Pleiotropic Control of Secondary Metabolism and Morphological Development by KsbC,a Butyrolactone Autoregulator Receptor Homologue in Kitasatospora setae

The γ-butyrolactone autoregulator signaling cascades have been shown to control secondary metabolism and/or morphological development among many Streptomyces species. However, the conservation and variation of the regulatory systems among actinomycetes remain to be clarified. The genome sequence of Kitasatospora setae, which also belongs to the family Streptomycetaceae containing the genus Streptomyces, has revealed the presence of three homologues of the autoregulator receptor: KsbA, which has previously been confirmed to be involved only in secondary metabolism; KsbB; and KsbC. We describe here the characterization of ksbC, whose regulatory cluster closely resembles the Streptomyces virginiae barA locus responsible for the autoregulator signaling cascade. Deletion of the gene ksbC resulted in lowered production of bafilomycin and a defect of aerial mycelium formation, together with the early and enhanced production of a novel β-carboline alkaloid named kitasetaline. A putative kitasetaline biosynthetic gene cluster was identified, and its expression in a heterologous host led to the production of kitasetaline together with JBIR-133, the production of which is also detected in the ksbC disruptant, and JBIR-134 as novel β-carboline alkaloids, indicating that these genes were biosynthetic genes for β-carboline alkaloid and thus are the first such genes to be discovered in bacteria.     

Molecular Analysis of a Gene Encoding a Cell-Bound Esterase from Streptomyces chrysomallus

A gene (estA) encoding a 42-kDa cell-bound esterase, EstA, was found to be located 75 bp upstream of the cyclophilin A gene (cypA) of Streptomyces chrysomallus. Western blot analysis revealed the presence of EstA (42 kDa) in cell extracts of S. chrysomallus X2 and Streptomyces lividans. EstA specifically hydrolyzes short-chain p-nitrophenyl esters. EstA formation starts at the end of growth phase, and its activity level remains constant throughout stationary phase. Expression of estA from the melanin (mel) promoter in plasmid pIJ702 led to a substantial increase of total esterase activity in streptomycetes.     

Identification of a Structural Gene Encoding a Metallothionein-like Domain that Includes a Putative Regulator Protein for Streptomyces Protease Gene Expression

An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation anaysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.     

Disruption of the ste22 Gene Encoding a Glycosyltransferase and Its Function in Biosynthesis of Ebosin in Streptomyces sp. 139

Streptomyces sp.139 produces an exopolysaccharide (EPS) designated Ebosin with remarkable anti-rheumatic arthritis activity in vivo. The ste (Streptomyces eps) gene cluster required for Ebosin biosynthesis has been identified. According to similarities with other proteins in the database, ste22 shows high homology with glycosyltransferases originated from different microorganisms. In this study, the ste22 gene was disrupted by double crossover via homologous recombination. The EPS produced by the mutant strain Streptomyces sp.139 (ste22⁻) has a different monosaccharide composition profile in comparison with that of Ebosin. This derivative of Ebosin retained the original antagonistic activity of IL-1R in vitro but lost the bioactivities of anti-inflammation and pain relief in vivo.     

Cytosine Deaminase as a Negative Selection Marker for Gene Disruption and Replacement in the Genus Streptomyces and Other Actinobacteria

We developed a novel negative selection system for actinobacteria based on cytosine deaminase (CodA). We constructed vectors that include a synthetic gene encoding the CodA protein from Escherichia coli optimized for expression in Streptomyces species. Gene disruption and the introduction of an unmarked in-frame deletion were successfully achieved with these vectors.     

Replacement of the Arginine Biosynthesis Operon in Xanthomonadales by Lateral Gene Transfer

The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the γ-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.     

链霉菌UDP-葡萄糖脱氢酸编码基因Ste6的克隆表达和性质研究

链霉菌139能够产生一种全新的胞外多糖——依博素（139A）,该多糖体内具有显著抗类风湿性关节炎活性。其生物合成基因簇（GenBank Accession Number：AYl31229）已被鉴定约31．3kb,包含22个开放阅读框（ste1—ste22）。以pET-30a为载体,克隆并在大肠杆菌BL21（DE3）中进行了ste6基因的表达,对该基因的克隆、表达与性质进行了研究。亲和层析法证实,纯化后重组蛋白具有催化UDP-葡萄糖脱氢变成UDP-葡萄糖醛酸的活性。这表明ste6编码产物是葡萄糖脱氢酶。为了证实ste6基因与依博素生物合成的关系,采用单交换基因破坏策略构建了ste6基因阻断突变株。结果初步显示ste6和依博素生物合成相关。