产毒和非产毒霍乱弧菌在甘露醇发酵液和Luria-Bertani培养液中的基因表达差异

[目的]分析霍乱弧菌产毒株和非产毒株在甘露醇发酵液和LB(Luria-Bertani)培养液中生长的基因表达谱和代谢差异特征.[方法]提取甘露醇发酵液和LB培养液中霍乱弧菌甘露醇慢发酵株(产毒株)N16961和快发酵株(非产毒株)93097生长第一小时的总RNA,应用霍乱弧菌N16961基因组芯片分析各菌株在不同培养液中的表达差异基因.[结果]筛选出产毒株N16961在甘露醇发酵液和LB中表达差异基因142个,非产毒株93097有418个,这些表达差异基因主要分属于6个不同的功能类群,主要是转运结合、能量代谢以及蛋白质合成代谢功能.[结论]甘露醇发酵液和LB中产毒株和非产毒株的许多功能基因的转录水平有显著差异,这些表达差异基因可能与霍乱弧菌在甘露醇发酵液中代谢产酸有关,这为进一步分析霍乱弧菌代谢甘露醇的机制、以及分析产毒株与非产毒株的甘露醇发酵快慢机制提供了基础.     

霍乱弧菌甘露醇PTS操纵子中mtlR为转录抑制基因

产毒和非产毒的El Tor生物型霍乱弧菌对甘露醇发酵利用的速率有明显差别,在霍乱致病株的快速判断中有重要的参考价值。通过比较快发酵菌株(非产毒株)和慢发酵菌株(产毒株)mtlR缺失突变株与野生株在含0.2%甘露醇的M9培养液及甘露醇发酵液中生长、产酸等的变化,定性地证明了mtlR基因的抑制作用;另外通过定量RT-PCR进一步验证了MtlR蛋白在mtlCBA转录水平发挥负调控作用。但是mtlR还不是引起快慢发酵菌株对甘露醇发酵差异的直接原因。本研究也为我们研究霍乱弧菌甘露醇快慢发酵差异机制提供了必要的参考依据。     

El Tor型霍乱弧菌分泌性蛋白初步分析

摘要：【目的】利用高通量蛋白质组分析技术,初步探讨El Tor型霍乱弧菌的分泌性蛋白组成,为进一步的功能研究打下基础。【方法】选取El Tor型霍乱弧菌流行株N16961和非流行株92-3,用双相电泳和激光辅助解析-飞行时间串联质谱（Matrix Assisted Laser Desorption Ionization-Time of Flight,MALDI-TOF）技术对培养上清的全部蛋白质组份进行鉴定和分析。【结果】从N16961株培养上清中可检测到206个蛋白点,并鉴定出49种蛋白;从92-3株培养上清中可检测到236个蛋白点,并鉴定出42种蛋白,两株菌共鉴定蛋白68种,其中经预测含有信号肽的蛋白占总数的55.88%（38/68）。按功能不同将所鉴定出的蛋白分为10类,其中代谢酶、蛋白折叠/伴侣蛋白、蛋白合成以及信号传导相关蛋白占全部培养上清蛋白数的36.76%,转运蛋白占14.71%,鞭毛蛋白占11.76%,降解酶占10.29%,外膜蛋白占5.88%,毒素占1.47%,在所鉴定出的蛋白中有11种具有信号肽的假想蛋白和2种功能未知的蛋白为首次被实验证实可出现在霍乱弧菌培养上清中,占19.12%。【结论】初步获得了El Tor型霍乱弧菌流行株和非流行株的分泌性蛋白谱及其组成特征,并提示与霍乱弧菌致病关系密切的鞭毛蛋白在胞外释放机制、红血球凝集素/蛋白酶在非流行株中的高表达特征等值得高度关注。     

