粤蓝链霉菌代谢产物的抗菌抗肿瘤活性及相关基因的初步研究

粤蓝链霉菌(Streptomyces vietnam ensis)是新近报道的物种,能在多种培养基中产生水溶性紫罗兰蓝色素。本研究发现发酵液乙酸乙酯粗提物对革兰氏阳性细菌有较强的广谱抗菌活性,对部分革兰氏阴性菌也有不同程度的抑制作用,TLC生-物显影试验进一步表明,两个蓝色组分B1、B2是主要的抗菌活性成分;同时,粗提物在20μg/mL的浓度下对HeLa细胞的生长抑制率达96.7%,显示了极强的抗肿瘤活性。利用简并引物扩增PKS/NRPS基因保守区域,获得相关序列4条,其中两条所代表的PKS基因簇有可能是粤蓝链霉菌产生次级代谢产物的重要途径,这些新基因的发现为组合生物合成提供了更多的基因资源。     

圈卷产色链霉菌尼可霉素生物合成基因sanA的克隆、结构和功能

圈卷产色链霉菌是尼可霉素产生菌,经紫外线诱变后,以赤星灰霉为指示菌筛选到遗传稳定的尼可霉素生物合成阻断突变株,选择其中的NBB19为受体,以质粒pIJ702为克隆载体,从野生型圈卷产色链霉菌的DNA文库中克隆到了6kb的DNA片段,能互补NBB19使之恢复尼可霉素的产生能力.对6kbDNA片段中的部分片段进行了序列分析,结果表明2710bpDNA片段包含一个1365bp的完整开放阅读框架,编码一个由454个氨基酸残基组成的蛋白质,该基因命名为sanA.蛋白序列数据库比较结果表明,sanA编码的蛋白质氨基酸序列与Pyrococcus horikoshii的甲基转移酶有较高的同源性,353个氨基酸残基中有25%的一致性.用基因破坏策略研究了sanA的功能,野生型菌株sanA基因被破坏后导致失去合成尼可霉素的能力,表明sanA是尼可霉素生物合成中的一个新的重要基因.     

黑暗链霉菌中tbmA基因的功能研究

PCR获得tbmA基因内部863 bp片段,构建基因阻断穿梭载体pSPU112-1,经接合转移导入Strepto-myces tenebrariusH6,筛选单交换阻断变株,并用Southern blot验证阻断变株的tbmA已经被破坏。经发酵产物分析,阻断变株不再合成氨甲酰妥布霉素,只合成安普霉素。首次从分子水平证明了tbmA只参与氨甲酰妥布霉素生物合成,而不参与安普霉素的生物合成。     

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     

圈卷产色链霉菌尼可霉素生物合成基因——sanH和sanI的研究

通过反向遗传学方法克隆到圈卷产色链霉菌尼可霉素生物合成基因簇中约7.0kb的DNA片段。该片段除含有尼可霉素生物合成基因sanF外,对sanF上游约22kb的BglⅡDNA片段进行序列测定及分析表明,还含有两个完整的开放阅读框(ORF)。ORF1由1233个核苷酸组成,ORF2由195个核苷酸组成,它们分别编码由410个氨基酸残基和64个氨基酸残基组成的蛋白质,依次命名为sanH和sanI。蛋白序列数据库比较结果表明,SanH和SanI与浅灰链霉菌(\%Streptomyces griseolus)\%中共转录的细胞色素P450(cytochrome P450)和铁氧还蛋白(ferredoxin)有较高的同源性,一致性分别为46%和56%,相似性分别为62%和70%。基因功能研究表明,sanH基因的破坏虽不影响圈卷产色链霉菌产生的尼可霉素的生物活性,但该基因可能参与了尼可霉素羟基化反应的生物合成。     

链霉菌Streptomyces tenebrarius H6中与抗生素有关的糖生物合成基因的克隆

链霉菌S.tenebrarius H6产生多种氨基糖甙类抗生素,主要有阿普霉素、妥普霉素及卡那霉素B,其中阿普霉素因含有8碳糖的一种特殊结构令人注目,它的抗菌谱广,特别是对革兰氏阴性菌有较强的抗菌活性,不容易产生耐药性,对已有的耐药菌产生的氨基糖苷转移酶等失活酶仍有抵抗力.主要用于牛、猪、鸡等的大肠杆菌、沙门氏菌和支原体所引起的白痢、腹泻和肺炎等疾病.迄今有关八碳糖生物合成基因簇的研究在国内外尚无报道,在该菌株开展有关糖合成代谢基因的研究有着一定的意义.     

