我国主要生态区域绿豆慢生根瘤菌的遗传多样性和系统发育研究

利用16SrRNAPCR-RFLP、16SrRNA序列分析以及16S-23SrRNAIGS(IntergeneticSpacer)PCR-RFLP技术对分离自中国主要生态区域的44株慢生型绿豆根瘤菌和5株参比菌株进行了遗传多样性和系统发育研究。16SrRNAPCR-RFLP分析表明:在76%的相似水平上,所有供试菌株可分为三大类群:群I由LYG1等13株慢生根瘤菌组成,该群在系统发育上与B.japonicum和B.liaoningense的参比菌株存在一定的差异;群Ⅱ由XJ1等21株供试菌株、B.japonicum和B.liaoningense的代表菌株组成;群Ⅲ由10株来自广东和广西的菌株和B.elkanii的代表菌株组成。16S-23SrRNAIGSPCR-RFLP分析将供试菌株分为A、B两大群。群A由34株供试菌株、B.japonicum和B.liaoningense的代表菌株组成。在85%的相似性水平上,可再分为AⅠ、AⅡ和AⅢ3个亚群。群B由10株分离自广西和广东的菌株和B.elkanii的代表菌株组成。在85%的相似性水平上,可再分为BI和BⅡ两亚群,表现出一定的多样性。与16SrRNAPCR-RFLP相比,16S-23SrRNAIGSPCR-RFLP具有更高的解析度,供试菌株表现出更加丰富的遗传多样性。分离自中国新疆、广东和广西等地的菌株在分群上具有较为明显的地域特征。     

淋病奈瑟菌临床菌株porB基因分型及其突变与耐药相关性研究

了解淋病奈瑟菌临床菌株porB基因型及其产物120和121位氨基酸突变与耐药的相关性。采用多重PCR(mPCR)检测淋病奈瑟菌临床菌株porB基因型,扩增产物测序后分析其编码蛋白的120和121位氨基酸突变情况,二倍平皿稀释法检测临床菌株对青霉素和四环素的耐药性。184株淋病奈瑟菌临床菌株中,99.5%(183/184)检出porB基因,其中61株(33.3%)为porB1A基因型,122株(66.7%)为porB1B基因型。122株porB1B基因型菌株中,117株(95.9%)porB基因120和/或121位氨基酸发生突变,5株(4.1%)porB1B及所有porB1A基因型菌株porB基因120和/或121位氨基酸未突变。117株porB基因120和/或121位氨基酸突变的porB1B基因型菌株中,97.4%(114/117)和95.7%(112/117)分别对青霉素和四环素耐药,2.6%菌株(3/117)对青霉素和四环素敏感。61株porB1A基因型菌株中,仅有2株(3.3%)对青霉素和四环素耐药。研究中采用的mPCR能快速、准确地对淋病奈瑟菌临床菌株porB基因进行分型,这些菌株主要携带porB1B基因且该基因型菌株对青霉素和四环素耐药率显著高于porB1A基因型(P<0.01),该耐药性与porB1B基因120和/或121位氨基酸突变密切相关。     

内生菌株B16发酵条件优化及其对人参锈腐病的防效

【目的】优化人参病害拮抗菌株B16的发酵条件,提高发酵液的活菌含量和抗菌活性,检测该菌对人参病害的防效。【方法】采用单因子试验、正交试验优化菌株B16的发酵培养基及发酵条件,于室内盆栽条件下研究其对人参锈腐病的防效。【结果】菌株B16发酵最适培养基为:蔗糖1.00%、酵母膏0.50%、Mg SO4·7H2O 0.05%、Fe SO4·7H2O 0.06%、Na Cl 1.00%;最佳发酵条件:p H 7.5、温度35°C、接种量5%、装液量40 m L/250 m L、摇床转速170 r/min、发酵周期48 h。菌株B16发酵液对人参锈腐病的保护作用和治疗作用防效分别达到64.8%和58.6%。【结论】菌株B16具有很强的生防潜力。     

杜氏盐藻促生菌株的分离与鉴定

拟通过探究藻际微生物对微藻生长及代谢产物积累的影响,筛选出促进微藻生长的促生菌株。以杜氏盐藻(Dunaliella salina)Ds-SXYC-2为试材,分离鉴定盐藻藻际环境中的共生菌株,进一步构建藻菌(1∶1)共培养体系、测试盐藻生长及代谢产物积累等表型。结果显示,从杜氏盐藻藻际环境分离获得5株共生菌株,经16S rDNA分子鉴定,属于3个菌属。菌株B1与B2为涅斯捷连科氏菌(Nesterenkonia),菌株B3与B4为盐单胞菌(Halomonas),菌株B5为海杆菌(Marinobacter)。5株共生菌株对杜氏盐藻的生长均有促进作用,菌株B3能显著促进杜氏盐藻生长及代谢产物的积累。共培养15 d后,杜氏盐藻生物量达到2.3 g/L,比对照组增加了28.9%,叶绿素a的含量达到4.61 mg/L,比对照组增加了36.3%,β-胡萝卜素比对照组提高了56.4%。盐藻多糖、蛋白质、总脂含量分别比对照组增加了34.8%、71.2%和37.6%。菌株B3盐单胞菌可以作为促进杜氏盐藻生长及代谢产物累积的优势菌株,进一步构建共培养体系可应用于杜氏盐藻的商业生产。     

