蛭弧菌J26转化山梨糖生产2—酮基—L—古龙酸发酵条件的研究

对新分离得到的能产生维生素C前体2-酮基-L-古龙酸（2-KGA）的优良菌株蛭弧菌J26,进行了一系列摇瓶发酵条件试验,通过添加一些其它有机碳源或其它氮源,可使蛭弧菌J26摇瓶发酵产生2－KGA由50-60mg／ml上升到70mg／ml以上,采用SMA探针技术的流加发酵摇瓶试验产生2－KGA可以达到83．9－91．7mg／ml。本文还对发酵时种子液的接种量、发酵初始精浓度、初始pH值等对产酸的影响,进行了比较研究,对摇瓶发酵全过程的单一菌体形态特征进行了电子显微镜观察。     

噬菌蛭弧菌生物特性研究进展

本文介绍了噬菌蛭弧菌的培养特性,温度对噬菌蛭弧菌的影响,噬菌蛭弧菌的生化特征,感染宿主的机制,在死的宿主菌中生长的特性,宿主的特异性以及噬菌蛭弧菌对致病菌的生物净化作用等生物特性的研究进展。研究认为,噬菌蛭弧菌是自然环境(水、土壤)中致病微生物的生物拮抗体,且极有可能利用它的寄生和溶解宿主菌细胞的特殊性,对环境水体的生物净化。     

蛭弧菌BDE-1的生物特性及促进其蛭质体形成的研究

【背景】蛭弧菌是众多海洋益生菌中的一类较新成员,应用前景十分广阔。但由于蛭弧菌特殊的繁殖方式和周期,它的应用效果受寄生宿主特性和生物活性的影响,因而优选寄生宿主,维持或者提高蛭弧菌微生态制剂的应用活性是关键。【目的】筛选出能够裂解枯草芽孢杆菌的蛭弧菌,以增进其益生性能;研究提高蛭弧菌的蛭质体密度,以利于保存。【方法】从海南取回海泥样后,以枯草芽孢杆菌作为宿主菌,通过稀营养肉汤(Dilute nutrient broth,DNB)双层平板法分离获得蛭弧菌,并对目标菌株进行透射电镜形态鉴定和16S rRNA基因序列分析;然后进行生物学特性研究,同时开展氨苄青霉素、吲哚、Ca~(2+)和Mg~(2+)影响蛭质体形成的研究。【结果】分离出一株以枯草芽孢杆菌作为宿主的蛭弧菌并命名为BDE-1,其最适温度、盐度和pH分别为25℃、2.0%和7.0;BDE-1可裂解24株试验菌,占总试验菌株数(28株)的85.7%,其中对试验弧菌(13株)的裂解率达92.3%;吲哚、氨苄青霉素、Ca~(2+)和Mg~(2+)4种因子对BDE-1蛭质体的形成均有促进作用,其中吲哚和Ca~(2+)的促进作用显著。【结论】研究结果不仅为蛭弧菌寄生宿主的优化选择提供了可行性解决思路,而且为维持或提高蛭弧菌微生态制剂的应用活性提供了理论依据。     

一株广盐性蛭弧菌的生物学特性

【背景】蛭弧菌有裂解水产养殖中常见致病菌的能力,具有重要的潜在应用价值,但在实际应用中,存在着菌株生长条件与应用环境不相符而导致效果差乃至无效果等问题。因此获得适应范围宽的蛭弧菌甚为关键。【目的】筛选出一株广盐性蛭弧菌以利推广应用;提升蛭弧菌蛭质体密度以利保存。【方法】以枯草芽孢杆菌为宿主,于浅滩水域分离纯化出一株广盐性蛭弧菌;对该广盐性蛭弧菌菌株进行分子生物学鉴定;之后探究其生物学特性及裂解性能,并研究谷氨酸钠、Ca~(2+)和Mg~(2+)、吲哚等影响蛭质体密度的因素。【结果】分离获得一株广盐性蛭弧菌BDN-1,其适宜温度、盐度和pH范围分别为20-30°C、0.5%-3.0%、6.0-8.5;BDN-1对30株受试菌的裂解率为86.7%,对其中16株受试弧菌裂解率为87.5%;谷氨酸钠、吲哚、Ca~(2+)和Mg~(2+)对BDN-1蛭质体密度有提升作用。【结论】研究结果提供了一株裂解能力强且海淡水均可应用的蛭弧菌菌株,查明了其生物学特性及影响其蛭质体密度的因素,为蛭弧菌的高效利用奠定了基础。     