克隆链霉菌的抗生素生物合成基因;产生杂种抗生素

人们最初克隆了一些识别相对简便的,在受体中以“显性”可选择的表型出现的链霉菌的一些基因,例如下列一些基因;决定次甲基霉素抗性的(8),硫链菌素和新霉素的抗性(19)以及紫霉素和红霉素的抗性(18)。     

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程序进行的分析揭示,此基因编码一个肌氨酸单体氧化酶。基因功能研究结果表明,此基因与圈卷产色链霉菌尼可霉素生物合成直接相关,命名为sanK。     

阿维链霉菌bkdAB的基因中断对阿维菌素合成的影响

以阿维菌B组分菌株StreptomycesavermitilisBjbm0 0 0 6为出发菌株 ,用PCR的方法构建bkdAB基因簇(Branched_chainα_ketoaciddehydrogenasegeneAB)的基因置换质粒pHJ582 1(pHZ13 58∷bkdAB&erm) ,并对其进行基因中断 ,得到重组菌株Bjbm582 1。Bjbm582 1的发酵产物经HPLC检测发现 ,除了产生B1a和B2a外 ,还产生一种原菌株没有的新组分 ,3个组分的总含量只有出发菌株Bjbm0 0 0 6的 2 5%。结果表明bkdAB的中断不仅部分阻断了阿维菌素的合成 ,还阻断了阿维菌素b组分的合成 ,可以推测bkdAB的产物在阿维菌素合成途径中主要承担了α酮基异戊酸脱氢酶 (α_ketoisovalericaciddehydrogenase)角色     

广州地区嗜肺军团菌环境分离株的基因序列分型分析

摘要:【目的】研究广州市嗜肺军团菌的基因特征,对来自不同水域环境的嗜肺军团菌进行分子分型研究。【方法】选择嗜肺军团菌的7个基因flaA、asd、mip、pilE、mompS、proA和neuA 作为目的基因, 对在2006-2009年间广州地区分离的44株嗜肺军团菌进行PCR扩增和测序,并将核苷酸序列上传至欧洲军团菌病感染工作组（EWGLI）数据库进行比对,得到基因型别（Sequence type, ST）,对结果进行基因序列分型（Sequence-Based Typing, SBT）和系统进化分析。【结     

Distinct difference of flaA genotypes of Legionella pneumophila between isolates from bath water and cooling tower water

To investigate the genetic difference of Legionella pneumophila in human‐made environments, we collected isolates of L. pneumophila from bath water (n = 167) and cooling tower water (n = 128) primarily in the Kanto region in 2001 and 2005. The environmental isolates were serogrouped and sequenced for a target region of flaA. A total of 14 types of flaA genotypes were found: 10 from cooling tower water and nine from bath water. The flaA genotypes of isolates from cooling tower water were quite different from those of bath water.     

Structure-function analysis of the C-terminus of IcmT of Legionella pneumophila in pore formation-mediated egress from macrophages

