RESUMO
We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.
Assuntos
Antibacterianos/biossíntese , Genes Bacterianos , Ácidos Nucleicos Peptídicos/genética , Interferência de RNA , RNA Antissenso/genética , Streptomyces coelicolor/genética , Antraquinonas/antagonistas & inibidores , Antraquinonas/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/metabolismoAssuntos
Antibacterianos/biossíntese , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Macrolídeos , Micromonosporaceae , Família Multigênica/genética , Ácido 4-Aminobenzoico/antagonistas & inibidores , Antibacterianos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Macrolídeos/farmacologia , Micromonosporaceae/genética , Micromonosporaceae/metabolismoRESUMO
Verrucosispora maris AB-18-032 is a marine actinomycete that produces atrop-abyssomicin C and proximicin A, both of which have novel structures and modes of action. In order to understand the biosynthesis of these compounds, to identify further biosynthetic potential, and to facilitate rational improvement of secondary metabolite titers, we have sequenced the complete 6.7-Mb genome of Verrucosispora maris AB-18-032.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Micromonosporaceae/genética , Antineoplásicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Micromonosporaceae/isolamento & purificação , Micromonosporaceae/metabolismo , Dados de Sequência Molecular , Netropsina/análogos & derivados , Netropsina/metabolismo , Análise de Sequência de DNA , Microbiologia da ÁguaRESUMO
The Streptomyces produce a plethora of secondary metabolites including antibiotics and undergo a complex developmental cycle. As a means of establishing the pathways that regulate secondary metabolite production by this important bacterial genus, the model species Streptomyces coelicolor and its relatives have been the subject of several genetic screens. However, despite the identification and characterization of numerous genes that affect antibiotic production, there is still no overall understanding of the network that integrates the various environmental and growth signals to bring about changes in the expression of biosynthetic genes. To establish new links, we are taking a biochemical approach to identify transcription factors that regulate antibiotic production in S. coelicolor. Here we describe the identification and characterization of a transcription factor, designated AtrA, that regulates transcription of actII-ORF4, the pathway-specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. Disruption of the corresponding atrA gene, which is not associated with any antibiotic gene cluster, reduced the production of actinorhodin, but had no detectable effect on the production of undecylprodigiosin or the calcium-dependent antibiotic. These results indicate that atrA has specificity with regard to the biosynthetic genes it influences. An orthologue of atrA is present in the genome of Streptomyces avermitilis, the only other streptomycete for which there is a publicly available complete sequence. We also show that S. coelicolor AtrA can bind in vitro to the promoter of strR, a transcriptional activator unrelated to actII-ORF4 that is the final regulator of streptomycin production in Streptomyces griseus. These findings provide further evidence that the path leading to the expression of pathway-specific activators of antibiotic biosynthesis genes in disparate Streptomyces may share evolutionarily conserved components in at least some cases, even though the final activators are not related, and suggests that the regulation of streptomycin production, which serves an important paradigm, may be more complex than represented by current models.
Assuntos
Actinas/genética , Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Proteínas de Helminto/genética , Streptomyces coelicolor/genética , Fatores de Transcrição/fisiologia , Antraquinonas/análise , Antraquinonas/metabolismo , Antibacterianos/análise , Proteínas de Bactérias/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Genes Bacterianos , Mutagênese Insercional , Fases de Leitura Aberta , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Streptomyces griseus/genética , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Site-directed mutagenesis of nonribosomal peptide synthetase (NRPS) adenylation (A) domains was investigated as a means to engineer new calcium-dependent antibiotics (CDA) in Streptomyces coelicolor. Single- and double-point mutants of the CDA NRPS module 7, A-domain were generated, which were predicted to alter the specificity of this domain from Asp to Asn. The double-point mutant produced a new peptide CDA2a-7N containing Asn at position 7 as expected. However, in both the single- and the double-point mutants, significant hydrolysis of the CDA-6mer intermediate was evident. One explanation for this is that the mutant module 7 A-domain activates Asn instead of Asp; however, the Asn-thioester intermediate is only weakly recognized by the upstream C-domain acceptor site (a), allowing a water molecule to intercept the hexapeptidyl intermediate in the donor site (d).