Pfk基因过表达对乳酸乳球菌N8产nisin速率的影响

【目的】通过基因工程手段增加糖酵解途径中编码限速酶6-磷酸果糖激酶基因Pfk在乳酸链球菌素(nisin)产生菌Lactococcus lactis N8中的表达,增快nisin的产生,从而提高单位时间内nisin的产量,缩短发酵周期。【方法】将pfk基因及编码以c AMP为依赖的蛋白激酶催化亚基基因pka C克隆到表达质粒p MG36e上,将共表达重组质粒转入L.lactis N8中,使Pfk-pka C基因过量表达,得到重组菌株L.lactis N8-p MG36epfk-pka C,并比较该重组菌株与野生菌的生长曲线、胞内6-磷酸果糖激酶活性、发酵上清液的抑菌活性及效价,并从转录水平分析两株菌nis A及pfk-pka C的转录差异,比较野生菌与重组菌在不同葡萄糖含量下培养产nisin的变化。【结果】Pfk基因与pka C基因的过表达对重组菌的生长速度没有明显的影响,却能提高重组菌产nisin的速度,在发酵10 h时nisin的产量比野生菌提高了20%,使得发酵周期缩短近2 h。野生菌及重组菌在不同葡萄糖含量下培养发酵上清液的nisin效价没有明显的变化。【结论】糖酵解途径中6-磷酸果糖激酶基因Pfk的过表达可以加快乳酸乳球菌N8产nisin的速率,缩短发酵周期。     

内生链霉菌SAT1转录组分析和抑菌代谢产物的鉴定

【背景】植物内生链霉菌Streptomyces sp.SAT1分离自药用植物荠苨根部,对多种植物病原真菌和病原细菌具有强抑菌活性,在农林业生物防治领域应用潜力巨大。【目的】揭示该菌在不同培养基条件下的抑菌效果和抑制细菌的活性物质类型,为该菌生物防治应用提供理论基础和技术支撑。【方法】通过测定发酵液和菌体萃取物的抑菌活性,研究培养基成分对抑菌活性物质生物合成的影响;选择抑制细菌活性高和无抑菌活性的培养基进行发酵,通过转录组测序分析差异表达基因的功能,并利用紫外吸收光谱和UPLC-MS/MS鉴定活性物质的成分。【结果】所选用的7种链霉菌常用发酵培养基中,无论发酵液还是菌体萃取物,TSB、GS和R5培养基无抑制细菌活性;PDB、ISP2、MS和H有较强的抑菌活性。对PDB、ISP2和TSB发酵菌体进行转录组测序分析,共发现差异表达基因3 567个,KEGG富集分析发现差异基因多集中在global and overview maps、氨基酸代谢和碳水化合物代谢等通路上,而且与TSB比,PDB和ISP2分别有18个和5个上调基因定位于moenomycin类物质的生物合成基因簇上。以标准品为对照,...     

两株低温沼气产酸细菌的分离鉴定及产酸特性

摘要:【目的】分离筛选低温沼气产酸细菌并研究其发酵特性。【方法】利用筛选培养基分离产酸细菌;通过形态学观察、16S rRNA进行鉴定;利用糖发酵、淀粉水解、明胶液化、过氧化氢酶等实验考察发酵特性;采用气相色谱法测定挥发性脂肪酸(Volatile fatty acid,VFA),对菌株的产酸特性进行研究。【结果】从霍林河市户用沼液中分离出两株在4 ℃产酸较高的菌株FJ-8和FJ-15,经16S rRNA系统发育树分析分别属于Pseudomonas sp．和Shewanella sp.。两株菌均能够水解淀粉、液化明胶,且过氧化氢酶反应均呈阳性。它们最适的产酸温度分别是15 ℃和20 ℃,在4 ℃液体发酵10 d后所产总VFAs分别为2593 mg/L和2687 mg/L。其中乙酸含量分别为792 mg/L和966 mg/L。另外,相同条件下5 L模拟发酵结果显示处理组(添加所筛两株菌混合发酵液)的周产气量比对照组高出59%。【结论】分离出的两株产酸细菌FJ-8和FJ-15在低温条件下具有较好的产酸性能,在提高低温条件下沼气产气量方面具有很强的应用前景。     

