千层塔内生真菌的分离与鉴定

目的：为从千层塔中分离具有药用价值的内生真菌奠定基础。方法：新鲜千层塔茎段,经酒精和升汞消毒后,接种于PDA平板培养基上进行内生真菌的分离、纯化;根据菌落形态和孢子等形态特征,结合核糖体基因居间序列（ITS序列）进行菌株鉴定。结果：从千层塔的茎中分离出4株内生真菌。内生真菌I菌落形态和孢子特征与枝状枝孢霉属的特征相符合,ITS序列与GenBank中多条属于枝状枝孢霉的ITS序列相似,鉴定该菌株属于枝状枝孢霉;内生真菌II菌落形态和孢子特征与黄青霉的特征相符合,鉴定该菌株属于黄青霉;内生真菌III菌落形态和孢子特征与尖孢镰刀菌的特征相符合,ITS序列与GenBank中多条属于尖孢镰刀菌的ITS序列相似程度高,鉴定该菌株属于尖孢镰刀菌;内生真菌Ⅳ菌落形态与盾壳霉相似,ITS序列与GenBank中6条属于盾壳霉的ITS序列具有较高的相似性,鉴定该菌株属于盾壳霉。结论：从千层塔中分离和鉴定出4株内生真菌,分别属于枝状枝孢霉、黄青霉、尖孢镰刀茵和盾壳霉。     

盾壳霉在油菜菌核病菌生物防治中的应用

油菜核病菌(Sclerotiniasclerotiorum)是一种世界性病原菌,其分布广、危害大、难根治。盾壳霉(Coniothyriumminitans)是该病原菌的破坏性寄生真菌,可以有效、专一地降低病原菌菌核的形成与萌发,在该病原菌的生物防治方面具有较大的应用潜力。从油菜核盘菌的致病过程与特点、盾壳霉的生长特性、盾壳霉和油菜核盘菌间相互作用的规律及途径等几个方面阐述了盾壳霉对油菜核盘菌的生防特性,讨论了盾壳霉在生产实践中的应用潜力及存在问题,并提出了一些解决问题的可能途径及需要进一步研究的内容与方向 。     

植病生防菌盾壳霉的研究进展

盾壳霉是世界性病原真菌核盘菌的主要拮抗真菌之一。许多研究表明盾壳霉具有控制温室和田间多种作物菌核病的潜力。目前欧洲一些国家已有盾壳霉商品制剂销售。为了加速盾壳霉产业化进程和更好地发挥其控制菌核病的作用 ,以及为了使人们对盾壳霉的研究不断深入 ,综述了盾壳霉生态学特性、遗传改良、对核盘菌的生防机制、在菌核病防治上的应用及基因工程研究等方面的研究进展。     

盾壳霉产生几丁质酶的条件研究

本实验采用摇瓶培养研究了盾壳霉(Coniothyrium minitans)产生几丁质酶的条件。改良的天然马铃薯葡萄糖培养基(mPDB)较合成培养基(SMCS)更适宜作为盾壳霉产生几丁质酶的基础培养基。添加9种不同碳源试验表明葡萄糖较适宜于盾壳霉产几丁质酶;氮源试验表明硝酸钾是产酶的较适宜氮源。盾壳霉形成几丁质酶的培养时间以15 d为佳,培养的适宜pH值为6.5。     

华南地区瓜类疫霉对甲霜灵的田间抗药性

【目的】了解华南地区瓜类疫霉(Phytophthora melonis)对甲霜灵的田间抗药性。【方法】2007-2010年从广西、广东两省(区)9个市冬瓜和黄瓜产区采集疫病样品,分离纯化瓜类疫霉,分别采用菌落生长速率法和叶盘漂浮法测定瓜类疫霉对甲霜灵的敏感性,并用药剂驯化方法从敏感性菌株诱导瓜类疫霉抗甲霜灵突变体。【结果】从9个市24个样点共分离纯化获得193株瓜类疫霉,抗药性检测结果表明,敏感菌株、中等抗性菌株和抗性菌株分别占测试菌株的29.0%、18.1%和52.8%;不同地区、不同寄主分离的菌株的抗性频率和抗性水平差异较大,来源于广东的菌株抗性频率和抗性水平一般高于来源广西的菌株,分离自黄瓜的菌株高于分离自冬瓜的菌株,大部分样点抗性菌株占据优势群体,个别菌株的抗性指数高达4226.9,叶盘漂浮法测定结果和菌落生长速率法相似;在含药平板上对敏感菌株进行甲霜灵抗性诱导结果表明,从60%的敏感菌株中成功诱导出对甲霜灵抗性稳定的突变体,突变体的抗性水平为敏感性亲本的189-407倍;9株来源于未施用过甲霜灵等苯基酰胺类杀菌剂样点的菌株均为敏感性菌株,其EC50值为0.0429-0.5461μg/mL,将它们EC50的平均值0.3200±0.1617μg/mL确定为华南地区瓜类疫霉对甲霜灵的敏感性基线;对两个样点的监测结果表明,瓜类疫霉抗甲霜灵菌株的频率及抗性指数有逐年增高趋势。【结论】华南广西和广东两省(区)瓜类疫霉对甲霜灵抗性普遍发生,瓜类疫霉对甲霜灵抗药性产生与其和药剂的接触密切相关。瓜类疫霉敏感性基线的建立,可为今后瓜类疫霉抗甲霜灵的评价和进一步监测提供科学依据。     