灭钉螺微生物的选育及抑螺效果观察

【目的】筛选出一种高效、持久、安全及价廉的灭螺微生物,对其进行鉴定并观察其抑螺功效。【方法】从钉螺孳生的土壤中筛选出4株灭螺活性较强的菌株(B8、B27、B36、B59),显微镜观察菌株形态和革兰氏染色均为G+杆菌。不同分离胶浓度的SDS-PAGE分析其灭螺活性成分;优势菌株经16s rRNA扩增后,PCR产物测序,序列比对,构建系统发育树鉴定其种属。【结果】灭螺结果表明,各试验组中发酵上清液各菌株间灭螺效果差异有统计学意义(χ2=21.286,P=0.002);细菌发酵液各菌株间差异也有统计学意义(χ2=17.298,P=0.008);菌体悬液各菌株间差异无统计学意义(χ2=7.579,P=0.271);此外,B59菌株的灭螺效果优于其它菌株,尤其是其发酵上清液浸泡48 h和72 h钉螺死亡率高达73.3%和96.7%。SDS-PAGE发现在B59细菌上清液中无蛋白带出现,推测其灭螺活性物质可能是其他成分;分子系统发育分析结果显示B59菌株位于Bacillus cereus (CP001746)分支上,一致性达100%。【结论】B59菌株的发酵上清液灭螺效果最好,其灭螺活性物质可能不是蛋白质,B59菌株被鉴定为Bacillus cereus。     

我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

PCR结合酶切-序列比对法鉴定B群脑膜炎奈瑟菌菌株

目的用PCR结合酶切-序列比对法对B群脑膜炎奈瑟菌菌株进行鉴定。方法用玻片凝集法对不同来源的15株B群脑膜炎奈瑟菌菌株进行初步检定,再用PCR结合酶切-序列比对法对上述15株菌株进行进一步鉴定,即用PCR结合酶切法扩增菌株的唾液酸转移酶sia D基因并对PCR产物进行酶切后,用BLAST软件将PCR产物测序结果与Gene Bank中原始sia D序列比对。结果 15株菌株玻片凝集结果均为阳性;15株菌株的PCR产物片段大小均为460 bp;TaqⅠ酶切后,13株菌株的酶切产物片段大小仍为460 bp,其PCR产物测序比对结果与B群脑膜炎奈瑟菌原始sia D序列同源性均达到99%;其余2株酶切产物片段大小约200 bp,与C群脑膜炎奈瑟菌sia D原始基因序列同源性分别为98%和99%。结论 15株菌株经PCR结合酶切-序列比对法鉴定,13株为B群脑膜炎奈瑟菌菌株,2株为C群脑膜炎奈瑟菌菌株;该方法可准确鉴定B群脑膜炎奈瑟菌菌株。     

甘蔗茎内两个内生菌株16S rRNA 序列分析及其生长特性

从两个甘蔗品种GT11和RB86-7515表面灭菌的茎中分离到两株具有固氮活性的菌株,分别编号为B11S和B8S.利用16S rRNA序列分析对其进行鉴定,并对两个菌株的生物学特性进行了比较.结果表明:B11S菌株与Stenotrophomonas maltophili菌株处在同一个分支,其序列与多个Stenotrophomonas maltophili菌株的序列相似性都达到98%以上,菌株B8S与多个土壤杆菌属细菌的序列相似性达到100%;两个菌株在温度31℃、pH为6左右时生长量最高;温度31℃、pH为6.5～7.0时固氮酶活性最高;相同浓度的蔗糖生长量大于葡萄糖,且表达固氮酶活性也最强;添加一定量的氮素有利于细菌生长和固氮酶活性的表达,但随着N含量的增加,抑制作用越来越明显.     

