RESUMO
A DNA fragment containing part of the salinomycin biosynthetic gene cluster from industrial strain Streptomyces albus CCM 4719 was cloned. Sequence analysis of the 25.809-kbp fragment revealed the presence of 8 open reading frames (ORFs), including two large ORFs encoding three modular sets of oligoketide synthase, followed by three genes (salRI, salRII, salRIII) encoding transcriptional regulators. The first two regulators, SalRI and SalRII, belonged to the novel LAL family of large transcriptional regulators. SalRIII was highly similar to the NysRIV, AmphRIV, and FscRI transcriptional regulators from the oligoene macrolides nystatin, amphotericin, and R008/candicidin clusters, respectively.
Assuntos
Genes Reguladores , Policetídeo Sintases/metabolismo , Piranos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Biblioteca Gênica , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura Aberta , Policetídeo Sintases/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Streptomyces/enzimologiaRESUMO
Transcription from the chiC promoter, directing expression of the chitinase gene, chiC, in Streptomyces coelicolor, was analyzed using xylE reporter gene and high-resolution S1-nuclease mapping. The transcription from the chiC promoter was induced by chitin, and this induction was dramatically reduced in the S. coelicolor chiR-disrupted strain. This indicated a dependence of chiC expression upon the chiR gene encoding a response regulator protein. To investigate this relationship, the S. coelicolor ChiR was overproduced using Escherichia coli T7 RNA polymerase expression system. However, gel mobility shift-assay with such a purified ChiR showed no binding in the chiC promoter region, which indicates a lack of specific phosphorylation of E. coli overproduced ChiR that is necessary for DNA-binding activity of response regulators.
Assuntos
Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptomyces/enzimologia , Fatores de Transcrição , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Quitinases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Mapeamento por Restrição , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Streptomyces/genéticaRESUMO
We cloned a new gene, sigH, encoding an alternative sigma factor in Streptomyces coelicolor A3(2). The deduced protein of 354 amino acids with an M(r) of 39486 showed greatest similarity to the sporulation sigma factor (sigma(F)) of S. coelicolor, general stress-response sigma(B) of Bacillus subtilis, and stationary-phase stress-response sigma(F) of Mycobacterium tuberculosis. Sequence analysis of the upstream region revealed an ORF encoding a protein (UshX) similar to several anti-sigma factors, and short ORF (UshY) containing zinc-finger DNA binding motif. Transcriptional analysis revealed that all three genes are located on the same polycistronic transcript in order ushY, ushX, and sigH. Expression of the operon was directed by four promoters differentially expressed in the course of differentiation. The first (P1) constitutive promoter was located upstream of ushY. The other three promoters (P2, P3, and P4) were located upstream of ushX, and were differentially induced after various stress conditions. The magnitude of the induction was greatest after osmotic stress and heat shock.
