热带假丝酵母细胞内pH的测定及其与生长代谢活性的关系

应用荧光探针5(6)-双醋酸羧基荧光素 (Carboxyfluorescein diacetate) 测定了产长链二元酸热带假丝酵母 (Candida tropicalis) 细胞内pH (pHi) 值,确定了该探针载入C. tropicalis细胞的适宜条件。用摇瓶培养C. tropicalis细胞,考察了细胞外pH和生长碳源对pH_I的影响,实验结果表明：细胞外pH对pH_I略有影响,而生长碳源对pH_I的影响略为明显。利用5L发酵罐进一步研究了细胞生长代谢活性与pHi的关系,结果表明：细胞比生长速率、CO₂比生产速率和葡萄糖比消耗速率与pHi变化密切相关,pH_I的增加伴随着细胞生长活力的增加,反之亦然。在pH6.0条件下用葡萄糖和醋酸钠共作碳源培养C. tropicalis细胞时,测得的pH_I值维持在5.72～6.15范围内。     

百里香提取物抑菌特性的研究

以百里香水和酒精提取物及百里香芳香油作为抑菌剂进行抑菌试验 ,结果表明 ,所用抑菌物对供试菌金黄色葡萄球菌 (Staphalococcusaureus)、枯草芽孢杆菌 (Bacillussubtilis)和大肠杆菌 (Escherichiacoli)均有不同程度的抑制作用 ,可望将其用于食品工业作为防腐和抑菌剂。     

芽孢杆菌属的一个新种

从苏芸金芽孢杆菌戈氏变种(Bacill thuringiensis var. Kurstaki)的混杂培养物中,分离得到编号为55B的三株菌。与已知的芽孢杆菌相比,在形态、生理生化和营养特征方面均不相同。其主要特征为：同时具有圆形和椭圆形两种芽孢,在芽孢游离之前,二者比例为1／3一1／2;游离后,比例降至1／10。好氧。在5％NaCI中生长,而在10％NaCl中不生长。不从葡萄糖产酸。不还原硝酸盐。产脲酶和酪氨酸酶,但苯丙氨酸不脱氨。不水解淀粉,也不产生二羟基丙酮。 以酪朊为唯一碳、氮源时,生长需要外源吡哆醛。其DNA中G+。含量为42 3—43．2克分子％。我们将其定为新种,根据菌落极易扩散的特点,命名为扩散芽孢杆菌(Bacillus diffusus Cai ＆ Wang sp. Nov.)。     

芽孢杆菌原生质体作为质粒DNA转化的受体

枯草芽孢杆菌(Bacillus subtilis) B3F 7658,短小芽孢杆菌(B. pumilus) AS 1.940,巨大芽孢杆菌(B．Megaterium) AS 1.941,和多粘芽孢杆菌(B. polymyxa) AS 1.878等菌株,既不能作为染色体DNA的转化受体,也不能作为质粒DNA的转化受体。用不同量的溶菌酶处理这些菌株形成原生质体,然后加pUB110质粒DNA,经聚乙二醇6000(PEG)诱导,在含新霉素(400μg／ml)的DM-3再生培养基上恢复细胞壁,培养48小时后,转化子数为1.0 x 103一4.6×105／μg DNA。若同时用PEG和Ca2+ 离子诱导,转化子数可提高2—3倍。质粒pUBll0用EcoRI酶切后,转化子数大大下降(2.0×102转化子／μg DNA)。Eco RI酶切后,用T4连接酶连成环状,转化子数有所增加(1．7×103转化子／μg DNA)。     

地衣芽孢杆菌产生碱性蛋白酶的动力学研究

应用自动控制发酵设备,首先进行分批发酵试验摸索了地衣芽孢杆菌2709生长与代谢的基本规律。然后采用补料分批发酵方法限制生长基质浓度,测定了一系列(S_I,μ_I)、(μ_j,q_pj)数据,获得K_S、μ_max、α、β等参数的值,并且推导出了细胞生长与产物合成的动力学公式,从而证明了用Monod方程描述地衣芽孢杆菌2709生长速率与基质浓度关系的合理性和合成碱性蛋白酶的发酵属于生长部分关联型。     

