RESUMEN
Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sigmaH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sigmaH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2-egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sigmaH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2-egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sigmaH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN , Factor sigma/genética , Streptomyces/fisiología , Factores de Transcripción , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Etanol/farmacología , Regulación Bacteriana de la Expresión Génica , Proteínas Fluorescentes Verdes , Respuesta al Choque Térmico , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Mutación , Operón , Regiones Promotoras Genéticas , Factor sigma/efectos de los fármacos , Factor sigma/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Esporas Bacterianas , Streptomyces/efectos de los fármacos , Streptomyces/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes. DNA complementing whiH mutants was located immediately upstream on hrdB, which encodes the principal sigma factor of S. coelicolor. Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype. Four whiH mutants contained base changes or a frameshift in this gene. The deduced product of whiH related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate-containing intermediates in carbon metabolism. Transcription of whiH was initiated at a single promoter, PwhiH. Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable. Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production. PwhiH was directly dependent on the sigma factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Proteínas Represoras/genética , Streptomyces/genética , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Prueba de Complementación Genética , Homeostasis , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Fenotipo , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Factor sigma/genética , Esporas Bacterianas , Streptomyces/metabolismo , Streptomyces/fisiología , Transcripción GenéticaRESUMEN
whiE is a complex locus that specifies the polyketide spore pigment in Streptomyces coelicolor A3(2). Two divergently oriented promoters, whiEP1 and whiEP2, were identified in the whiE gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated; whiEP1 and whiEP2 transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known "early" whi genes required for sporulation septum formation (whiA, -B, -G, -H, -I, and -J). Mutation of the late sporulation-specific sigma factor gene, sigF, had no effect on the activity of whiEP1 but blocked transcription from whiEP2. However, sigmaF-containing holoenzyme was not sufficient to direct transcription of whiEP2 in vitro. The whiEP2 promoter controls expression of whiE ORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of whiE ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779-2791, 1995). Consistent with these observations, construction of a sigF null mutant of S. coelicolor M145 caused the same change in spore color, showing that disruption of sigF in S. coelicolor changes the nature of the spore pigment rather than preventing its synthesis altogether.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Complejos Multienzimáticos/genética , Péptidos , Pigmentos Biológicos/genética , Streptomyces/genética , Proteínas Bacterianas/biosíntesis , Color , ARN Polimerasas Dirigidas por ADN , Genes Bacterianos , Modelos Genéticos , Morfogénesis , Familia de Multigenes , Pigmentos Biológicos/biosíntesis , Regiones Promotoras Genéticas , Factor sigma/genética , Esporas Bacterianas , Streptomyces/enzimología , Transcripción GenéticaRESUMEN
In the past two years, the isolation of extracellular factors involved in the initiation of aerial mycelium formation, the identification of metabolic defects in certain developmental mutants, and the characterisation of three further bld genes and several gamma-butyrolactone receptor genes have led to new ideas about the mechanisms that initiate aerial mycelium formation in Streptomyces. The emerging picture suggests the integration of numerous signals from both inside and outside the cell.
Asunto(s)
Streptomyces/crecimiento & desarrollo , Streptomyces/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Transducción de Señal , Streptomyces/metabolismoRESUMEN
whiG and sigF encode RNA polymerase sigma factors required for sporulation in the aerial hyphae of Streptomyces coelicolor. Their expression was analysed during colony development in wild-type and sporulation-defective whi mutant strains. Each gene was transcribed from a single promoter. Unexpectedly, whiG mRNA was present at all time points, including those taken prior to aerial mycelium formation; this suggests that whiG may be regulated post-transcriptionally. Transcription of whiG did not depend upon any of the six known 'early' whi genes required for sporulation septum formation (whiA, B, G, H, I and J), placing it at the top of the hierarchy of whi loci. sigF expression appeared to be regulated at the level of transcription; sigF transcripts were detected transiently when sporulation septa were observed in the aerial hyphae. Transcription of sigF depended upon all six of the early whi genes, including whiG. The sigF promoter does not resemble the consensus sequence established for sigma WhiG-dependent promoters and E sigma WhiG did not transcribe from the sigF promoter in vitro. Consequently, the genetic dependence of sigF upon whiG is very likely to be indirect. These results show that there is a hierarchical relationship between sigma factors required for Streptomyces sporulation and also that at least five other genes are involved in this transcriptional network.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica , Factor sigma , Proteínas Bacterianas/metabolismo , Secuencia de Bases , División Celular , Coenzimas , Medios de Cultivo , Cartilla de ADN , ADN Bacteriano , ARN Polimerasas Dirigidas por ADN , Datos de Secuencia Molecular , ARN Bacteriano/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Esporas Bacterianas , Streptomyces/genética , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismo , Transcripción GenéticaRESUMEN
A gene (sigF) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial sigma 70 family. Homologues were present in other Streptomyces spp., and that of the genetically well studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 (S. coelicolor) and 295 (S. aureofaciens) amino acid residues, both showing greatest similarity to sigma B of Bacillus subtilis. However, while sigma B is involved in stationary-phase gene expression and in the general stress response in B. subtilis, sigma F affects morphological differentiation in Streptomyces. Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. sigma F is proposed to control the late stages of spore development in Streptomyces.
Asunto(s)
Proteínas Bacterianas/fisiología , Genes Bacterianos/genética , Factor sigma/fisiología , Streptomyces/crecimiento & desarrollo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Clonación Molecular , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Factor sigma/genética , Esporas Bacterianas/genética , Streptomyces/genética , Streptomyces/fisiologíaRESUMEN
Streptomyces coelicolor A3(2) J1668 sporulated ectopically in the substrate hyphae (the Esp phenotype) with the same time course as sporulation in the aerial hyphae. Examination of related strains implied that the Esp phenotype was caused by the deletion of DNA that lies close to, but is distinct from, the glucose kinase gene (glkA). Co-transduction of the Esp phenotype with the deletion present in J1668 confirmed this hypothesis. The size of the deletion was found to be 7.4 kb. Construction of a strain carrying both the J1668 deletion and a whiG mutation demonstrated that the Esp phenotype depends on at least one of the genes required for the differentiation of aerial hyphae into spores.
Asunto(s)
Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano/genética , Proteínas de Unión al ADN , Glucoquinasa/genética , Eliminación de Secuencia/fisiología , Factor sigma , Streptomyces/fisiología , Mapeo Cromosómico , Genes Bacterianos/fisiología , Mutación , Esporas Bacterianas/genética , Streptomyces/genética , Factores de Tiempo , Transducción GenéticaRESUMEN
The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase-coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome-mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.
Asunto(s)
Genes Bacterianos , Metiltransferasas/genética , Streptomyces/efectos de los fármacos , Streptomyces/genética , Tilosina/farmacología , Secuencia de Bases , Sitios de Unión , Secuencia de Carbohidratos , Mapeo Cromosómico , Cartilla de ADN/genética , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Streptomyces/metabolismo , Transcripción Genética , Tilosina/química , Tilosina/metabolismoRESUMEN
Aminoglycoside-resistance genes (grm) were cloned from a gentamicin producer Micromonospora purpurea and a sisomicin producer Micromonospora rosea. The nucleotide (nt) sequences of both genes were determined and the similarity between them was very high (90.4% identity). In either case, the transcription start point was localised to about 11 nt upstream from the likely translation start codons of grm, which is expressed as a polycistronic transcript. In studies to be reported elsewhere, it has been established that the M. purpurea grm gene encodes a ribosomal RNA methyltransferase. Here, we confirmed that the similarity of the two genes exists not only at the structural but also at the functional level.