RESUMEN
Cloning and characterization of the gor gene of the lactic acid bacterium Streptococcus thermophilus, encoding glutathione reductase, are described in this paper. This enzyme is a part of the enzymatic defences against oxidative stress in eukaryotic cells and in Gram-negative bacteria, but was never found in Gram-positive bacteria before this study. The amino acid sequence shares extensive similarities with glutathione reductases from other organisms, e.g. 62% amino acid identity with Escherichia coli protein. Northern blot analysis and glutathione reductase enzyme assays gave evidence that the gene is expressed in aerobically growing cells.
Asunto(s)
ADN Bacteriano/química , Genes Bacterianos/genética , Glutatión Reductasa/genética , Streptococcus/genética , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Glutatión Reductasa/farmacocinética , Técnicas In Vitro , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Streptococcus/enzimología , Streptococcus/crecimiento & desarrolloRESUMEN
From a cosmid clone of Streptomyces ambofaciens containing the dnaA and gyrAB genes, a 2.7-kb self-replicating DNA fragment containing the chromosome replication origin oriC was isolated. This cosmid was previously maped physically to a region near the middle of the 8-Mb linear chromosomal DNA. A pulsed-field gel electrophoresis time-course analysis revealed that sequences flanking oriC were overrepresented relative to the rest of the chromosomal DNA during rapid growth, indicating that this origin is active. In addition, the terminal regions of the chromosomal DNA showed a slight overrepresentation at the onset of stationary phase.
Asunto(s)
Replicación del ADN/genética , ADN Bacteriano/genética , Origen de Réplica/genética , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Cósmidos/química , ADN Bacteriano/química , Densitometría , Electroforesis en Gel de Campo Pulsado , Electroporación , Dosificación de Gen , Biblioteca de Genes , Datos de Secuencia Molecular , Mapeo Restrictivo , Factores de TiempoRESUMEN
The recA gene was isolated from Streptomyces ambofaciens DSM40697. Its nucleotide sequence predicted a protein of 372 residues. Two recA mutants, NSAR1001 and NSAR57, obtained by gene disruption encoded a RecA protein lacking respectively 30 and at least 62 amino acids from the C-terminal end. NSAR1001 showed a wild-type sensitivity to UV light and oxolinic acid. In contrast, NSAR57 was highly sensitive to these agents and the loss of the inserted DNA restored the wild-type phenotype. Western blot analysis using antiserum to Escherichia coli RecA showed that overproduction of RecA was correlated with overtranscription of recA in an S. ambofaciens amplified mutant derived from genetic instability.
Asunto(s)
Rec A Recombinasas/genética , Streptomyces/genética , Western Blotting , Daño del ADN/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Mutagénesis/fisiología , Plásmidos , ARN Bacteriano/genética , Rec A Recombinasas/análisis , Streptomyces/química , Transcripción Genética/genéticaRESUMEN
Streptomyces ambofaciens RP181110 produces the macrolide polyketide spiramycin. Like many other Streptomyces species, the RP181110 strain is prone to genetic instability involving genomic rearrangements (deletions and/or amplifications) in the large unstable region of the genome. It has previously been demonstrated that the amplification of a particular locus (AUD205) affects spiramycin biosynthesis and, conversely, the loss of this amplification is correlated with the restoration of antibiotic production. This report focuses on a 0.93 kb reiterated fragment specific for the AUD205 locus. Sequencing of 3596 bp including this reiteration revealed the presence of an ORF (orfPS) whose potential product was highly homologous to the EryA and Raps proteins, responsible for the biosynthesis of erythromycin in Saccharopolyspora erythraea and rapamycin in Streptomyces hygroscopicus, respectively. orfPS encodes a protein with at least four successive domains: ketoacyl synthase, acyltransferase, ketoreductase and acyl carrier protein. This organization is very similar to most eryA and rap modules. The reiterated sequence corresponds to the acyltransferase domain. orfPS was transcribed during rapid growth and stationary phase in RP181110 and overtranscribed in the amplified mutant. Both these results suggest that the gene encodes a type I polyketide synthase and its reorganization is responsible for the loss of spiramycin production in the amplified strains.