细菌超低温冻结保藏的研究

本文报道10属19种19株细菌超低温冻结保藏试验的结果。从细胞存活率看,冻结保藏8个月,10％甘油、10%二甲基亚砜保护剂保藏效果优于蒸馏水作保护剂,少数菌株三种保护剂保藏效果相近。快速冻结与慢速冻结对细咆存活率影响不显著。恶臭醋杆菌混浊变种(Acelobacter rancens var. turbidans) AS 1.41,产氨短杆菌 (Brevibacterium ammoniagenes) AS1．844,细胞悬液浓度大,细胞存活率有增高趋势。电镜观查,超低温冻结细胞死亡率高的产气气杆菌(Aerobacter aerogenes) AS 1.489有胞壁破裂、胞质溢出现象,是细胞死亡原因之一。冻结融化后直接测定,植物乳杆菌(Lactobacillus plantarum) AS 1.557乳酸生成力下降3．4—13.8％,溶壁小球菌(Micrococcus lysodeikticus) AS 1.634对溶菌酶敏感性下降14—23％。冻结融化后移接2代测定,钝齿棒杆菌(Corynebactertum crenatum) AS 1.998 产 L-异亮氨酸,大肠埃希氏菌(Escherichia coli) AS 1.76产青霉素酰化酶酶活力,铜绿假单胞菌(Pseudomonas aeruginosa) AS 1．647产2-酮基-L-古龙酸,植物乳杆菌产乳酸均与冻结前相近。     

微囊藻毒素对典型微生物生长及生理生化特性的影响

研究了不同浓度微囊藻毒素对典型微生物大肠杆菌和枯草芽孢杆菌生长及生理生化特性的影响。微囊藻毒素对大肠杆菌和枯草芽孢杆菌的生长和细胞活性具有一定的剂量效应,较高浓度微囊藻毒素对其生长和活性有短时间的抑制作用,随着处理时间的延长,细胞的生长和活性逐渐恢复。细胞内可溶性糖和可溶性蛋白的含量,处理组和对照组相比均有先上升后下降的趋势。结果表明,微囊藻毒素的处理对大肠杆菌和枯草芽孢杆菌具有一定的胁迫作用,细胞通过调节细胞内可溶性蛋白和可溶性糖的含量来抵抗外界胁迫,但随着处理时间的延长,细菌逐渐适应了这种胁迫,恢复正常的生长。     

谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成

为阐明谷氨酸棒杆菌的乙醛酸循环与菌体的生长以及谷氨酸合成之间的关系 ,以谷氨酸棒杆菌基因组测序用典型菌株Corynebacteriumglutamicum ATCC 130 32为出发菌株 ,构建了乙醛酸循环途径缺失的谷氨酸棒杆菌突变株Corynebacteriumglutamicum WTΔA。该菌株没有异柠檬酸裂解酶活性 ,不能在以乙酸盐为唯一碳源的基本培养基上生长。与出发菌株ATCC13032相比 ,WTΔA在以葡萄糖为唯一碳源的培养基上生长时不受影响 ,说明谷氨酸棒杆菌并不需要乙醛酸循环途径提供菌体生长所需的能量和生物合成反应所需的中间产物。但是 ,与出发菌株ATCC13032相比 ,WTΔA的谷氨酸合成能力大幅下降。     

芽孢杆菌E2菌株纤维素酶形成条件的研究

芽孢杆菌E,菌株(Bacillus sp.strain E2)能在55℃下良好生长并在培养液中大量积累胞外纤维素酶(190 mu／ml培养液),所产生的纤维素酶为单一的CMCa se。对芽孢杆茁E2菌株产酶条件进行了研究． 该菌产酶的最适培养基装量为200ml/500mI三角瓶,最适起始pH为6 5,最适产酶温度为45℃,产酶高峰在培养时间8—12小时。E2菌株不能利用单一的无机氮源形成纤维素酶。酪蛋白是试验过的供E2菌株形成纤维素酶的最好氮源,其用量为3g／L。CMC—Na,纤维二糖,能作为碳源供芽孢杆菌E2菌株形成纤维素酶。高浓度的葡萄糖(8g／L)对芽孢杆菌E2菌株纤维素酶的形成有抑制作用。天然纤维素不能作为芽孢杆菌E2菌株形成纤维素酶的碳源。     

