RESUMEN
Gamma-butyrolactones (GBLs) produced by several Streptomyces species have been shown to serve as quorum-sensing signaling molecules for activating antibiotic production. The GBL system of Streptomyces chattanoogensis L10, a producer of antifungal agent natamycin, consists of three genes: scgA, scgX, and scgR. Both scgA and scgX contribute to GBL production, while scgR encodes a GBL receptor. ΔscgA and ΔscgX mutants of S. chattanoogensis behaved identically: they had a growth defect in submerged cultures and delayed or abolished the morphological differentiation and secondary metabolites production on solid medium. ScgR could bind to the promoter region of scgA and repress its transcription. Moreover, scgA seems also to be controlled by a GBL-mediated negative-feedback system. Hence, it is apparent that GBL biosynthesis is tightly controlled to ensure the correct timing for metabolic switch. An additional direct ScgR-target gene gbdA was identified by genomic SELEX and transcriptional analysis. Comparative proteomic analysis between L10 and its ΔscgA mutant revealed that the GBL system affects the expression of more than 50 proteins, including enzymes involved in carbon uptake system, primary metabolism, and stress response, we thus conclude that scgR-scgA-scgX constitute a novel GBL regulatory system involved in nutrient utilization, triggering adaptive responses, and finally dictating the switch from primary to secondary metabolism.
Asunto(s)
4-Butirolactona/metabolismo , Regulación Bacteriana de la Expresión Génica , Percepción de Quorum , Streptomyces/fisiología , Proteínas Bacterianas/análisis , Eliminación de Gen , Genes Bacterianos , Redes y Vías Metabólicas/genética , Datos de Secuencia Molecular , Familia de Multigenes , Regiones Promotoras Genéticas , Unión Proteica , Proteoma/análisis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Análisis de Secuencia de ADN , Streptomyces/genética , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismoRESUMEN
In this work, we report a case of episodic sitewise positive selection acting on the highly conserved SRP protein Ffh in Actinobacteria. An elevated non-synonymous to synonymous mutation ratio (ω) was observed along the non-terminal branches of the species tree, which contained 11 Actinobacteria species, where positively selected residues were frequently observed within the signal sequence-binding domain. Together with the estimated lineage-specific ω ratios for several core components in the major protein translocation systems, our data suggest that this positive selection might be partially driven by the diversification of signal sequences.
Asunto(s)
Actinobacteria/genética , Proteínas Bacterianas/genética , Evolución Molecular , Selección Genética , Partícula de Reconocimiento de Señal/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mutación , Transporte de Proteínas/genéticaRESUMEN
A novel Streptomyces strain, L10, which is capable of producing natamycin, was isolated from a soil sample collected from Zhejiang province, China. On the basis of phylogenetic analysis of rpoB gene and 16S rDNA sequences, as well as phenotypic comparison, strain L10 (CGMCC 2644) is proposed to be a previously uncharacterized strain of S. chattanoogensis. By screening a cosmid library of strain L10 and primer walking, a partial sequence of scnRI and the entire sequence of scnRII were obtained, which are orthologues to the pathway-specific positive regulator genes of natamycin biosynthesis in S. natalensis. The engineered S. chattanoogensis Dl, generated by inserting an additional copy of scnRII into the chromosome of strain L10, increased its natamycin production by 3.3 fold in YSG medium and 4.6 fold in YEME medium without sucrose.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Genes Reguladores , Natamicina/biosíntesis , Streptomyces/aislamiento & purificación , Streptomyces/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , China , Clonación Molecular , ADN Bacteriano/genética , ADN Ribosómico/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Alineación de Secuencia , Microbiología del Suelo , Streptomyces/clasificación , Streptomyces/genéticaRESUMEN
FtsY, the Signal Recognition Particle (SRP) receptor in bacteria, is known to facilitate the cotranslational protein targeting by recruiting SRP-protein complex to secYEG. We show in this work that deletion of the ftsY gene in Streptomyces coelicolor would also lead to complete blockage of sporulation process and reduced production of antibiotic actinorhodin. These defects cannot be complemented by only the NG domain of FtsY, while full-length ftsY was able to restore spore generation and increase production of actinorhodin in ftsY-disrupted strains. Further transcriptional analysis on sporulation controlling genes, i.e., whiG, whiB, whiH, and prox, indicated that the regulation of sporulation by ftsY is likely to take effect through whiH.
Asunto(s)
Proteínas Bacterianas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Streptomyces coelicolor/fisiología , Antraquinonas/metabolismo , Proteínas Bacterianas/genética , Eliminación de Gen , Prueba de Complementación Genética , Receptores Citoplasmáticos y Nucleares/genética , Esporas Bacterianas/genética , Esporas Bacterianas/fisiología , Streptomyces coelicolor/genéticaRESUMEN
This study indicated that the N-terminus was dispensable for FtsY GTPase activity, and that the N-domain plays an essential role in the GTPase activity of the NG domain. In addition, the S.scoelicolor FtsY was able to restore function in an E. coli mutant. However, its NG domain was unable to play any roles.
Asunto(s)
Proteínas Bacterianas/química , GTP Fosfohidrolasas/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Streptomyces coelicolor/química , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Magnesio/farmacología , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , TemperaturaRESUMEN
Fifty-four homolog (Ffh) and FtsY are the central components of the signal recognition particle secretory pathway of bacteria. In this study, the core domain and active sites of FtsY and Ffh from Streptomyces coelicolor, which are responsible for guanosine triphosphate (GTP) hydrolysis, were identified using site-directed mutagenesis. Mutations were introduced to the conserved GXXGXGK loop of the putative GTP binding site. Mutation of the Lys residue to Gly in both FtsY and Ffh NG domains significantly decreased the GTPase activity and GTP binding affinity. Furthermore, a structural model of the ternary complex of FtsY/Ffh NG domains and the non-hydrolyzable GTP analog guanylyl 5'-(beta,gamma-methylenediphosphonate) also revealed that each Lys residue in GXXGXGK of FtsY and Ffh provides the predicted hydrogen bond required for GTP binding. However, in FtsY not in Ffh, mutation of the first Gly residue in the GXXGXGK loop disrupted the GTPase activity. In addition, protease-digesting test demonstrated that NG protein with the mutation of Lys residue was decomposed more easily. Western blot analysis suggested that in Streptomyces coelicolor, FtsY is present in the membrane fraction and Ffh in the cytosol fraction during the mid-log phase of growth. These results indicated that Lys residue in the putative GTP binding loop was the crucial residue for the GTPase activity of NG domain.