NsdB——天蓝色链霉菌A3(2)中一个负调控抗生素产量蛋白的研究

TPR(tetratricopeptide repeat)是在很多蛋白中均被发现到的一个含有34个氨基酸的蛋白重复序列,其基本功能是参与蛋白间的相互作用。天蓝色链霉菌A3(2)中有70个蛋白含有类TPR结构域,NsdA是其中的一个,研究发现该蛋白对天蓝色链霉菌的产孢和产素都有负调控作用。本研究中发现基因SCO7252和SCO1593编码含TPR结构的蛋白,中断SCO7252基因后菌株放线紫红素和钙依赖抗生素产量均提高,但形态分化没有明显变化,基因SCO1593中断后菌株在产孢产素及形态等各方面均未受到影响。基因SCO7252被命名为nsdB,RT-PCR分析表明,该基因在生长30h时开始表达。通过生物信息分析表明,天蓝色链霉菌的70个含类TPR结构的蛋白中有32个仅含该结构域,有25个另外含有DNA结合区域,这些暗示着它们可能直接控制基因的表达。     

负调节基因nsdA在链霉菌中同源性及激活沉默抗生素合成基因簇的研究

nsdA基因是在天蓝色链霉菌中发现的抗生素合成负调控基因。以nsdA基因片段为探针,通过Southern杂交发现nsdA存在于多种链霉菌中。根据天蓝色链霉菌和阿维链霉菌的nsdA序列设计PCR引物,扩增多种链霉菌中nsdA基因并测序。发现在不同链霉菌中nsdA基因的相似性高达77%~100%。其中变铅青链霉菌与天蓝色链霉菌A3(2)的nsdA序列100%一致。变铅青链霉菌通常不合成放线紫红素,中断nsdA获得的突变菌株WQ2能够合成放线紫红素;在WQ2中重新引入野生型nsdA,又失去产抗生素能力。表明nsdA的中断可以激活变铅青链霉菌中沉默的放线紫红素生物合成基因簇的表达;nsdA的广泛存在及其序列高度保守则提示可以尝试用于这些菌种的抗生素高产育种。     

Transcriptome analysis of an antibiotic downregulator mutant and synergistic Actinorhodin stimulation via disruption of a precursor flux regulator in Streptomyces coelicolor

Through microarray analysis of an antibiotic-downregulator-deleted Streptomyces coelicolor ΔwblA ΔSCO1712 mutant, 28 wblA- and SCO1712-dependent genes were identified and characterized. Among 14 wblA- and SCO1712-independent genes, a carbon flux regulating 6-phosphofructokinase SCO5426 was additionally disrupted in the ΔwblA ΔSCO1712 mutant and further stimulated actinorhodin production in S. coelicolor, implying that both regulatory and precursor flux pathways could be synergistically optimized for antibiotic production.     

Role of phosphopantetheinyl transferase genes in antibiotic production by Streptomyces coelicolor

The phosphopantetheinyl transferase genes SCO5883 (redU) and SCO6673 were disrupted in Streptomyces coelicolor. The redU mutants did not synthesize undecylprodigiosin, while SCO6673 mutants failed to produce calcium-dependent antibiotic. Neither gene was essential for actinorhodin production or morphological development in S. coelicolor, although their mutation could influence these processes.     

Cloning and analysis of a gene cluster from Streptomyces coelicolor that causes accelerated aerial mycelium formation in Streptomyces lividans.

We describe the cloning and analysis of two overlapping DNA fragments from Streptomyces coelicolor that cause aerial mycelium to appear more rapidly than usual when introduced into Streptomyces lividans on a low-copy-number plasmid vector. Colonies of S. lividans TK64 harboring either clone produce visible aerial mycelia after only 48 h of growth, rather than the usual 72 to 96 h. From deletion and sequence analysis, this rapid aerial mycelium (Ram) phenotype appears to be due to a cluster of three genes that we have designated ramA, ramB, and ramR. Both ramA and ramB potentially encode 65-kDa proteins with homology to ATP-dependent membrane-translocating proteins. A chromosomal ramB disruption mutant of S. lividans was found to be severely defective in aerial mycelium formation. ramR could encode a 21-kDa protein with significant homology to the UhpA subset of bacterial two-component response regulator proteins. The overall organization and potential proteins encoded by the cloned DNA suggest that this is the S. coelicolor homolog of the amf gene cluster that has been shown to be important for aerial mycelium formation in Streptomyces griseus. However, despite the fact that the two regions probably have identical functions, there is relatively poor homology between the two gene clusters at the DNA sequence level.     

Expression of the Streptomyces coelicolor A3(2) ptpA gene encoding a phosphotyrosine protein phosphatase leads to overproduction of secondary metabolites in S. lividans

Abstract A DNA fragment that caused pigment production in Streptomyces lividans was isolated from a gene library of Pst I-digested chromosomal fragments of S. coelicolor A3(2). Subcloning and nucleotide sequencing proved the identity of the cloned gene to ptpA encoding a low-molecular-mass phosphotyrosine protein phosphatase. The S. lividans transformant containing ptpA on pIJ41 with a copy number of 3–4 per genome produced large amounts of undecylprodigiosin and A-factor, in addition to the pigmented antibiotic actinorhodin, whereas the transformant containing ptpA on an SCP2* derivative with a copy number of 1–2 did not. The PtpA protein produced as a fusion to the maltose binding protein in Escherichia coli showed phosphatase activity toward o -phosphotyrosine, but not toward o -phosphoserine or o -threonine. Introduction of a mutant ptpA gene encoding an inactive protein with serine instead of the 9th cysteine caused no pigmentation. Disruption of the chromosomal ptpA gene of S. coelicolor A3(2), however, appeared to cause no detectable effect on the production of the pigmented antibiotics or A-factor and the ptpA disruptants developed aerial mycelium and spores normally.     

