RESUMO
Three open reading frames (ORFs) have been located downstream of cefE in the cephamycin C gene cluster of Streptomyces clavuligerus. ORF13 (pcd) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (cmcT) encodes a 523-amino-acid protein (MW, 54,232) analogous to Streptomyces proteins for efflux and resistance to antibiotics. ORF15 (pbp74) encodes a high molecular weight penicillin-binding protein (MW, 74, 094).
Assuntos
Ácido 2-Aminoadípico/biossíntese , Proteínas de Bactérias , Cefamicinas/metabolismo , Família Multigênica , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Streptomyces/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Bacteriano , Dados de Sequência Molecular , Fases de Leitura Aberta , Prolina/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
A regulatory gene (ccaR), located within the cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S. clavuligerus. The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs. Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize cephamycin and clavulanic acid. Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites. ccaR was expressed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resulted in a two- to threefold increase in the production of cephamycin and clavulanic acid.
Assuntos
Cefamicinas/biossíntese , Ácidos Clavulânicos/biossíntese , Genes Reguladores , Streptomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Ácido Clavulânico , Clonagem Molecular , Amplificação de Genes , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Recombinação Genética , Streptomyces/metabolismoRESUMO
Sequencing of ORF10 (gene cmcH) of the Nocardia lactamdurans cephamycin gene cluster proved that it encodes a protein with a deduced molecular mass of 57,149 Da. This protein showed significant similarity to the putative O-carbamoyltransferases (O-Cases) encoded by the nodU genes of Rhizobium fredii and Bradyrhizobium japonicum, involved in the synthesis of nodulation factors. The carbamoyl-phosphate (CP)-binding amino-acid sequence of human OTCase is conserved in the cmcH product. A similar cmcH (80% identify in a 160-nt fragment) in the cephamycin (CmC) cluster of cmc genes of Streptomyces clavuligerus was partially sequenced. The cmcH gene is closely linked to and in the same orientation as cefF in both organisms. Both cmcH were subcloned in pIJ702 and expressed in Streptomyces lividans. Extracts of transformants could carbamoylate decarbamoylcefuroxime. A similar cmcH was found by Southern hybridization in Streptomyces cattleya, but not in Streptomyces griseus or Streptomyces lipmanii which produce non-carbamoylated CmC.
Assuntos
Carboxil e Carbamoil Transferases , Cefamicinas/biossíntese , Genes Bacterianos/genética , Nocardia/genética , Streptomyces/genética , Transferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Dados de Sequência Molecular , Nocardia/enzimologia , Fases de Leitura Aberta/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologiaRESUMO
Three genes related to beta-lactam resistance have been found in the cluster of genes for cephamycin C biosynthesis in Nocardia lactamdurans. The cmcT gene encodes a hydrophobic protein located in the cytoplasmic membrane. The sequence of CMCT has a 21-31% identity in amino acids to proteins involved in antibiotic export from other antibiotic producing microorganisms. The pbp gene encodes a penicillin binding protein. Nocardia lactamdurans is rather sensitive to penicillins, but no to cephalosporins or cephamycin C. A third gene, bla, encodes a type A beta-lactamase. Both the beta-lactamase and the PBP protein, might form a system for the sensing and hydrolysis of penicillin intermediates which are released into the medium during the lysis of antibiotic producing cells.