鱼病蛭弧菌的微生态学初步研究

本次以鲫鱼出血性腹水病病原菌点状产气单胞菌（Ａｅｒｏｍｏｎａｓ Ｐｕｎｃｔａｔａ）为宿主菌。从自然界分离获得６株噬菌蛭弧菌菌株,它们具有噬菌蛭弧菌的生物学特性。研究表明,这６株蛭弧菌的最适培养条件为温度２５￣２８℃,ｐＨ７．２,有溶菌性,能裂解多种鱼类病原菌。其中对蛭弧菌Ｂｄｓ－４菌株在实验条件下对病原菌的自然净化作用等方面进行了研究。说明蛭弧菌是精养鱼糖水体中某些致病菌的自然净化的重要生物因素之     

异育银鲫肠道蛭弧菌的分离及其生物学特性的研究

以一株具有致病力的温和气单胞菌作为筛选宿主菌,从异育银鲫肠道中分离到一株蛭弧菌,暂命名为BDF-H16。通过光学显微镜、相差显微镜、电子显微镜对蛭弧菌BDF-H16进行形态观察,并研究了其部分生物学特性。结果表明:蛭弧菌BDF-H16为革兰氏阴性菌,杆形或弧杆形,端生一根鞭毛,菌体大小多为0．2μm～0．5μm×0．8μm～1．2μm;蛭弧菌BDF-H16对实验所选用的革兰氏阴性菌和部分革兰氏阳性菌均有裂解作用;以大肠杆菌为宿主菌,其最佳生长条件为宿主菌浓度6．75×10~9cfu/mL、pH7．0～7．5、温度28℃;在NaCl含量0．85%～5．00%的培养基中能够生长;恩诺沙星、诺氟沙星对其有抑制作用。     

异育银鲫肠道蛭弧菌的分离及其生物学特性的研究

以一株具有致病力的温和气单胞菌作为筛选宿主菌,从异育银鲫肠道中分离到一株蛭弧菌,暂命名为BDF-H16。通过光学显微镜、相差显微镜、电子显微镜对蛭弧菌BDF-H16进行形态观察,并研究了其部分生物学特性。结果表明:蛭弧菌BDF-H16为革兰氏阴性菌,杆形或弧杆形,端生一根鞭毛,菌体大小多为0．2μm～0．5μm×0．8μm～1．2μm;蛭弧菌BDF-H16对实验所选用的革兰氏阴性菌和部分革兰氏阳性菌均有裂解作用;以大肠杆菌为宿主菌,其最佳生长条件为宿主菌浓度6．75×10⁹cfu/mL、pH7．0～7．5、温度28℃;在NaCl含量0．85%～5．00%的培养基中能够生长;恩诺沙星、诺氟沙星对其有抑制作用。     

不同条件下蛭弧菌裂解河流弧菌的研究

选择四种不同培养液,控制温度、pH和Ca＋＋Mg＋＋离子的不同水平,进行蛭弧菌HD94－12－7对河流弧菌WY91－24－3的裂解实验,结果表明,在灭菌蒸馏水、灭菌自来水、灭菌池塘水及Trisbuffer培养液中蛭弧菌HD94－12－7均表现出良好的裂解活性;温度在20—35℃时,蛭弧菌的裂解活性最大,低于15℃时,蛭弧菌的裂解能力明显减弱;pH值在6.5—8．1之间,蛭弧菌的裂解强度最大,pH低于5．6或高于8．1时,不利于蛭弧菌的生存。Ca＋＋,Mg＋＋离子能明显提高蛭弧菌的裂解活性。本研究证实了在实验条件下蛭弧菌对鱼类致病菌一河流弧菌具有明显地清除作用。     

大豆根瘤菌蛭弧菌的发现*

从我国的一些大豆产区土壤中分离出四种大豆根瘤菌的噬菌蛭弧菌,对它们的培养特征、宿主范围以及其它生物学特性进行了初步研究,发现:大豆根瘤菌蛭弧菌具有典型的蛭弧菌特征,有极广泛的寄主范围和很强的裂解大豆根瘤菌的能力。它是影响结瘤效果和降低大豆产量的主要生物因子之一。     