产黑色素海洋放线菌的分离、鉴定和产色素条件优化

【目的】从连云港高公岛沿岸海底泥样中分离筛选到一株产黑色素的海洋放线菌。【方法】对筛选得到的菌株HT-18所产的色素进行紫外-可见吸收光谱、红外光谱分析。通过形态特征、培养特征、生理生化测定以及16S rRNA序列的系统发育学分析确定菌株HT-18的分类学地位。采用单因素对产色素发酵条件进行优化。【结果】菌株HT-18所产色素为黑色素。鉴定该菌株属链霉菌属(Streptomyces)。最佳产色素条件为:初始pH7.0,装液量为70/250 mL,温度31°C,发酵时间为3 d。【结论】海洋链霉菌HT-18是一株具有研究和应用潜力的产黑色素菌株。     

清香型白酒中乳酸菌和酵母菌的相互作用

【目的】探究清香型白酒中不同乳酸菌和酵母菌的相互作用,了解不同菌株的发酵性能,为更深入地认识白酒发酵机理、实现发酵过程优化提供理论基础。【方法】利用程序控温和固态发酵模拟清香型白酒酿造环境,测定纯培养和共培养中菌株的理化指标、活菌数以及主要代谢产物的变化。【结果】Saccharomyces cerevisiae YJ1糖消耗快产乙醇和酯类物质多,Lactobacillus plantarum JMRS4糖消耗快产酸较多。共培养中乳酸菌对Saccharomyces cerevisiae YJ1的生长和产乙醇抑制较大,对Candida aaseri MJ7产乙醇几乎无影响。乳酸菌对Pichia kudriavzevii MJ14的生物量和乙醇代谢抑制作用较小,还对其产己酸乙酯、乙酸乙酯和异戊醇等代谢产物有促进作用;而反过来Pichia kudriavzevii MJ14对3株乳酸菌产乳酸均有抑制作用,对产乙酸则有促进作用。【结论】建立了一种固态培养方法,结合清香型白酒发酵温度变化规律,有效模拟了实际发酵环境。Pichia kudriavzevii MJ14在与乳酸菌共培养中受到的抑制较小并能有效抑制乳酸菌产乳酸,Saccharomyces cerevisiae YJ1能代谢产生多种风味物质,对清香型白酒酿造有重要意义。     

巴斯德毕赤酵母甲醇诱导表达磷脂酶A2的转录组学分析

【目的】基于转录组学技术研究表达磷脂酶A_2的毕赤酵母重组菌在甲醇诱导表达外源蛋白时的基因表达差异,从而解析外源蛋白高效诱导表达机制,为进一步工程菌株的改造提供理论支撑。【方法】以一株产磷脂酶(PLA_2)的毕赤酵母为出发菌株,采用RNA-Seq二代测序方法,研究在甘油培养和甲醇诱导两种条件下,重组毕赤酵母转录组基因表达差异情况。【结果】重组毕赤酵母中共鉴定到5225个转录本。甘油培养与甲醇诱导相比,共有857个基因发生显著变化。依据代谢途径分类,差异基因集中在核糖体成分、甲醇代谢、磷酸戊糖途径、糖酵解途径、柠檬酸循环、乙醛酸循环以及蛋白质加工过程。【结论】通过分析甲醇诱导前后的差异表达基因,结果表明碳源改变对胞内代谢会产生全局影响。本研究结果为进一步研究毕赤酵母表达外源蛋白的机制提供了基础。     

