Gene controlled by promoter--P_(TH4) depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library of Streptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_(TH4) as probe. The experiments revealed that this DNA fragment consists of saw D gene and a 1.4 kb Pvu Ⅱ fragment which can accelerate mycelium formation of S. ansochromogerms. The nucleofide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was desiguated as samfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded by hppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) in Rhodococcus globerulus. The function of samfR gene was studied using strategy of gene disruption, and the resulting samfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped     

In vivo transcription of two promoters, PTH4 and PTH270 involved in regulation ofStreptomyces differentiation

The promoters, PTH4 and P-TH270 involved in the regulation of Streptomyces coelicolor differentiation were subcloned into Streptomyces promoter, i.e. probe plasmid pIJ4083, and the recombinant plasmids, pIJ4470 and pIJ4471, were constructed. Two promoters could drive the expression of reporter gene encoding catechol dioxygenase when pIJ4470 and pIJ4471 were introduced into some white mutants (C85, C70, C71, C17 and C119). The total RNA was isolated from these strains containing recombinant plasmid. Probes were prepared by labelling 5 -ends of PTH4 AND PTH270 DNA fragments using radioisotope. DNA - RNA hybridization was carried out with the probes and RNAs isolated from different strains. The S1 mapping result showed that all RNAs from strains of C85/pIJ4470, C85/4471, C70/pIJ4470, C70/pIJ4471 and C17/pIJ4470 as well as C17/pIJ4471 gave rise to strong positive hy-bridization signal, whereas RNAs from C71/pIJ4470 and C71/pIJ4471 did not give any positive signal. RNAs from C119/pIJ4470 and C119/pIJ4471 gav     

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。     

链霉菌胞外多糖139A生物合成中引导糖基转移酶基因的克隆和鉴定

链霉菌139能够产生一种新的胞外多糖139A,该多糖具有抗类风湿性关节炎的活性。为研究多糖139A的生物合成基因簇,首要策略是克隆到在多糖139A的生物合成中起关键作用的引导糖基转移酶基因。根据其他几个种属的糖基转移酶氨基酸序列的两个保守区域设计简并引物,通过PCR方法扩增出相应的DNA片段作为探针,从链霉菌139基因组文库中分离到引导糖基转移酶基因ste5,并定位于约32kb的基因簇上。序列分析发现其蛋白序列与引导糖基转移酶具有较高的同源性,其C-端含有A,B和C3个保守区,N-端具有5个跨膜区。引导糖基转移酶基因阻断突变株不能够产生多糖139A表明其参与多糖139A的生物合成。     

圈卷产色链霉菌7100分化相关基因——sawE的结构与功能

以天蓝色链霉菌的whiB基因为探针,从圈卷产色链霉菌7100的总DNA部分文库中克隆了含有whiB同源序列的28kb DNA片段,并对其中的14kb片段进行了序列测定。序列分析表明,该片段含有一个完整的开放阅读框—sawE。预测的蛋白质结构及同源性分析显示,sawE与天蓝色链霉菌孢子形成早期的关键基因whiB高度同源,编码产物为一个调控蛋白。sawE的破坏使圈卷产色链霉菌7100的分化终止在气生菌丝阶段,在延长培养时间的情况下仍保持白色的表型,菌丝不能分隔,不能形成成熟的灰色孢子,结果表明sawE基因是一个与圈卷产色链霉菌分化有关的重要基因。     

普那霉素生物合成相关基因Spr1的功能

以与普那霉素生物合成密切相关的新基因Afsk-like为探针,从始旋链霉菌F618基因组文库中筛选得到含有约8 kb的DNA片段.经测序分析表明,其上含有1个具有1 146个核苷酸的完整可阅读框,该基因被命名为Spr1(HQ450023),推测其编码1个含381个氨基酸的蛋白质产物.经Blastp程序进行分析得知,该基...     

The Streptomyces coelicolor ssgB gene is required for early stages of sporulation

ssgB was identified as a novel early sporulation gene in Streptomyces coelicolor. An ssgB deletion mutant failed to sporulate, over-produced actinorhodin, and its colonies were significantly larger than those of the parental strain, suggesting an important role for the ssgB gene product in the process of growth cessation prior to sporulation-specific cell division. This places ssgB temporally before the paralogous sporulation gene ssgA. Analysis of ssgB mutant hyphae by electron microscopy and by confocal fluorescence microscopy showed that it was defective in the initiation of sporulation, as no sporulation septa could be identified, and DNA segregation had not yet been initiated in the mutant.     

圈卷产色链霉菌尼可霉素生物合成基因-sanK的克隆及功能研究

利用染色体步移策略,以尼可霉素生物合成相关的基因片段为探针,从圈卷产色链霉菌中克隆到了一个大约１０ｋｂ的ＤＮＡ片段。对其中１．８ｋｂ的ＰｖｕⅡ－ＳａｃⅡ片段进行了序列分析,结果表明：此片段中含有一个具有１１７０个核苷酸的完整开放阅读框,起始密码子为４４７位的ＡＴＧ,终止密码子为１６１４位的ＴＧＡ,推测其编码一个３８９个氨基酸的蛋白质产物。利用ＢＬＡＳＴＸ程序进行了分析揭示,此基因编码一个肌氨酸单体     

毒性T淋巴细胞相关抗原-4(CTLA-4)在链霉菌中的表达研究

应用两种链霉菌新型信号肽--vsi和gpp在常用工程菌变铅青链霉菌（Streptomyces lividans）中进行了CTLA-4的分泌表达研究,vsi信号肽与CTLA-4的融合片段克隆至链霉素-大肠杆菌穿梭质粒pUWL-219,同时gpp信号肽与CTLA-4片段在质粒pLNSP中融合,分别转化S.lividans TK24,获得重组菌株S.lividans[pUWL219-VC]和S.lividans[pLNSP/CTLA-4]。重组菌株的发酵上清液经SDS-PAGE及Western blotting分析结果表明：应用不同信号肽构建的两株工程菌均能表达分子量为13000重组蛋白,具有免疫活性。     

