枯草芽孢杆菌抗菌肽生物合成的研究进展

革兰氏阳性菌模式生物--枯草芽孢杆菌能分泌多种肽类及由肽类衍生的抗菌活性物质,按合成途径不同,可分为核糖体肽和非核糖体肽。其中,非核糖体肽分子量较小,一般为3000Da以下,其生物合成是通过多功能复合酶系--非核糖体肽链合成酶来完成的,多发生在菌体生长停止之后;而核糖体肽分子量较大,其合成多于菌体快速生长时期。非核糖体肽链合成酶和核糖体肽的合成及其调控均需基因参与,而这一系列基因就构成了各种抗菌肽生物合成的基因簇。对核糖体肽和非核糖体肽的生物合成及其相关调控机制进行了综述。     

单核细胞增多性李斯特菌谷胱甘肽合成酶调控鞭毛形成研究

[目的] 本试验旨在构建单核细胞增多性李斯特菌谷胱甘肽合成酶基因gshF缺失株和互补株并研究其在细菌运动和鞭毛形成中的调控作用。[方法] 采用同源重组的方法构建得到gshF缺失株后,测定野生株及缺失株的运动性和体外生长能力;同时利用荧光定量PCR方法检测gshF缺失株中鞭毛相关基因转录水平的变化。[结果] 缺失gshF后细菌在体外培养基中的生长能力未受影响,但缺失株的运动性及鞭毛形成能力显著降低。此外,缺失株中鞭毛形成重要调控基因gmaR以及鞭毛结构元件基因flaA的转录水平显著低于野生株,而其他鞭毛相关基因的转录水平变化不明显。[结论] 研究首次表明单增李斯特菌谷胱甘肽合成酶通过调控鞭毛重要基因的转录进而影响细菌的运动性和鞭毛形成,研究有助于深入理解重要食源性胞内菌通过精确调控鞭毛形成以适应外界和宿主环境的分子机制。     

非核糖体肽合成酶工程改造研究进展

非核糖体肽合成酶合成的非核糖体肽类天然产物具有丰富的结构和多样的功能,在医药、农业、工业等领域具有广泛的应用潜力。利用合成生物技术工程改造非核糖体肽合成酶,在微生物细胞工厂中组合生物合成新型非核糖体肽分子顺应绿色化学的发展理念,是国内外学者关注的热点。文中归纳了3种不同的非核糖体肽合成酶工程改造策略,并对近年来相关领域的研究进展进行综述。     

海绵共生萎缩芽孢杆菌C89 PPTase bap基因的异源表达

【背景】磷酸泛酰巯基乙胺基转移酶(PPTase)催化非核糖体肽合成酶(NRPS)中肽酰载体蛋白(PCP)从无活性的脱辅基形态转化为有活性的全辅基形态,从而启动非核糖体肽类化合物的生物合成。【目的】鉴定贪婪倔海绵共生萎缩芽孢杆菌C89中Sfp型PPTase Bap,验证Bap激活NRPS中PCP的能力。【方法】通过BLAST和氨基酸多序列比对鉴定萎缩芽孢杆菌C89中Sfp型PPTase Bap。将bap基因在sfp基因突变株枯草芽孢杆菌168中异源表达,通过重组菌枯草芽孢杆菌168-bap的代谢物检测非核糖体肽类化合物Surfactin。【结果】Bap为Sfp型PPTase,检测到重组菌枯草芽孢杆菌168-bap中Surfactin的产生。【结论】本研究为海洋萎缩芽孢杆菌中NRPS基因簇的异源表达奠定了基础。     

非核糖体肽合成酶装配机制研究进展CSCD

非核糖体多肽(nonribosomal peptide,NRP)是天然生物活性产物一大类群,组成结构多样,具有多种重要的药用价值。在微生物中催化非核糖体多肽生物合成的是非核糖体肽合成酶(nonribosomal peptide synthetase,NRPS),NRPS是一类模块酶系,模块的组装在非核糖体多肽合成及其环化中起着关键作用。本文主要对非核糖体肽合成酶常规模块组装模式及3种非常规合成模式进行综述,为深入了解和应用非核糖体肽合成酶在抗生素类生物活性物质中的作用提供理论依据。     