We have recently shown an essential role of the 32 amino acids C-terminus domain of IcmT of Legionella pneumophila in bacterial egress from macrophages. Mutants expressing an IcmT protein with a truncation in the C-terminus, replicate intracellularly but are defective in pore formation-mediated egress. The C-terminus domain of IcmT is the only hydrophilic domain of IcmT that is predicted to be in the cytoplasm while the rest of the protein is in the cytoplasmic membrane. In order to characterize the structure-function of the C-terminus of IcmT in the pore-forming activity and bacterial egress, we constructed 10 icmT missense mutant alleles differing by a single amino acid in the C-terminus of icmT and introduced them into the null icmT mutant. The H58Q, W69L, R71I, R79I and R86I icmT mutant alleles showed significantly lower pore-forming activity as measured by hemolysis of sRBC. The Y59S, R68L and S77L mutant alleles showed significantly lower cytopathogenicity to U937 macrophages. All 10 mutant alleles enabled the icmT null mutant to replicate intracellularly as efficiently as icmT null mutant harboring the wild-type icmT. Seven of the icmT alleles enabled the icmT null mutant to egress from infected macrophages as efficiently as icmT null mutant harboring the wild-type icmT. The other 3 substitutions conferred a partial defect in hemolysis and two of them also conferred a defect in egress from macrophages. Thus, two amino acid residues in the C-terminus of IcmT are required for both pore formation and bacterial egress. However, certain single amino acid substitutions in the C-terminus reduce the pore-forming activity when tested in vitro, but may or may not have a detectable effect on egress of L. pneumophila from U937 macrophages.     

嗜肺军团菌SBT、PFGE、AFLP分子分型方法的比较

比较SBT、PFGE、AFLP三种分子分型方法在嗜肺军团菌分型研究中的分辨力,探讨SBT方法在嗜肺军团菌分型中的可应用性。收集石家庄市6所医院冷却塔水中分离的32株嗜肺军团菌,对其中的24株血清I型嗜肺军团菌进行SBT分型研究,并与PFGE和AFLP分型结果进行了比较。24株LP1型嗜肺军团菌共分为4个ST型,分辨系数为0.239 1。PFGE方法将32株菌株共分为15个PFGE型,分辨系数为0.925 4。AFLP方法将32株菌株分为23个AFLP型,分辨系数为0.973 7。通过比对EWGLI网站SBT数据库,ST1021型和ST345型为本地区独特型别且属于同一克隆系;ST1型为优势型别并在我国长期流行;由于缺失neuA而未分型的嗜肺军团菌株与其他23株菌分属于不同的克隆系。SBT方法的分型能力不及PFGE方法和AFLP方法。但SBT分型方法能够通过全球比对数据库得到更多的关于菌株遗传进化和流行分布的资料,在研究菌株分子流行病学及进化方面优于PFGE和AFLP方法。     

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae

Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.     

The pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase

The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis. Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond. Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs. Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively. Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.     

Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius

The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from Streptomyces tenebrarius ATCC 17920. A genomic library of S. tenebrarius was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-scyllo-inosose (DOI) synthase, and L-glutamine:DOI aminotransferase and L-glutamine:scyllo-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, tbmA, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.     

An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius

Genes homologous to 2-deoxystreptamine (DOS) biosynthetic genes were isolated from aminoglycoside producers, Micromonospora and Streptomyces spp., using PCR primers based on the core sequences of 2-deoxy-scyllo-inosose (DOI) synthase and L-glutamine: scyllo-inosose aminotransferase (GIA). Identities of 40-45% were observed for DOI synthases, and 65-75% were observed for GIAs. The gene cluster of tobramycin biosynthesis was isolated from the genomic library of Streptomyces tenebrarius using DOI synthase as a probe. Sequencing of 33.9 kb revealed 24 putative open reading frames including the tobramycin biosynthetic gene cluster (13.8 kb) and a transport protein. This cluster encodes proteins homologous to 2-deoxystreptamine biosynthetic enzymes, glycosyltransferase and other aminocyclitols biosynthetic enzymes. Sequence analysis revealed the evolution of DOI synthases from 3-dehydroquinate (DHQ) synthases in actinomycetes. DOI synthases and GIA are therefore useful for cloning biosynthetic genes of DOS-containing aminocyclitol antibiotics or for screening such metabolites producers.     

Legionella pneumophila serogroup 1 isolates from cooling towers in Japan form a distinct genetic cluster

Thirty-one epidemiologically unrelated Legionella pneumophila serogroup 1 isolates (10 from cooling towers, 10 from public spas and/or hot spring baths, and 11 from patients) were analyzed by pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) using 6 loci, flaA, pilE, asd, mip, mompS, and proA. The results of PFGE and SBT analysis indicated that all 10 isolates from cooling towers clustered into a unique type, which was distinct from strains of other environmental sources.