紫外诱变耐糖选育D-核糖高产菌株

以D-核糖产生菌枯草芽胞杆菌B2502为出发菌株,经紫外诱变、耐糖选育,得到1株可在D-核糖浓度达6%的肉汤液体培养基中生长的菌株B2502-2。其D-核糖产量提高37%,菌体生长达到对数期的时间缩短3 h,耐糖性比出发菌株提高了50%。     

以综合能力为标准筛选草莓灰霉病生防细菌

在草莓叶表、果表分离筛选出3株枯草芽孢杆菌(Bacillus subtilis)B1、B17和B18。3菌株对灰葡萄孢(Botrytis cinerea)孢子萌发的抑制作用达到100%、99%和85%,平板对峙抑菌距离达到6.6、7.0和6.7mm。在适宜病原菌生长的22℃、pH5条件下,3菌株生长良好。在草莓叶片、花和果实上定植能力B1>B17>B18,定植周期13d。     

Spore pool glutamic acid as a metabolite in germination

Spore glutamic acid pools were examined in dormant and germinating spores using colorimetric and (14)C analytical procedures. Germination of spores of Bacillus megaterium (parent strain), initiated by d-glucose, was accompanied by a rapid drop in the level of spore pool glutamate, from 12.0 mug/mg of dry spores to 7.7 mug/mg of dry spores after 30 sec of germination. Similar decreases in extractable spore pool glutamate were observed with l-alanine-initiated germination of B. licheniformis spores. On the other hand, glutamate pools of mutant spores of B. megaterium, with a requirement of gamma-aminobutyric acid for spore germination, remained unchanged for 9 min of germination, at which time more than 50% of the spore population had germinated. Evidence for conversion of spore pool glutamate to gamma-aminobutyric acid during germination of spores of B. megaterium (parent strain) was obtained.     

Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus

The folC gene from mutant strain SF4 was cloned into a pUC19 plasmid. Expression of the mutant gene from the lac promoter of the plasmid complemented the auxotrophy for methionine of the SF4 strain. The only difference in sequence between the mutant and wild-type genes was a G925A base change resulting in an A309T amino acid change. The mutant enzyme had a 30-fold higher Km for 10-formyltetrahydrofolate as well as a 60-fold higher Km for glutamate and a 200-fold higher Km for dihydropteroate of the dihydrofolate synthetase activity. Site-specific mutagenesis was used to substitute other amino acids at codon 309. Mutants with glycine, isoleucine, and valine substitutions at this position, when expressed from multicopy plasmids, complemented the SF4 strain. The glycine mutant had properties similar to the wild-type enzyme, whereas the isoleucine and valine mutants had properties similar to the threonine mutant, SF4. Mutant genes with arginine, glutamate, and leucine substitutions, which did not complement the SF4 strain, could complement a folC deletion strain, but produced smaller colonies on complex plates and did not grow on minimal medium. In the deletion strain, an increasing requirement for folate product supplements was observed as the folylpolyglutamate synthetase-dihydrofolate synthetase activities of the complementing mutants decreased.     

Enhancement of bacitracin biosynthesis by branched-chain amino acids in a regulatory mutant ofBacillus licheniformis

DL-4-Azaleucine-resistant mutant of Bacillus licheniformis azlr-1 isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, was a better bacitracin producer than the parent strain. In the minimal medium, the antibiotic biosynthesis was 4 times higher in the mutant than in the parent strain and less dependent on L-leucine addition. In the complex fermentation medium, the yield was 18-20% higher in the mutant strain. Transaminase B activity measured in the crude extract revealed that the branched-chain amino acid biosynthetic enzymes were 5-10 times derepressed supplying bacitracin synthetase with enhanced quantity of isoleucine and leucine, the building units of bacitracin molecule.     

Regulation of carbon and nitrogen flow by glutamate synthase in Neurospora crassa

A glycine-resistant Neurospora crassa mutant (am-132;glyr), derived from the am-132 mutant, was isolated and characterized. [am-132 itself has a deletion in the structural gene for NADP-dependent glutamate dehydrogenase (GDH).] This new mutation also conferred resistance to serine and methionine sulphoximine (MS), which are inhibitors of glutamine synthetase (GS). In addition, the mutant obtained grew better on ammonium than the am-132 parental strain. Resistance to glycine was not due to increased synthesis of glutamine by an altered or induced GS, nor to increased glutamate synthesis by induction of the catabolic NAD-dependent GDH, nor to NADH-dependent glutamate synthase (GOGAT), which was as sensitive to inhibitors as the GOGAT from the parental strain. The glycine-resistance mutation lowered but did not abolish the carbon flow; this resulted in a lower content of tricarboxylic acid cycle intermediates. GOGAT activity was inhibited in vitro by several organic acids and methionine sulphone (MSF). The higher growth rate of the glycine-resistant mutant on ammonium or on ammonium plus glycine, serine or MS was explained by an increased capacity of GOGAT to synthesize glutamate in vivo due to a lower content of inhibitory tricarboxylic acid cycle intermediates; the higher glutamate content overcomes the effect of the GS inhibitors and explains the MSF resistance of the mutant.     

