadeia da Polimerase , RNA Bacteriano/análise , RNA Mensageiro/análise , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética , Especificidade da EspécieRESUMO
We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.
Assuntos
Elementos de DNA Transponíveis/genética , Staphylococcus/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Hibridização de Ácido NucleicoRESUMO
Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).
Assuntos
Isomerases de Aminoácido/metabolismo , Ácido Glutâmico/biossíntese , Staphylococcus/enzimologia , Transaminases/metabolismo , Isomerases de Aminoácido/biossíntese , Isomerases de Aminoácido/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , D-Alanina Transaminase , DNA Bacteriano/genética , Escherichia coli/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Proteínas Recombinantes/biossíntese , Staphylococcus/genética , Transaminases/biossíntese , Transaminases/genéticaRESUMO
DNA probes consisting of pUC19 containing cloned Staphylococcus aureus chromosomal fragments were constructed from two methicillin-resistant S. aureus strains with different DNA sequences 5' to mecA, the gene that mediates methicillin resistance. The probe from one strain, BMS1, contained a portion of the regulatory sequences (the terminal 641 bp of mecR1 and all of mecI) associated with the induction and repression of mecA transcription (pGO195). The second probe, from strain COL (pGO198), contained DNA not found in strain BMS1. This DNA was within the sequences added at the site of a mecR1 deletion. Genomic digests of 14 S. aureus isolates recovered between 1961 and 1969 all hybridized with pGO198. In contrast, 78% (36 of 46) of the S. aureus organisms isolated since 1988 hybridized with pGO195 but not with pGO198; the remainder hybridized with pGO198. No S. aureus isolates hybridized with both probes. Staphylococcus epidermidis digests hybridized with pGO198 (46%), pGO195 (14%), or both probes (35%); all 20 Staphylococcus haemolyticus isolates hybridized with pGO198. The restriction fragment length polymorphism patterns of all pGO198-hybridizing regions in S. aureus were identical to those in strain COL. In addition, the mecR1 deletion junction nucleotide sequences of eight S. aureus and six S. epidermidis isolates were identical. However, 21 of 23 S. epidermidis and all 20 S. haemolyticus isolates had from 5 to more than 20 additional chromosomal bands that hybridized with pGO198; none of 21 S. aureus isolates had additional hybridizing bands. These data suggest that the additional DNA responsible for the mecR1 deletion was part of a repetitive, and possibly mobile, element resident in coagulase-negative staphylococci but not in S. aureus. These data also support a hypothesis that the deletion event occurred in a coagulase-negative staphylococcus with subsequent acquisition of the interrupted sequences by S. aureus.
Assuntos
DNA Bacteriano/genética , Resistência a Meticilina/genética , Staphylococcus/genética , Sequência de Bases , Clonagem Molecular , Sondas de DNA , Elementos de DNA Transponíveis , DNA Bacteriano/análise , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Infecções Estafilocócicas/microbiologia , Staphylococcus/química , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus epidermidis/química , Staphylococcus epidermidis/genéticaRESUMO
D-Glutamic acid is an essential component of bacterial cell wall peptidoglycan in both gram-positive and gram-negative bacteria. Very little is known concerning the genetics and biochemistry of D-glutamate production in most bacteria, including Escherichia coli. Evidence is presented in this report for the roles of two distinct genes in E. coli WM335, a strain which is auxotrophic for D-glutamate. The first gene, which restores D-glutamate independence in WM335, was mapped, cloned, and sequenced. This gene, designated dga, is a previously reported open reading frame, located at 89.8 min on the E. coli map. The second gene, gltS, is located at 82 min. gltS encodes a protein that is involved in the transport of D- and L-glutamic acid into E. coli, and the gltS gene of WM335 was found to contain two missense mutations. To construct D-glutamate auxotrophs, it is necessary to transfer sequentially the mutated gltS locus, and then the mutated dga locus into the recipient. The sequences of the mutant forms of both dga and gltS are also presented.