RESUMEN
In Streptomyces ambofaciens, white papillae that genetic instability events generate during aerial mycelium growth, give rise to Pig-pap mutants which are unable to sporulate and devoid of large genome rearrangement. Knowing that genetic and environmental factors can influence the number of papillae per colony, we investigated the effect of nutrient limitated conditions of growth on the formation of white papillae. We observed that under nitrogen limitation and, most particularly, under amino acid limitation, the number of papillae per colony dramatically increased. Most of the Pig-pap mutants deriving from such papillae displayed a mutation in the whiG gene, which encodes the sigma factor sigma(whiG) which is absolutely required for the sporulation process. In most cases, the mutation led to a loss of function. We showed that the Pig-pap mutants deriving from papillae appearing under usual growth conditions also frequently displayed null mutation of whiG too. As the whiG mutation ratio among the Pig-pap mutants isolated with or without nitrogen limited conditions did not change, the results described in this paper suggest that the production of papillae could constitute a response of S. ambofaciens to an amino acid limitation.
Asunto(s)
Genes Bacterianos/genética , Inestabilidad Genómica/genética , Micelio/crecimiento & desarrollo , Nitrógeno/deficiencia , Streptomyces/crecimiento & desarrollo , Streptomyces/genética , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Proteínas Bacterianas/química , Secuencia de Bases , Recuento de Colonia Microbiana , Genoma Bacteriano/genética , Datos de Secuencia Molecular , Mutación/genética , Polimorfismo Conformacional Retorcido-Simple , Alineación de Secuencia , Esporas Bacterianas/metabolismo , Streptomyces/clasificación , Streptomyces/metabolismoRESUMEN
The genetic instability of Streptomyces ambofaciens affects the pigmentation of colonies and generates a variety of mutants the majority of which display large genome rearrangements. Among them, the Pig-pap mutants, which probably result from a mutational event occurring during aerial mycelium growth, display specific features, since they are unable to sporulate and do not harbor any large detectable genome rearrangements. To identify the mutational event causing their phenotype, three Pig-pap mutants originating from three independent mutational events were characterized. These mutants exhibited a whiG-like phenotype which was suppressed by the introduction of one copy of Streptomyces coelicolor whiG. Their own whiG gene was devoid of mutations and appeared to be transcribed at a level similar to that of the WT. However, whiH, the expression of which depends on sigma(WhiG), was not transcribed in any of the three Pig-pap mutants, suggesting that the sigma(WhiG) was absent or inactive. This suggests that in these Pig-pap mutants, the regulation of sigma(WhiG) might be affected. Finally, the introduction of S. coelicolor whiG in one of these Pig-pap mutants restored not only pigmentation and sporulation, but also the ability to once again form white papillae. Analyses of transgene whiG in these papillae revealed that it constitutes a mutational target during aerial mycelium formation when integrated into the genome of this Pig-pap mutant.
Asunto(s)
Mutación , Factor sigma/genética , Streptomyces/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Conjugación Genética , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Campo Pulsado , Regulación del Desarrollo de la Expresión Génica , Prueba de Complementación Genética , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Micelio/genética , Micelio/crecimiento & desarrollo , Micelio/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Factor sigma/biosíntesis , Factor sigma/metabolismo , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismoRESUMEN
In Streptomyces, the linear chromosomal DNA is highly unstable and undergoes large rearrangements usually at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, AUD ( amplifiable unit of DNA). In Streptomyces ambofaciens, two amplifiable regions (AUD6 and AUD90), located approximately 600 kb and 1,200 kb from the right chromosomal end respectively, have been characterized. Here, the isolation and molecular characterization of a new S. ambofaciens mutant strain exhibiting a green-pigmented phenotype is described; the wild-type produces a gray pigment. In this mutant, both chromosome ends were deleted, which probably led to circularization of the chromosome. These deletions were associated with amplification of a sequence belonging to the chromosomal terminal inverted repeats (TIRs), which might constitute the new fragment generated by the chromosomal circularization.