人粪便中分离的噬菌蛭弧菌生物学特性的研究

本文研究了人粪便中分离的噬菌蛭弧菌的生物学特性,测定了它们的生长曲线,并利用微孔滤膜过滤和机械振荡的方法,研究了它们的吸附和穿入动力学,发现链霉素、庆大霉素和卡那霉素能抑制蛭弧菌的吸附;青霉素不影响蛭弧菌的吸附和穿入,但抑制蛭弧菌在宿主内的生长过程。从人粪中分出的噬菌蛭弧菌不仅能裂解大部分需氧的革兰氏阴性菌,而且在微氧条件下也能裂解厌氧菌中的二株脆弱拟杆菌。我们发现一株人粪便中分出的噬菌蛭弧菌能形成蛭弧菌囊体——它的休眠态,它对高热、紫外线照射和真空干燥的耐受力较相应的繁殖体强,看来它是蛭弧菌保持生命期限的一种方式。     

Respiration of Bdellovibrio bacteriovorus Strain 109J and Its Energy Substrates for Intraperiplasmic Growth

Measurements of oxidation rates, respiratory quotients (RQ), and release of (14)CO(2) from uniformly labeled substrates showed that glutamate, alpha-ketoglutarate, and synthetic and natural amino acid mixtures are oxidized by suspensions of Bdellovibrio bacteriovorus strain 109J. The oxidation of these substrates largely suppress the endogenous respiration of the Bdellovibrio cells and may or may not cause a small increase, 20 to 50%, in their rate of oxygen consumption. The failure of respired substrates to increase markedly the respiration rate of the Bdellovibrio cells over the endogenous value is discussed. Carbon from these substrates is incorporated into the Bdellovibrio cells during oxidation. Acetate is also oxidized, but its oxidation inhibits endogenous respiration by only about 40% and no acetate is assimilated. The RQ of the Bdellovibrio cells changes from a value characteristic of endogenous respiration to that characteristic of the oxidation of glutamate or of a balanced amino mixture very shortly after the attack of the Bdellovibrio cells on their prey, and the latter RQ is maintained during intraperiplasmic growth. Glutamate, or a mixture of amino acids in the external environment, contributes to the carbon dioxide produced by the Bdellovibrio cells growing intraperiplasmically. It is concluded from these data that amino acids, derived from the breakdown of the protein of the prey, serve as a major energy source during intraperiplasmic growth of B. bacteriovorus 108J. Insofar as they were tested, B. bacteriovorus strains 109D and A. 3. 12 were similar in respiration to strain 109J.     

Chemotaxis of Bdellovibrio bacteriovorus toward pure compounds.

Positive chemotaxis by Bdellovibrio bacteriovorus strain UKi2 was measured for 139 compounds. Twenty-one compounds were attractants; sensitive attraction was elicited by acetate, propionate, thioacetate, malonate, cis-oxalacetate, D-glucose-6-phosphate, acetyl coenzyme A, ammonium ion, barium ion, manganous ion, and potassium ion. Several of the attractants for B. bacteriovorus strain UKi2 also were attractants to strains 6-5-S and 114; however, strains 109D and 109J were not attracted by the compounds tested. Of 33 compounds tested, 8 were repellents for B. bacteriovorus strain UKi2: n-caproate, alanine, isoleucine, leucine, phenylalanine, tyrosine, cobaltous chloride, and hydronium ion. None of the organic repellents for strain UKi2 elicited repulson from strains 114 or 109D. However, all three strains of Bdellovibrio show aerotaxis. Several compounds were tested for their effects on viability and predacious growth of B. bacteriovorus strain UKi2. No simple correlation was found between attraction or repulsion and benefit or harm to bdellovibrios. The data are consistent with the view that in nature, the greatest survival value of chemotaxis for bdellovibros may be in aerotaxis, attraction to certain inorganic ions and acetate, and repulsion by hydronium ion.     

Verification of the protein in the outer membrane of Bdellovibrio bacteriovorus as the OmpF protein of its Escherichia coli prey.

Two research groups showed that several Bdellovibrio strains incorporated into their outer membranes intact OmpF porin proteins derived from their Escherichia coli prey. These results could not be reproduced by another group using Bdellovibrio bacteriovorus 109J. They showed that a major protein appearing in the Bdellovibrio Triton X-100-insoluble outer membrane was coded for by the bdellovibrios. We reconciled these results by examining the strain used by this group and by reviving a freeze-dried culture of strain 109J which had been stored for almost 9 years. B. bacteriovorus 109J failed to acquire substantial amounts of the OmpF protein from E. coli ML35, and a protein coded for by the bdellovibrios was expressed in its place. However, B. bacteriovorus 109J incorporated the OmpF protein from rough K-12 strains of E. coli, and the revived 9-year-old culture of B. bacteriovorus 109J incorporated more of the OmpF protein from the smooth E. coli ML35 than did its contemporary counterpart. The protein isolated from the outer membrane of the bdellovibrios was identified as the OmpF protein of E. coli by its protease peptide profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot analysis. This confirmed that bdellovibrios relocalize outer membrane proteins from their prey, but relocalization may be an unstable trait which can be influenced by the prey.     

