RESUMEN
The region located upstream of the alpha-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993amino acid (aa) protein with a calculated molecular weight of 107.054kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1, 4-alpha-D-glucan glucanohydrolase family. amlC is transcribed as a unique 3kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700kb from a chromosomal end of S. lividans TK24, 100kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI-II fragment of the chromosome.
Asunto(s)
Proteínas Bacterianas , Genes Bacterianos/genética , Glicósido Hidrolasas/genética , Streptomyces/genética , alfa-Amilasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Bacterianos/genética , Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glicósido Hidrolasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/genética , Mutación/genética , Sistemas de Lectura Abierta/genética , Fenotipo , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN , Streptomyces/química , Streptomyces/enzimologíaRESUMEN
A regulatory gene, reg1, was identified in Streptomyces lividans. It encodes a 345-amino-acid protein (Reg1) which contains a helix-turn-helix DNA-binding motif in the N-terminal region. Reg1 exhibits similarity with the LacI/GalR family members over the entire sequence. It displays 95% identity with MalR (the repressor of malE in S. coelicolor), 65% identity with ORF-Sl (a putative regulatory gene of alpha-amylase of S. limosus), and 31% identity with CcpA (the carbon catabolite repressor in Bacillus subtilis). In S. lividans, the chromosomal disruption of reg1 affected the expression of several genes. The production of alpha-amylases of S. lividans and that of the alpha-amylase of S. limosus in S. lividans were enhanced in the reg1 mutant strains and relieved of carbon catabolite repression. As a result, the transcription level of the alpha-amylase of S. limosus was noticeably increased in the reg1 mutant strain. Moreover, the induction of chitinase production in S. lividans was relieved of carbon catabolite repression by glucose in the reg1 mutant strain, while the induction by chitin was lost. Therefore, reg1 can be regarded as a pleiotropic regulatory gene in S. lividans.
Asunto(s)
Amilasas/genética , Proteínas Bacterianas , Quitinasas/genética , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Proteínas Represoras/genética , Streptomyces/genética , Secuencia de Aminoácidos , Amilasas/biosíntesis , Secuencia de Bases , Quitinasas/biosíntesis , Clonación Molecular , Medios de Cultivo , Genes Bacterianos , Glucosa/metabolismo , Maltosa/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Mapeo Restrictivo , Streptomyces/enzimologíaRESUMEN
Physical maps of the chromosomes of three strains of Streptomyces ambofaciens were constructed by ordering Asel fragments generated from the genomic DNA as a single linear chromosome of about 8 Mb. The physical maps of the three strains were very similar. For strain DSM40697, a Dral map was obtained by positioning the Dral sites relative to the Asel map. Eighteen genetic markers as well as the deletable and amplifiable region were assigned to the Asel and Dral fragments in this strain. The resulting genetic map resembled that of Streptomyces coelicolor A3(2). The two terminal Asel fragments exhibited retarded pulsed-field gel electrophoresis mobility, demonstrating that proteins are covalently bound at this position. A restriction map of this region was made using four additional endonucleases. Repeated sequences present at both ends of the chromosome were mapped as long terminal inverted repeats stretching over 210 kb. This corresponds to the longest terminal inverted repeats so far characterized. The deletable region of S. ambofaciens was localized at the chromosomal extremities.
Asunto(s)
ADN Bacteriano/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Mapeo Restrictivo , Streptomyces/genética , Cromosomas Bacterianos/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Biblioteca de Genes , Marcadores Genéticos/genética , Streptomyces/aislamiento & purificaciónRESUMEN
The genome of four Streptomyces ambofaciens strains from different geographical origins (ATCC15154, DSM40697, ETH9247 and ETH 11317) was analysed by pulsed-field gel electrophoresis (PFGE). The PFGE technique has allowed the study of the extrachromosomal content of these strains and the characterization of their genomic DNA by restriction analyses. Electrophoretic migration of undigested DNA allowed us to detect a 80 kb-length linear molecule with concatemeric forms in S. ambofaciens ATCC15154. These extrachromosomal molecules were shown to be homologous to the circular plasmid pSAM1 (80 kb) suggesting that pSAM1 could exist not only in circular form but also in linear form. In the same way a 45 kb-length linear molecule was detected in S. ambofaciens ETH9427 and ETH11317. In contrast, no extrachromosomal DNA could be detected in S. ambofaciens DSM40697. The analysis of the macrorestriction patterns using the rate-cutting enzymes AseI and DraI indicated a close relationship between the DSM- and ETH- strains. Indeed, three types of restriction patterns were distinguished: while S. ambofaciens ETH9427 and ETH11317 were characterized by the same pattern and share more than 75% of comigrating fragments with the strain DSM40697, S. ambofaciens ATCC15154 exhibited a restriction pattern different from the other three. The total genome sizes of S. ambofaciens ATCC15154, DSM40697, ETH9427 and ETH11317 were estimated to be about 6500, 8000, 8200 and 8200 kb, respectively.
Asunto(s)
ADN Bacteriano/análisis , Genes Bacterianos , Plásmidos , Streptomyces/genética , Southern Blotting , Enzimas de Restricción del ADN/metabolismo , ADN Bacteriano/genética , Densitometría , Electroforesis , Homología de Secuencia de Ácido NucleicoRESUMEN
A chromosomal linkage map of ten markers was established for Streptomyces ambofaciens by the four-factor cross method and allele-gradient analysis. Mutants were obtained by nitrous acid treatment as well as UV mutagenesis. The fertility of crosses was enhanced over 100-fold by pSAM2, a plasmid present in some strains of S. ambofaciens, and over 1000-fold by the conjugative plasmid pIJ303.
Asunto(s)
Streptomyces/genética , Alelos , Mapeo Cromosómico , Cruzamientos Genéticos , Intercambio Genético , Genotipo , Mutación , Recombinación GenéticaRESUMEN
DNA analysis of several genetically unstable strains of the fungus Ascobolus immersus revealed the presence of at least seven different plasmids. These plasmids ranged from 2 to 20 kb in size, and showed homology to one of them, pA1. In 18 stocks directly isolated from nature, two-thirds harboured plasmids ranging from 3 to 17 kb. Plasmids with homology to pA1 had similar molecular masses (about 8.5 kb). A possible mechanism of plasmid formation from chromosomal DNA is discussed.