不同胁迫条件下产志贺毒素大肠杆菌生长及产毒能力异质性分析

【目的】本文旨在探究产志贺毒素大肠杆菌(Shiga toxin-producing Escherichia coli, STEC)在不同胁迫条件下的生长情况以及产毒状况的变化,为STEC菌株在胁迫压力下应激机制研究提供基础数据参考。【方法】选取3株STEC菌株分别在不同pH值、不同盐浓度、不同温度下进行适应性传代培养,使用一级生长模型Gompertz模型分析其在胁迫环境下的生长异质性;通过Vero细胞毒性实验分析胁迫条件下菌株的产毒状况。【结果】除了pH 5.0条件下的ST462菌株,在pH5.0和9.0、较高盐浓度1.5%NaCl和2.5%NaCl以及低温10℃胁迫条件下STEC菌株都表现出最大比生长速率(μmax)降低、迟缓期(λ)延长,差异显著(P<0.01);不同胁迫条件下提取的STEC菌株毒素侵染细胞后测得的细胞存活率均低于对照最适条件,而pH 5.0条件下相反。【结论】STEC菌株在碱性、较高浓度盐以及低温环境胁迫压力下响应应激机制,生长虽受到抑制,但其所产毒素对Vero细胞造成的损伤加重,毒性增强。本研究通过胁迫环境下生长和产毒状况的异质性分析,将有助于全面了解ST...     

Genetic diversity of toxigenic and nontoxigenic Vibrio cholerae serogroups O1 and O139 revealed by array-based comparative genomic hybridization

Toxigenic serogroups O1 and O139 of Vibrio cholerae may cause cholera epidemics or pandemics. Nontoxigenic strains within these serogroups also exist in the environment, and also some may cause sporadic cases of disease. Herein, we investigate the genomic diversity among toxigenic and nontoxigenic O1 and O139 strains by comparative genomic microarray hybridization with the genome of El Tor strain N16961 as a base. Conservation of the toxigenic O1 El Tor and O139 strains is found as previously reported, whereas accumulation of genome changes was documented in toxigenic El Tor strains isolated within the 40 years of the seventh pandemic. High phylogenetic diversity in nontoxigenic O1 and O139 strains is observed, and most of the genes absent from nontoxigenic strains are clustered together in the N16961 genome. By comparing these toxigenic and nontoxigenic strains, we observed that the small chromosome of V. cholerae is quite conservative and stable, outside of the superintegron region. In contrast to the general stability of the genome, the superintegron demonstrates pronounced divergence among toxigenic and nontoxigenic strains. Additionally, sequence variation in virulence-related genes is found in nontoxigenic El Tor strains, and we speculate that these intermediate strains may have pathogenic potential should they acquire CTX prophage alleles and other gene clusters. This genome-wide comparison of toxigenic and nontoxigenic V. cholerae strains may promote understanding of clonal differentiation of V. cholerae and contribute to an understanding of the origins and clonal selection of epidemic strains.     

Proteins involved in difference of sorbitol fermentation rates of the toxigenic and nontoxigenic Vibrio cholerae El Tor strains revealed by comparative proteome analysis

Background  

The nontoxigenic V. cholerae El Tor strains ferment sorbitol faster than the toxigenic strains, hence fast-fermenting and slow-fermenting strains are defined by sorbitol fermentation test. This test has been used for more than 40 years in cholera surveillance and strain analysis in China. Understanding of the mechanisms of sorbitol metabolism of the toxigenic and nontoxigenic strains may help to explore the genome and metabolism divergence in these strains. Here we used comparative proteomic analysis to find the proteins which may be involved in such metabolic difference.     