福氏2a志贺氏菌2457T株argT基因突变体的构建

目的：构建福氏2a志贺氏菌2457T株argT基因缺失突变体和ArgT蛋白非降解突变体,以进行后续ArgT功能研究。方法：根据福氏2a志贺氏菌2457T株基因组全序列,采用λ-Red重组系统对argT基因进行缺失,并经PCR验证;采用定点突变的方法构建ArgT非降解株,并经SDS-PAGE验证;对野生株、argT缺失突变株和ArgT非降解突变株37℃时的生长曲线及生化反应进行比较研究。结果：构建了2457T的argT缺失突变株和ArgT非降解突变株;2种突变株初始生长均较慢,但最终和野生株状态一致;2种突变株利用甘露醇的能力都比野生株强,而利用葡萄糖的能力降低。结论：获得了福氏2a志贺氏菌2457T株argT基因缺失突变体和ArgT蛋白非降解突变体。     

高亲和性铁离子渗透酶Ftr1和Ftr2调控白念珠菌生长和形态

摘要:【目的】通过分析FTR1、FTR2基因缺失株在不同培养条件下的生长情况以及菌丝生长能力,明确高亲和性铁离子渗透酶在白念珠菌生长和形态发生中的功能。【方法】将不同基因型的菌株分别置于不同的培养基和培养温度下进行培养,对其生长速度以及菌丝的生长状态进行观察,获取相应的实验结果。【结果】FTR1或FTR2单基因缺失对于白念珠菌的生长没有显著的影响,但是FTR1、FTR2双基因缺失使白念珠菌在Spider培养基中不能生长,铁离子的增加能够恢复该双基因缺失株的生长能力。FTR1、FTR2双基因缺失株在营养贫瘠的合成培养基上生长速度也较慢。此外,ftr1/frt1菌株的菌丝生长能力增强,而ftr2/ftr2菌株的菌丝生长能力减弱。双突变株ftr1/ftr1 ftr2/ftr2的菌丝生长能力能够恢复到野生对照株的水平。【结论】Ftr1与Ftr2对白念珠菌在微量铁元素环境中的生存有着重要的作用,还参与了白念珠菌对碳源N-乙酰葡萄糖胺、乙醇和甘油等的利用。此外,Ftr1对白念珠菌菌丝生长起负调控作用,Ftr2对菌丝生长起正调控作用。因此,ftr1/ftr1 ftr2/ftr2双基因突变株的菌丝生长能力能够恢复到野生对照株的水平。     

非核糖体肽合成酶装配机制研究进展

非核糖体多肽(nonribosomal peptide,NRP)是天然生物活性产物一大类群,组成结构多样,具有多种重要的药用价值。在微生物中催化非核糖体多肽生物合成的是非核糖体肽合成酶(nonribosomal peptide synthetase,NRPS),NRPS是一类模块酶系,模块的组装在非核糖体多肽合成及其环化中起着关键作用。本文主要对非核糖体肽合成酶常规模块组装模式及3种非常规合成模式进行综述,为深入了解和应用非核糖体肽合成酶在抗生素类生物活性物质中的作用提供理论依据。     

志贺氏菌福氏2a 301株ipaH4.5突变株的构建及生物学功能分析

利用λ-Red重组系统对福氏2a志贺氏菌301株ipaH4.5基因进行缺失突变,构建了福氏2a志贺氏菌301株ipaH4.5基因缺失突变株?ipaH4.5,利用低拷贝质粒构建ipaH4.5缺失突变株的回复突变株△ipaH4.5HF。PCR方法证实了ipaH4.5基因的缺失和回复。对野生株、突变株和回复突变株的生长代谢及细胞侵袭能力进行比较;ELISA方法检测3株菌侵袭鼠J774巨噬细胞后培养上清中炎性因子的水平。生长代谢实验表明缺失和回复ipaH4.5不影响志贺氏菌的生长速度,侵袭实验表明缺失和回复ipaH4.5也不影响志贺氏菌对HeLa细胞和鼠J774巨噬细胞的侵袭能力,表明ipaH4.5基因与志贺氏菌的生长代谢和侵袭能力无关;鼠J774巨噬细胞培养上清中细胞因子水平的改变提示该基因在志贺氏菌侵入细胞后抑制宿主细胞炎症反应。     

苏云金素合成基因簇中非核糖体肽合成酶基因thu2功能的初步分析

对苏云金素生物合成基因簇中编码非核糖体肽合成酶基因thu2进行基因缺失插入失活的研究。用温敏型质粒pHT304-TS构建基因thu2的插入缺失质粒pEMB1434,电转化苏云金芽胞杆菌菌株CT-43后,通过抗性筛选和PCR验证得到thu2基因同源双交换基因敲除突变株CT-43-22。HPLC(高效液相色谱,High Performance Liquid Chromatography)检测发现CT-43-22没有苏云金素特征吸收峰;用pHT304构建得到含有完整thu2基因的回补质粒pEMB1435,电转化CT-43-22后得到互补重组菌CT-43-22b,发现其恢复了苏云金素的产生。显微镜观察突变株和互补重组菌均能产生正常的晶体和芽胞。thu2的基因敲除和基因互补实验证明,thu2基因为CT-43苏云金素生物合成的必需基因,但对晶体和芽胞的形成没有影响。     

Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic human pathogen Aspergillus fumigatus. As gliotoxin exerts immunosuppressive effects in vitro and in vivo, a role as a virulence determinant in invasive aspergillosis has been discussed for a long time but evidence has not been provided until now. Here, by the use of different selection marker genes A. fumigatus knock-out strains were generated that are deficient for the non-ribosomal peptide synthetase GliP, the putative key enzyme of the gliotoxin biosynthesis. Deletion of the gliP gene resulted in loss of gliotoxin production, as analysed by high performance liquid chromatography and tandem mass spectrometry. No differences in morphology or growth kinetics between wild-type and gliP-deletion strains were observed. In vitro, the culture supernatant of the gliP-deficient strains showed a reduced cytotoxic effect on both macrophage-like cells and T cell lines. In a low-dose murine infection model of invasive aspergillosis, gliotoxin was detected in the lung and absent when mice were infected with the gliP deletion strain. However, gliP deletion strains showed no difference in virulence compared with the corresponding wild-type strains. Taken together, the non-ribosomal peptide synthetase GliP is essential for gliotoxin production in A. fumigatus. Gliotoxin is not required for pathogenicity of the fungus in immunocompromised mice, despite the fact that a reduced cytotoxicity of the culture supernatant of gliP deletion strains was demonstrated.     

Siderophore synthesis in Magnaporthe grisea is essential for vegetative growth,conidiation and resistance to oxidative stress

The plant pathogenic fungus Magnaporthe grisea excretes siderophores of the coprogen-type for iron acquisition and uses ferricrocin for intracellular iron storage. In the present report we characterize mutants with defects in extracellular siderophore biosynthesis. Deletion of the M. grisea SSM2 gene, which encodes a non-ribosomal peptide synthetase, resulted in a loss of the production of all coprogens. The mutant strains had a reduced growth rate, produced fewer conidia and were more sensitive to oxidative stress. Ferricrocin production was not affected. Upon deletion of M. grisea OMO1, a gene predicted to encode an l-ornithine-N⁵-monooxygenase, no siderophores of any type were detected, the strain was aconidial, growth rate was reduced and sensitivity to oxidative stress was increased. Abundance of several proteins was affected in the mutants. The Δssm2 and Δomo1 mutant phenotypes were complemented by supplementation of the medium with siderophores or reintroduction of the respective genes.     

Creating and screening Cochliobolus heterostrophus non-ribosomal peptide synthetase mutants

An exhaustive characterization of the set of non-ribosomal peptide synthetase (NRPS) genes of the corn pathogen, Cochliobolus heterostrophus, and the small molecule peptides produced by the enzymes they encode, has been undertaken to ascertain the role of the peptide metabolites in the fungal cell. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycete fungi (and to a limited extent, for basidiomycete fungi) and for bacteria, only. In addition to structural diversity, non-ribosomal peptides have a broad spectrum of biological activities, many are useful in medicine, agriculture, industry, and biological research. However, to suggest that inter-organismal activities is their primary function is likely incorrect; in fact, the physiological significance of these peptides to the producing fungi is largely unknown. We document that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches and that some are conserved, but most are not.     

A Staphylococcus capitis strain with unusual bacteriocin production

Staphylococcus capitis is a member of the human and mammal skin microbiomes and is considered less harmful than Staphylococcus aureus. S. capitis subsp. urealyticus BN2 was isolated from a cat and expressed strong antibacterial activity against a range of Gram-positive species, most notably including S. aureus strains with resistance to methicillin (MRSA) and strains with intermediate resistance to vancomycin (VISA). These latter strains are normally relatively resistant to bacteriocins, due to cell wall and cell membrane modifications. Genomic sequencing showed that the strain harboured at least two complete gene clusters for biosynthesis of antagonistic substances. The complete biosynthetic gene cluster of the well-known lantibiotic gallidermin was encoded on a large plasmid and the mature peptide was present in isopropanol cell extracts. In addition, a chromosomal island contained a novel non-ribosomal peptide synthetase (NRPS) gene cluster. Accidental deletion of two NRPS modules and partial purification of the anti-VISA activity showed that this novel bacteriocin represents a complex of differently decorated, non-ribosomal peptides. Additionally, a number of phenol-soluble modulins (PSMs) was detected by mass spectrometry of whole cells. Producing these compounds, the strain was able to outcompete several S. aureus strains, including MRSA and VISA, in tube cultures.     