通过基因工程构建谷氨酸生产菌B44的研究

从大肠杆菌E.colik-12中通过PCR克隆出磷酸果糖激酶编码基因(pfkA),将其连到表达载体pCMVTNTTMvector。连接构建成重组质粒Ku-1,导入谷氨酸棒杆菌B4(已经诱变改造),并得到表达。酶活性测定表明Ku-1的pfkA基因在B44中得到表达(磷酸果糖激酶为128.6±0.86U/g蛋白)。解除磷酸果糖激酶对已经改造的谷氨酸的整个代谢途径的限制。同时,B44对糖转化率比B4(由出发菌株B1诱变而来)高10.64%,产酸率比B4高17.1%。     

Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.     

Metabolic changes in Saccharomyces cerevisiae strains lacking citrate synthases

The yeast, Saccharomyces cerevisiae, contains two citrate synthase isoenzymes, mitochondrial (CS1) and cytosolic (CS2). In this study, we have examined the metabolic consequences of the absence of CS1, CS2, and both isoenzymes in the respective mutant strains CS1-, CS2-, and CS1-CS2-. No significant differences were found in the growth rates of the parental, CS1-, or CS2- strains when grown in the single carbon sources galactose, glycerol, lactate, pyruvate, or glutamate. However, in nonfermentable carbon sources, the lag period in growth of CS1- was approximately 4 times that of the parental strain and the CS2- mutant. This difference was found even in glutamate. The CS1- mutant failed to grow on acetate in either complete or minimal liquid medium. Total cellular citrate concentration in the CS1- compared to the parental strain was higher when the cells were grown in lactate or pyruvate. On these same substrates, the malate concentration was 2-fold higher in the CS1-mutant when compared to the parental or CS2- strains. The production of 14CO2 by CS1- from [1-14C]acetate was 36% and that from [2-14C]acetate was 9.2% of the amount from the parental or CS2- strains. The 14CO2 production from [1-14C]glutamate was 28% and 20% in CS1- and CS1-CS2-, respectively, compared to the parental strain. Since these results are not easily explained solely by the absence of mitochondrial citrate synthase enzyme, we also determined the activity of some other enzymes of the citric acid cycle and electron transport chain. We found decreased activity of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and aconitase, while the rest of the citric acid cycle enzymes and oxidative enzymes did not change significantly. The same changes in enzyme activities were found in two different yeast strains carrying the same citrate synthase mutations.     

Production of l-Glutamic Acid from Hydrocarbon by Penicillin-resistant Mutants of Corynebacterium hydrocarboclastus

Penicillin-resistant mutants were derived from Corynebacterium hydrocarboclastus R-7. One of them produced 84 g/liter of l-glutamic acid from hydrocarbon, though its parent strain produced 26 g/liter.

The penicillin-resistant mutant had stronger activities of substrate consumption and oxygen absorption than the parent strain, and this was one of the reasons for the accumulation of a larger quantity of l-glutamic acid.

The interacellular content of phosphatidyl inositol mannoside (P.I.M) was related to the glutamate productivity, and the higher glutamate productivity of the penicillin-resistant mutant was supposed to be related to the remarkable diminution in the content of P.I.M.     

Formation of 2,5-Bis-(d-threo-trihydroxypropyl)pyrazine and Its 6-Isomer by the Oxidative Browning Reaction of d-Xylose with Ammonium Acetate in a Methanolic Medium

A pyruvate kinase-lacking mutant of Brevibacterium flavum produced 22.6 g/liter of l-aspartic acid with glutamic acid as a by-product, when cultured for 48 hr in a medium containing 100 g/liter of glucose. The production clearly depended on the amount of biotin added. This strain, 70, was derived by several steps of mutation from wild strain 2247 producing glutamate, successively via a citrate synthase-defective glutamate auxotroph, strain 214, a prototrophic revertant, strain 15-8, producing 10 g/liter of l-aspartic acid, and an S-(2-aminoethyl)-l-cysteine-resistant mutant, strain 1-231, having low pyruvate kinase and homoserine dehydrogenase and producing lysine. Strain 70, a methionine-insensitive revertant from strain 1-231, had a normal level of homoserine dehydrogenase but no pyruvate kinase. Its citrate synthase activity was about half that of the wild strain at saturated concentrations of the substrates with Michaelis constants for oxalacetate and acetyl-CoA of 110 and 6 times as high as those of the wild-type enzyme, respectively. The mutational step for these alterations in citrate synthase was strain 15-8. Phosphoenolpyruvate carboxylase of strain 70 showed 1.5-fold higher activity in the crude extract at saturated concentrations of phosphoenolpyruvate, a lower Michaelis constant (1.5mM).for the substrate, phosphoenolpyruvate, less sensitivity to the feedback inhibition by aspartate, and higher sensitivities to the activators, acetyl-CoA and fructose-1,6-bisphosphate, than those of the wild strain. The concentrations of aspartate giving 50% inhibition were 6.2- and 4.5-fold higher in the absence and presence of acetyl-CoA, respectively.