RESUMO
Nikkomycins are a group of peptidyl nucleoside antibiotics with potent fungicidal, insecticidal, and acaricidal activities. sanN was cloned from the partial genomic library of Streptomyces ansochromogenes 7100. Gene disruption and complementation analysis demonstrated that sanN is essential for nikkomycin biosynthesis in S. ansochromogenes. Primer extension assay indicated that sanN is transcribed from two promoters (sanN-P1 and sanN-P2), and sanN-P2 plays a more important role in nikkomycin biosynthesis. Purified recombinant SanN acts as a dehydrogenase to convert benzoate-CoA to benzaldehyde in a random-order mechanism in vitro, with respective Kcat/Km values of 3.8 mM-1s-1 and 12.0 mM-1s-1 toward benzoate-CoA and NADH, suggesting that SanN catalyzes the formation of picolinaldehyde during biosynthesis of nikkomycin X and Z components in the wild-type stain. These data would facilitate us to understand the biosynthetic pathway of nikkomycins and to consider the combinatorial synthesis of novel antibiotic derivatives.
Assuntos
Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Oxirredutases/metabolismo , Streptomyces/enzimologia , Aminoglicosídeos/análise , Antibacterianos/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Vias Biossintéticas , China , Clonagem Molecular , Expressão Gênica , Teste de Complementação Genética , Cinética , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/genética , Análise de Sequência de DNA , Microbiologia do Solo , Streptomyces/genética , Sítio de Iniciação de TranscriçãoRESUMO
In Bacilli, gerM is a very conservative gene. Primers were designed according to the gerM gene sequence of Bacillus cereus, and a 640bp DNA fragment was obtained from Bacillus thuringiensis subsp. kustaki 1. 175 by PCR. Using this fragment as a probe, a 4.5kb DNA fragment was cloned from the partial DNA library of Bacillus thuringiensis subsp. kustaki 1.175. Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 349-amino acid (aa) protein, which has high homology with GerM protein from Bacillus subtilis. This gene was designated gerM (GenBank Accession No. DQ537381 ) . RT-PCR analysis showed that gerM gene was only expressed in the process of sporulation, suggesting gerM is not required for the vegetative growth. The function of the gerM gene was studied by a strategy of gene disruption, and the resulting gerM disruption mutant did show normal growth and sporulation. However, gerM disruption mutant spores germinate slower than wild-type spores when triggered by L-alanine or inosine, indicating that gerM is required for the spore normal germination initiated by L-alanine or inosine in Bacillus thuringiensis.
Assuntos
Bacillus thuringiensis/genética , Genes Bacterianos/fisiologia , Bacillus thuringiensis/fisiologia , Clonagem Molecular , Esporos Bacterianos/fisiologiaRESUMO
The previous result showed that samR plays an important role in the development progress of Streptomyces ansochromogenes. It was reported that the differentiation progress of S. ansochromogenes was accelerated by a recombinant plasmid containing an extra copy of samR gene. However, the differentiation progress of S. ansochromogenes was not further accelerated by a multicopy plasmid containing samR gene. Electrophoresis mobility shift assay (EMSA) demonstrated that SamR binds to its own promoter region specifically. All these results hint that samR is an autoregulatory gene in Streptomyces ansochromogenes.
Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Streptomyces/genética , Proteínas de Bactérias/genética , Dosagem de Genes , Plasmídeos/genética , Esporos Bacterianos/fisiologia , Streptomyces/fisiologiaRESUMO
The development of the genetic transformation systems in extremely halophilic Archaea was reviewed in this paper. Included are the screening of selectable markers for resistance to antibiotics, the development of gene cloning and expression vectors, and the modifications of the host organisms.