A novel streptococcal integrative conjugative element involved in iron acquisition

In this study, we determined the function of a novel non-ribosomal peptide synthetase (NRPS) system carried by a streptococcal integrative conjugative element (ICE), ICESe2. The NRPS shares similarity with the yersiniabactin system found in the high-pathogenicity island of Yersinia sp. and is the first of its kind to be identified in streptococci. We named the NRPS product 'equibactin' and genes of this locus eqbA-N. ICESe2, although absolutely conserved in Streptococcus equi, the causative agent of equine strangles, was absent from all strains of the closely related opportunistic pathogen Streptococcus zooepidemicus. Binding of EqbA, a DtxR-like regulator, to the eqbB promoter was increased in the presence of cations. Deletion of eqbA resulted in a small-colony phenotype. Further deletion of the irp2 homologue eqbE, or the genes eqbH, eqbI and eqbJ encoding a putative ABC transporter, or addition of the iron chelator nitrilotriacetate, reversed this phenotype, implicating iron toxicity. Quantification of (55)Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbI and eqbJ genes are required for its associated iron import. In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production.     

Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus

Little is known about the process whereby the emetic toxin (or cereulide) of Bacillus cereus is produced. Two cereulide-producing strains of B. cereus were cloned and sequenced following polymerase chain reaction (PCR) amplification with primers that were specific for conserved regions of non-ribosomal peptide synthetase (NRPS) genes. The cloned regions of the B. cereus strains were highly homologous to conserved regions of other peptide synthetase nucleotide sequences. Primers were designed for two variable regions of the NRPS gene sequence to ensure specificity for the emetic strains. A total of 86 B. cereus strains of known emetic or non-emetic activity were screened using these primers. All of the emetic strains (n=30) displayed a 188 bp band following amplification and gel electrophoresis. We have developed an improved method of identifying emetic strains of B. cereus and provided evidence that cereulide is produced by peptide synthetases.     

A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product biosynthesis genes from metagenome libraries

The single-module non-ribosomal peptide synthetase BpsA from Streptomyces lavendulae has the unique ability to autonomously synthesize a coloured product (indigoidine) from a single substrate (l-glutamine), conditional upon activation by a 4'-phosphopantetheinyl transferase (PPTase) partner. We show that bpsA can be expressed in an entD PPTase gene deleted mutant of Escherichia coli to yield a sensitive reporter strain for recovery of PPTase genes from metagenome libraries. We also show that recombinant bpsA constructs, generated by substitution of the native peptidyl carrier protein domain followed by directed evolution to restore function, can be used to increase the diversity of PPTase genes recovered from a sample. As PPTases are essential for activation of non-ribosomal peptide synthetase and polyketide synthase enzymes, they are frequently associated with secondary metabolite gene clusters. Nearly half of the PPTases recovered in our screening of two small-insert soil metagenome libraries were genetically linked to recognizable secondary metabolite biosynthetic genes, demonstrating that PPTase-targeting functional screens can be used for efficient recovery of natural product gene clusters from metagenome libraries. The plasticity and portability of bpsA reporter genes can potentially be exploited to maximize recovery and expression of PPTase-bearing clones in a wide range of hosts.     

A glutamic acid 3-methyltransferase encoded by an accessory gene locus important for daptomycin biosynthesis in Streptomyces roseosporus

In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.     

Increase in Isoleucine Accumulation by α-Aminobutyric Acid-Resistant Mutants of Serratia marcescens

Several alpha-aminobutyric acid-resistant (Abu-r) mutants of Serratia marcescens were found to be superior to the parent strain in converting d-threonine to l-isoleucine. One of them accumulated 1.5 times more l-isoleucine that the parent strain. The level of acetohydroxy acid (AHA) synthetase in this mutant increased twofold above that of the parent strain. In the parent strain, AHA synthetase was repressed and l-isoleucine accumulation was decreased by either l-valine or l-leucine, whereas in the mutant the AHA synthetase level and l-isoleucine accumulation were not affected by these amino acids. AHA synthetase of the Abu-r mutant was feedback-inhibited by l-valine to the same extent as that of the parent strain. The level of d-threonine dehydratase in both strains was only slightly affected by several amino acids tested. l-Threonine dehydratase of the parent strain and of the mutant was almost completely inhibited by l-isoleucine. These results indicate that the increase in l-isoleucine accumulation by Abu-r mutants is due to the genetic derepression of AHA synthetase.