amrG1基因在谷氨酸棒杆菌乙酸活化中的作用

谷氨酸棒杆菌Corynebacterium glutamicum可以利用乙酸为碳源和能源进行生长. 乙酸代谢中涉及乙酸活化的两个酶为磷酸转乙酰酶PTA和乙酸激酶AK, 它们是由pta-ack操纵子经诱导表达产生的. 采用转座子挽救法, 我们从调控突变株C. glutamicum G25中获得了amrG1和amrG2两个目标基因. 经分析鉴定, amrG1基因(NCBI GenBank 接受号为AF532964)可能参与乙酸代谢调控, 编码作用于pta-ack操纵子的一个调控因子. 该调控因子基因序列全长732 bp, 开放阅读框含有243个氨基酸, 分子量约为27 kD. 通过基因定点缺失和过量表达技术, 在谷氨酸棒杆菌野生型菌株中分别构建了amrG1基因缺失菌株和表达菌株, 并研究了它们在含有葡萄糖和/或乙酸不同碳源的基本培养基上生长时产生的PTA和AK酶活性特征. 酶活性测定结果发现其中的amrG1基因缺失菌株和表达菌株存在着与野生型菌株不同的一系列酶学特征, 分析显示: 以野生型菌株为对照, amrG1基因缺失菌株在含有葡萄糖碳源的培养基上生长时表现出较高的PTA和AK酶活性, 并且在葡萄糖和乙酸两种碳源上生长时表现出与乙酸碳源上生长时几乎同样的PTA和AK酶活性; amrG1基因过量表达对葡萄糖碳源上生长产生的PTA和AK酶活性有一定程度的抑制, 即表现出与基因缺失情况相反的调控效应. 根据以上结果分析, amrG1可能编码了作用于pta-ack操纵子的一个阻遏因子或共阻遏因子.     

北京棒杆菌邻氨基苯甲酸合成酶基因的克隆、序列分析及表达

以北京棒杆菌(Corynebacterium pekinense)野生株AS1.299和突变株PD-67的基因组为模板,用PCR方法扩增了邻氨基苯甲酸合成酶(AS)基因(trpEG)片段和前端控制序列。核酸序列分析结果表明,该片段全长3374bp,包含3个ORF,推测分别为前导肽基因trpL、AS componentⅠ基因trpE和AS componentⅡ基因trpG。C.pekinense野生株AS1.299与突变株PD-67相比较,trpL基因完全一样;trpE基因有6个碱基的突变,导致了5个氨基酸残基的改变;trpG基因有1个碱基的突变,导致了1个氨基酸残基的改变;同时它们在-35序列处还有一个A→G的突变。通过同源性比较发现,C.pekinense AS1.299与Corynebacterium glutamicum ATCC13032和Brevibacterium lactofermentum的亲缘关系是很近的。trpL基因上游存在启动子区域,并能被Escherichiacoli的RNA聚合酶所识别,实现异源互补。野生型和突变型AS基因在C.pekinense AS1.299和PD-67中都得到表达,并且重组菌相对于宿主菌的酶活都有了很大提高。摇瓶发酵实验结果表明,带有突变型AS基因的PD-67重组菌生长比较慢,稳定期比PD-67推迟24h,但产生的L-色氨酸比PD-67高22.39%。     

On the control mechanism of bacterial growth by cyclic adenosine 3',5'-monophosphate

Inhibition of E. coli growth by cyclic adenosine monophosphate is observed in wild type strains cultured in glucose as carbon source, but not in a cyclic AMP receptor protein deficient mutant. A deletion mutant of the adenylate cyclase gene requires cyclic adenosine monophosphate for optimal growth. Using glucose as carbon source, 2 mM cyclic AMP promotes maximal rates of cell multiplication in this mutant; however higher concentrations of the nucleotide inhibit growth. Cell multiplication of wild type strains grown in glycerol is not affected by cyclic adenosine monophosphate. Nevertheless, in this carbon source the growth rate of the adenylate cyclase mutant is strongly inhibited by concentrations of this nucleotide beyond 0.1 mM. This suggests that growth inhibition by exogenous cyclic adenosine monophosphate is highly dependent on the intracellular levels of the nucleotide.     

北京棒状杆菌AS1.299谷氨酰胺合成酶活力调节的研究

北京棒状杆菌(Corynebacterium pekinense AS1.299)谷氨酰胺合成酶的转移酶活性依赖于Mn~(++),酶的生物合成酶活性依赖于Mg~(++),其他二价金属离子只能部分代替Mn~(++)和Mg~(++)的作用。Mn~(++)对ATP或ADP的克分子比对酶活力起调节作用。ATP、CTP,丙氨酸和甘氨酸对谷氨酰胺合成酶有较强的抑制作用;丝氨酸、谷氨酸和6-磷酸葡萄糖胺对酶活力的抑制作用分别是24,15和21％。效应物混合物对酶的作用被证明是累积性的抑制作用。     

Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate]

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.     

Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum

A corynebacterial clone, previously isolated by scoring repression of lacZYA fused to the aceB promoter of Corynebacterium glutamicum, was analyzed further. In the clone, an open reading frame designated glxR, consisting of 681 nucleotides and encoding a 24,957-Da protein, was found. The molecular mass of a native GlxR protein was estimated by gel filtration column chromatography to be 44,000 Da, suggesting that the protein formed dimers. The predicted amino acid sequence contained both cyclic AMP (cAMP)- and DNA-binding motifs and was homologous with the cAMP receptor protein family of proteins. The aceB-repressing activity of the glxR clone was markedly relieved in an Escherichia coli cya mutant, but the activity was restored in growth medium containing cAMP. In glucose medium, the intracellular cAMP concentration of C. glutamicum reached 22 nmol/mg of protein in the early exponential phase and then decreased further; but in acetate medium, the intracellular cAMP concentration was only 5 nmol/mg of protein and remained low throughout the growth phase. The expression of glxR was not affected by the carbon source. Binding of purified GlxR to the promoter region of aceB could be demonstrated only in the presence of cAMP. These data suggest that GlxR may form dimers which bind to the aceB promoter region in the presence of cAMP and repress the glyoxylate bypass genes.     

Catabolite repression of tryptophanase in Escherichia coli

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.     

Amino sugar assimilation by Escherichia coli

The carbon skeleton of glucose is extensively randomized during conversion to cell wall glucosamine by Escherichia coli K-12. Exogenous glucosamine-1-(14)C is selectively oxidized, and isotope incorporation into cellular glucosamine is greatly diluted during assimilation. A mutant unable to grow with N-acetylglucosamine as a carbon and energy source was isolated from E. coli K-12. This mutant was found to be defective in glucosamine-6-phosphate deaminase. Glucosamine-1-(14)C and N-acetylglucosamine-1-(14)C were assimilated during the growth of mutant cultures without degradation or carbon randomization. Assimilated isotopic carbon resided entirely in cell wall glucosamine and muramic acid. Some isotope dilution occurred from biosynthesis, but at high concentrations (0.2 mm) of added N-acetylglucosamine nearly all cellular amino sugar was derived from the exogenous source. Growth of the mutant was inhibited with 1 mmN-acetylglucosamine.     

Evidence against the involvement of adenosine 3',5'-cyclic monophosphate in glucose inhibition of beta-galactosidase induction in Bacillus megaterium.

When Bacillus megaterium cells are grown on D-galactose as the sole carbon source, the cells actively synthesize beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23). However, D-galactose, when added to a glucose-grown culture, did not induce beta-galactosidase, apparently because of the glucose inhibition of the transport of galactose. On the other hand, when glucose was added to a galactose-grown culture, the transport of galactose continued at a reduced but significate rate, whereas further synthesis of beta-galactosidase was halted. Adenosine 3',5'-cyclic monophosphate (camp) or guanosine 3',5'-cyclic monophosphate (Cgmp) did not relieve the glucose inhibition of beta-galactosidase synthesis in the preinduced culture. A method which gave a reproducible assay of c[32P]AMP in Escherichia coli did not detect cAMP or cGMP in a B. megaterium culture undergoing beta-galactosidase induction, but revealed the extracellular accumulation of two unknown phosphorylated compounds. Cell-free extracts prepared from galactose-grown cells did not catalyze the degradation of cAMP or cGMP.     

Characterization of glk, a gene coding for glucose kinase of Corynebacterium glutamicum

The glk gene from Corynebacterium glutamicum was isolated by complementation using Escherichia coli ZSC113 (ptsG ptsM glk). We sequenced a total of 3072 bp containing the 969-bp open reading frame encoding glucose kinase (Glk). The glk gene has a deduced molecular mass of 34.2 kDa and contains a typical ATP binding site. Comparison with protein sequences revealed homologies to Glk from Streptomyces coelicolor (43%) and Bacillus megaterium (35%). The glk gene in C. glutamicum was inactivated on the chromosome via single crossover homologous recombination and the resulting glk mutant was characterized. Interestingly, the C. glutamicum glk mutant showed poor growth on rich medium such as LB medium or brain heart infusion medium in the presence or absence of glucose, fructose, maltose or sucrose as the sole carbon source. Growth yield was reduced significantly when maltose was used as the sole carbon source using minimal medium. The growth defect of glk mutant on rich medium was complemented by a plasmid-encoded glk gene. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression, and it was found that glk was expressed constitutively under all tested conditions with different carbon sources.     

Inhibitory effect of Li+ on cell growth and pyruvate kinase activity of Escherichia coli.

Li+ inhibited growth of Escherichia coli when glucose, galactose, fructose, or glycerol was added as the sole source of carbon. Growth inhibition was not observed when lactate or a mixture of amino acids was used as the carbon source. A mutant possessing elevated activity of Li+ extrusion was not inhibited by Li+. These results suggested that intracellular Li+ inhibited the glycolytic pathway, most likely triose metabolism, without affecting gluconeogenesis. We also found that pyruvate kinase I was inhibited by Li+.