以蔗糖为底物利用重组大肠杆菌合成甘露醇

【目的】异型发酵乳酸菌可利用胞内产生的甘露醇脱氢酶将果糖高效转化为甘露醇,但果糖作为底物相对昂贵,不利于工业化生产。为了降低生产成本,必须选择廉价的底物。蔗糖相对便宜,并且大量存在于自然界中,能够被重组大肠杆菌利用产生甘露醇。蔗糖水解酶(Sucrose hydrolase)和甘露醇脱氢酶(Mannitol dehydrogenase)是发酵生产甘露醇中催化蔗糖转化成甘露醇的关键酶,构建蔗糖水解酶和甘露醇脱氢酶共表达菌株并进行相关研究是本文的主旨。【方法】利用PCR方法分别从植物乳杆菌(Lactobacillus plantarum)和布氏乳杆菌(Lactobacillus buchneri)基因组DNA中获得sac A和mdh基因,得到大小分别为1 502 bp和1 032 bp的目的基因,经序列分析后将其连接到表达载体p ET-28a(+)上,得到重组表达载体p ET28a-sac A-mdh。将重组质粒转化到大肠杆菌BL21(DE3)中,并用SDS-PAGE分析目的蛋白的表达情况并测定其酶活。【结果】SDS-PAGE显示表达蛋白的大小亚基分子量分别为55.1 k D和37.8 k D,与预期分子量一致,实现sac A和mdh基因的表达。蔗糖水解酶和甘露醇脱氢酶酶活分别为25.78 U/m L和14.56 U/m L。对重组菌株BL21(DE3)/p ET28a-sac A-mdh进行发酵条件优化,甘露醇质量浓度达到45.19 g/L,总糖转化率为37.66%。【结论】与乳酸菌利用蔗糖发酵生产甘露醇相比,产量提高了6倍,且具有发酵周期短、稳定性高等优点,菌株的成功构建为甘露醇工业化生产奠定了基础。     

Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain.

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and some properties of nontoxigenic derivatives of a strain of Clostridium tetani.

Nontoxigenic derivatives of a toxigenic strain of Clostridium tetani were isolated gy treatment with acridine orange, N-methyl-N'-nitro-soguanidine, rifampicin or ultraviolet light. The frequency of appearance fo non-toxigenic derivatives on these treatments was 0.8 to 3.2 per cent. The nontoxigenic derivatives peoduced all the same extracellular antigenic and protein components as the toxigenic parent strain, except the toxin and materials cross-reacting with the toxin. The nontoxigenic strains, like the toxigenic parent strain, were lyzed by trratment with mitomycin C. Bacteriophage was detected in the lysates of all the nontoxigenic derivatives produced with mitomycin C, and this bacteriophage was morphologically indistinguishable from that obtained from the toxigenic parent strain. The genetic factor controlling tetanus toxin production is discussed.     

Proteomic analysis of two isogenic Vibrio cholerae of the classical biovar with the alternative expression of virulence genes

At carrying out the proteomic analysis of two isogenic Vibrio cholerae Dakka35 of the classical biovar itwas revealed, that toxigenic (1 type) and nontoxigenic (2 type) clones differ from each other not only the expression ofgenes of exopolysaccharide, motility, and soluble haemagglutinin/protease, but also change of activity about other 60 genes. Among 11 identified proteins 5 are the enzymes participating in a metabolism cells. Besides it is revealed, that clones 2 types of Dakka35 strain synthesize in a more level of OmpU and TolC proteins, which provide their more significant stability to action of bile in comparison with clones of 1 type. It was shown, that bile serves as a signal from an environment for switching of gene expression of the genes, coding production of factors as virulence, and carrying out protective function of bacterial cell.     

Effect of lysogenic conversion of Corynebacterium diphtheriae strain on its iron assimilation and spectrum of cellular proteins

Growth of the isogenic pair of C. diphtheriae strains, the nontoxigenic strain C7 (-) and the toxigenic strain C7 (beta), was studied under conditions of limited availability of the iron source. The growth of the toxigenic strain was shown to depend on the concentration of iron in the medium to a lesser degree than the nontoxigenic one. Lysogenic conversion results in the synthesis of additional iron-dependent proteins, absent in C. diphtheriae initial nontoxigenic strain C7 (-). Special attention was paid to proteins with a mol. wt. 66 kD, synthesized by the toxigenic strain irrespective of the concentration of iron in the medium, while in the toxigenic strain these proteins were detected only under conditions of iron deficiency.     

Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.

The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.