利用天蓝色链霉菌转化7-木糖紫杉烷的研究

紫杉醇是目前临床治疗癌症的一线化疗药物,资源紧张,价格昂贵。7-木糖-10-去乙酰基紫杉醇(7-XDT)在红豆杉中含量可达紫杉醇的10倍,脱除木糖基后生成的10-脱乙酰紫杉醇(10-DAT)经乙酰化可生成紫杉醇。通过木聚糖平板对不同菌株进行筛选,从52株供试微生物中,发现27株在木聚糖平板上生长良好。经转化实验筛选,发现一株天蓝色链霉菌(Streptomyces coelicolor YUCM 410115)具有转化7-XDT为10-脱乙酰紫杉醇的能力。菌体细胞经破碎离心后,沉淀及上清液均无转化反应出现,而发酵液的硫酸铵沉淀物则可以转化7-XDT生成10-DAT,表明该菌株能产生一种胞外紫杉醇-7-木糖苷酶,发酵液酶活为6 268U。首次发现天蓝色链霉菌能够产生紫杉醇-7-木糖苷酶,为7-XDT转化生产紫杉醇提供了新的酶源。     

圈卷产色链霉菌san7324和san7324L基因阻断对形态分化和尼可霉素产生的影响

【目的】圈卷产色链霉菌全局性调控基因wblA阻断突变后,尼可霉素不再产生。RNA-seq和转录分析表明san7324基因在野生型菌株中可以正常转录,而在wblA阻断突变株(ΔwblA)中不能转录,为此本文旨在揭示san7324与尼可霉素产生的关系。【方法】利用同源双交换策略对san7324进行基因阻断,而后通过基因遗传回补及对尼可霉素生物合成相关基因的转录分析等方法研究san7324的功能。【结果】在相同培养条件下,阻断突变株Δsan7324与野生型菌株相比失去了合成尼可霉素的能力。我们通过同源比对发现圈卷产色链霉菌中还存在一个与san7324同源的基因san7324L,该基因的阻断导致尼可霉素产量降低。当san7324和san7324L两个基因同时被阻断后,得到的突变株Δsan7324-san7324L生长稀疏而且不能正常发育分化形成灰色表型的孢子或孢子链,只能形成白色表型的气生菌丝,同时也丧失了合成尼可霉素的能力。当这两个基因(san7324-san7324L)回补双突变株后,则恢复了野生型的表型(能形成孢子链并恢复尼可霉素的产生)。进一步的研究初步表明san7324和san7324L的阻断主要影响了尼可霉素生物合成基因簇中途径特异性调控基因sanG的转录水平,从而影响圈卷产色链霉菌的发育分化和尼可霉素的产生。【结论】该结果为链霉菌形态分化与生理代谢关系的研究提供了更多的证据,同时为多效调控基因wblA作用机制的阐明奠定了基础。     

吸水链霉菌谷氨酰胺转胺酶基因的阻断及其对细胞分化的影响

【目的】通过对吸水链霉菌(Streptomyces hygroscopicus)中谷氨酰胺转胺酶基因的阻断,以期深入了解谷氨酰胺转胺酶生理功能,并为谷氨酰胺转胺酶发酵优化提供新的研究思路。【方法】以温敏型质粒pKC1139为出发质粒,构建阻断吸水链霉菌谷氨酰胺转胺酶编码基因的重组质粒pKC1139-TG1,转化吸水链霉菌原生质体,通过抗性筛选和PCR验证,成功得到一株谷氨酰胺转胺酶阻断菌株,命名为S.h-△TG。【结果】以原始菌株为对照,重组子基内菌丝生长不受影响,但是由基内菌丝分化形成气生菌丝的过程受到影响,重组子基本不产气生菌丝。【结论】谷氨酰胺转胺酶对吸水链霉菌气生菌丝的形成有着重要的影响,参与链霉菌气生菌丝的形成。     

天蓝色链霉菌胞内蓝色素提取方法研究

对天蓝色链霉菌— 10 0胞内蓝色素提取方法进行了研究 ,结果表明碱提取法、SDS法、研磨法的色素提取得率分别为 90 2 %、95 2 %和 54 6 % ;酶水解法的色素提取得率 <30 % ;细胞在pH9缓冲液中自溶 ,浓度为 1/4原发酵浓度 ,4 0℃保温搅拌 4 8h ,色素提取得率为 33 8%。     

Structural basis for prokaryotic calcium- mediated regulation by a Streptomyces coelicolor calcium binding protein

The important and diverse regulatory roles of Ca²⁺ in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.     

大肠杆菌-链霉菌穿梭载体的构建及应用

pIJ6021和pIJ4123是链霉菌的高拷贝表达载体,它们携带有受硫链丝菌素诱导的强启动子P_tipA。分别在它们的合适位点插入大肠杆菌质粒的复制子和在大肠杆菌中选择用的抗性标记基因(bla),得到了两个能在大肠杆菌和链霉菌中穿梭复制、并保持结构稳定的链霉菌表达载体：pHZ1271和pHZ1272。将透明颤菌(Vitreoscillia sp.)血红蛋白基因(vhb)克隆到pHZ1272中,用它转化变铅青链霉菌(Streptomyces lividans),经Western blotting分析和CO结合实验表明,在变铅青链霉菌中表达出了有生物活性的透明颤菌血红蛋白,从而证明所构建的pHZ1272载体具有在链霉菌中表达外源基因的功能。