Genetic characterization of two S-adenosylmethionine-induced ABC transporters reveals their roles in modulations of secondary metabolism and sporulation in Streptomyces coelicolor M145

S-Adenosylmethionine (SAM) was previously documented to activate secondary metabolism in a variety of Streptomyces spp. and to promote actinorhodin (ACT) and undecylprodigiosin (RED) in Streptomyces coelicolor. The SAM-induced proteins in S. coelicolor include several ABC transporter components (SCO5260 and SCO5477) including BldKB, the component of a well-known regulatory factor for differentiations. In order to assess the role of these ABC transporter complexes in differentiation of Streptomyces, SCO5260 and SCO5476, the first genes from the cognate complex clusters, were individually inactivated by gene replacement. Inactivation of either SCO5260 or SCO5476 led to impaired sporulation on agar medium, with the more drastic defect in the SCO5260 null mutant (ASCO5260). ASCO5260 displayed growth retardation and reduced yields of ACT and RED in liquid cultures. In addition, SAM supplementation failed in promoting the production of ACT and RED in ASCO5260. Inactivation of SCO5476 gave no significant change in growth and production of ACT and RED, but impaired the promoting effect of SAM on ACT production without interfering with the effect on RED production. The present study suggests that SAM induces several ABC transporters to modulate secondary metabolism and morphological development in S. coelicolor.     

Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans.

A-factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone), an autoregulating factor originally found in Streptomyces griseus, is involved in streptomycin biosynthesis and cell differentiation in this organism. A-factor production is widely distributed among actinomycetes, including Streptomyces coelicolor A3(2) and Streptomyces lividans. A chromosomal pleiotropic regulatory gene of S. coelicolor A3(2) controlling biosynthesis of A-factor and red pigments was cloned with a spontaneous A-factor-deficient strain of S. lividans HH21 and plasmid pIJ41 as a host-vector system. The restriction endonuclease KpnI-digested chromosomal fragments were ligated into the plasmid vector and introduced by transformation into the protoplasts of strain HH21. Three red transformants thus selected were found to produce A-factor and to carry a plasmid with the same molecular weight, and a 6.4-megadalton fragment was inserted in the KpnI site of pIJ41. By restriction endonuclease mapping and subcloning, a restriction fragment (1.2 megadaltons, approximately 2,000 base pairs) bearing the gene which causes concomitant production of A-factor and red pigments was determined. The red pigments were identified by thin-layer chromatography and spectroscopy to be actinorhodin and prodigiosin, both of which are the antibiotics produced by S. coelicolor A3(2). The cloned fragment was introduced into the A-factor-negative mutants (afs) of S. coelicolor A3(2) by using pIJ702 as the vector, where it complemented one of these mutations, afsB, characterized by simultaneous loss of A-factor and red pigment production. We conclude that the cloned gene pleiotropically and positively controls the biosynthesis of A-factor, actinorhodin, and prodigiosin.     

圈卷产色链霉菌分化相关基因——saw1的结构和功能

用双脱氧链终止法进行了分化基因——saw1的双链测序.结果表明在1500bp的DNA片段中有一个完整的开读框架(ORF),其编码区是在419bp至1252bp处.其产物与已知的天蓝色链霉菌whiG的氨基酸序列有89%的同源性.当把1500bp的saw1DNA片段插入到链霉菌表达质粒载体pIJ702后,构建的重组质粒转化天蓝色链霉菌孢子形成缺陷突变株C71,可使C71形成孢子和灰色色素.用基因破坏的策略进一步研究了该基因的生物学功能,结果表明saw1在圈卷产色链霉菌气生菌丝到孢子形成的发育转变中有重要作用,是分化中控制孢子发育起始的一个重要基因.     

Interaction of SCO2127 with BldKB and its possible connection to carbon catabolite regulation of morphological differentiation in Streptomyces coelicolor

In Streptomyces coelicolor, the sco2127 gene is located upstream of the gene encoding for glucose kinase. This region restores sensitivity to carbon catabolite repression (CCR) of Streptomyces peucetius var. caesius mutants, resistant to 2-deoxyglucose (Dog(R)). In order to search for the possible mechanisms behind this effect, sco2127 was overexpressed and purified for protein-protein interaction studies. SCO2127 was detected during the late growth phase of S. coelicolor grown in a complex media supplemented with 100 mM glucose. Pull-down assays using crude extracts from S. coelicolor grown in the same media, followed by far-western blotting, allowed detection of two proteins bound to SCO2127. The proteins were identified by MALDI-TOF mass spectrometry as SCO5113 and SCO2582. SCO5113 (BldKB) is a lipoprotein ABC-type permease (～66 kDa) involved in mycelium differentiation by allowing the transport of the morphogenic oligopeptide Bld261. SCO2582, is a putative membrane metalloendopeptidase (～44 kDa) of unknown function. In agreement with the possible role of SCO2127 in mycelium differentiation, delayed aerial mycelium septation and sporulation was observed when S. coelicolor A3(2) was grown in the presence of elevated glucose concentrations (100 mM), an effect not seen in a Δ-sco2127 mutant derived from it. We speculate that SCO2127 might represent a key factor in CCR of mycelium differentiation by interacting with BldKB.