山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析

酮古龙酸杆菌Ketogulonigenium vulgare是维生素C二步混菌发酵过程中的产酸菌。山梨酮脱氢酶(L-sorbosone dehydrogenase,缩写为SNDH)作为维生素C直接前体2-酮基-L-古龙酸(2-KGA)合成的关键酶,其作用机制并不十分清楚。借助全基因组测序抽提2个山梨酮脱氢酶基因,分别位于基因组(缩写为sndhg)和质粒(缩写为sndhp)上。通过工程化改造技术在工业产酸菌中构建山梨酮脱氢酶功能模块,比较其对2-KGA产量的影响。研究发现sndhg过表达对菌株产酸影响不明显,sndhp过表达使菌株明显产生副产物。将sndhg和sndhp分别配合辅因子PQQ合成基因pqq A,分别构建sndhg-pqq A和sndhp-pqq A模块,得到的工程菌株产酸情况与之前的结果大致相同。将4株K.vulgare工程菌株分别与内生芽孢杆菌Bacillus endophyticus混合培养传代50 d后,分离菌株进行混菌发酵,其2-KGA的转化率分别提高了15.4%、179%、0.65%和125%。表明混菌适应性进化策略是一种增加功能模块与底盘细胞适配性,进而快速获得优良性状菌种的有效方法。     

A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-d-gluconic acid

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO₂, 0.05% pCO₂) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.     

Biotransformation of glucose to 5-keto-<Emphasis Type="SmallCaps">d</Emphasis>-gluconic acid by recombinant<Emphasis Type="Italic"> Gluconobacter oxydans</Emphasis> DSM 2343

For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a precursor of the industrially important l-(+)-tartaric acid, Gluconobacter strains were genetically engineered. In order to increase 5-KGA formation, a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreductase (gno) was overexpressed in G. oxydans strain DSM 2434. This enzyme is involved in the nonphosphorylative ketogenic oxidation of glucose and oxidizes gluconate to 5-KGA. As the 5-KGA reductase activity depends on the cofactor NADP⁺, the sthA gene (encoding Escherichia coli transhydrogenase) was cloned and overexpressed in the GNO-overproducing G. oxydans strain. Growth of the sthA-carrying strains was indistinguishable from the G. oxydans wild-type strain and therefore they were chosen for the coupled overexpression of sthA and gno. G. oxydans strain DSM 2343/pRS201-gno-sthA overproducing both enzymes showed an enhanced accumulation of 5-KGA.     

Three-Membered Parasitic System: a Bacteriophage, Bdellovibrio bacteriovorus, and Escherichia coli

Two bacteriophages for Bdellovibrio bacteriovorus were isolated. One of the phages (VL-1) was isolated on a host-independent Bdellovibrio strain, and the other (VL-2) was isolated on a host-dependent strain. Both phages grew on host-dependent as well as on host-independent Bdellovibrio strains. The development of the phages in host-dependent bdellovibrios occurred only when the phage-infected bdellovibrios parasitized cells of other bacteria. In the absence of other bacteria, the phages adsorbed to the bdellovibrios and killed them and in the process lost their own plaque-forming ability.     

Prey range characterization, ribotyping, and diversity of soil and rhizosphere Bdellovibrio spp. isolated on phytopathogenic bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 x 10(2) to 6 x 10(3) and 2.8 x 10(2) to 2.3 x 10(4) PFU g(-1). A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

A new model for the penetration of prey cells by bdellovibrios.

Bdellovibrio bacteriovorus 109J and most other bdellovibrios cause prey cells to round following penetration. Bdellovibrio sp. strain W does not cause rounding of the prey. Analysis of enzyme activities during the early stages of bdellovibrio attack indicated that strain W differs from most other bdellovibrios in that there is no glycanase activity produced during penetration. Likewise, heat-killed prey were penetrated normally by strain 109J, but the resulting bdelloplast did not become round and no glycanase was detected, indicating that glycanase is not essential for penetration. Peptidoglycan from prey cells penetrated by strain W was sensitive to lysozyme, but these cells were not susceptible to attack and penetration by strain 109J, indicating that peptidoglycan deacetylation is not the primary exclusion mechanism. We propose a model in which it is the peptidase activity of the bdellovibrios which allows them to breach the peptidoglycan of their prey and in which the glycanase activity exhibited by strain 109J and other bdellovibrios is responsible for the rounding of the bdelloplast.