极端污染环境草甘膦抗性菌株的分离、鉴定及特性

[目的]筛选高抗草甘膦菌株并对其进行鉴定和特性研究.[方法]从草甘膦极端污染土壤中分离高抗草甘膦菌株,并检测其草甘膦耐受能力,最适生长pH和抗生素抗性.通过生理生化特征和分子生物学特征的测定对该菌株进行鉴定.[结果]从草甘膦极端污染土壤中分离到一株高抗草甘膦的菌株SL06500,该菌株最高耐受草甘膦浓度为500 mmol/L,并且在200～500 mmol/L之间,菌株生长迅速,最适生长pH为4.0,具有氨苄青霉素、卡那霉素、四环素和氯霉素抗性.用16S rDNA的通用引物,经PCR扩增、测序得到SL06500的16S rDNA序列,该序列在GenBank的登录号为EU006066.将此序列经NCBI Blast进行核苷酸比对发现SL06500与无色杆菌属(Achromobacter)和产碱杆菌属(Alcaligenes)的Identity值均为99%.按照1994年版伯杰氏鉴定细菌学手册的命名规则,结合生理生化指标测定的结果,将菌株命名为木糖氧化产碱杆菌木糖氧化亚种SL06500 (Alcaligenes xylosoxidans subsp.xylosoxidans SL06500).[结论]该菌株的较高草甘膦抗性和嗜药性的特点值得我们进行进一步的研究.更重要的是,这是首次关于木糖氧化产碱杆菌木糖氧化亚种草甘膦抗性的报道.     

抗吡虫啉棉蚜种群对啶虫脒和噻虫胺的交互抗性及相关酶学机理

【目的】通过对乙酰胆碱受体β1亚基突变后的抗吡虫啉棉蚜Aphis gossypii (Glover)种群的继续筛选, 明确该种群的抗性发展规律以及对其他新烟碱类杀虫剂啶虫脒和噻虫胺的交互抗性及相关酶学机理。【方法】采用浸渍法连续对抗吡虫啉棉蚜进行室内筛选、 测定噻虫胺和啶虫脒对抗吡虫啉棉蚜种群的毒力; 选择LC₂₀剂量吡虫啉、 啶虫脒和噻虫胺处理抗性棉蚜, 采用生化分析法测定其体内羧酸酯酶、 谷胱甘肽-S-转移酶和乙酰胆碱酯酶的活性变化, 并观察其生物学特性的变化。【结果】本研究对抗性棉蚜突变种群用吡虫啉继续筛选至75代, 抗性倍数达到72.6倍, RF75停止用药筛选12代（RF₇₅₊₁₂）, 抗性仍达72.0倍。且RF₇₅₊₁₂对噻虫胺和啶虫脒的交互抗性可分别达11.9倍和20.1倍。噻虫胺对抗吡虫啉棉蚜的蜜露分泌和体重的抑制作用均大于吡虫啉和啶虫脒。噻虫胺对RF₇₅₊₁₂的羧酸酯酶、 谷胱甘肽-S-转移酶和乙酰胆碱酯酶均具有明显的抑制作用, 而啶虫脒的抑制作用较小。【结论】结果表明乙酰胆碱受体基因突变棉蚜种群对吡虫啉的抗性水平不仅升高, 且停止用药后其抗性可稳定遗传; 第二代新烟碱类的噻虫胺在抗吡虫啉棉蚜靶标突变种群的治理中具有较大的应用价值。     

须糖多孢菌Saccharopolyspora pogona的核糖体工程改造对丁烯基多杀菌素合成的影响

核糖体工程是以微生物的各类抗生素抗性突变为筛选标记,高效获得次生代谢产物合成能力提高的突变株的一种育种新方法。通过核糖体工程技术,使用链霉素对须糖多孢菌Saccharopolyspora pogona进行抗性选育,以获得高产丁烯基多杀菌素突变菌株。对原始菌株和所获得的突变菌株代谢产物的研究发现,相对于原始菌株,其中突变株S13的丁烯基多杀菌素产量提高幅度最大,相比原始菌株提高了1.79倍。经质谱测定表明,其代谢物中比原始菌株多了一种丁烯基多杀菌素组分Spinosynα1。对抗性突变株S13的DNA序列进行分析,发现在编码核糖体S12蛋白的rps L基因保守区域中出现点突变,第314位和第320位的胞嘧啶(C)分别突变为腺嘌呤(A)和胸腺嘧啶(T),对应的氨基酸残基分别由脯氨酸突变为谷氨酰胺,丙氨酸突变为缬氨酸。研究显示,突变株S13遗传稳定性良好。     

我国棉花主产区棉蚜对吡虫啉的抗性监测及抗性机理

【目的】由于生长周期短、繁殖率高,棉蚜Aphis gossypii容易对杀虫剂产生抗药性。本研究旨在明确我国棉花主产区棉蚜对吡虫啉的抗性水平及抗性机理。【方法】采用浸叶法测定了北京海淀,河北廊坊和邯郸,山东德州,河南许昌,以及新疆奎屯和阿克苏地区棉蚜对吡虫啉的抗性水平;测定了不同种群棉蚜3种解毒酶（多功能氧化酶、羧酸酯酶、谷胱甘肽S-转移酶）及乙酰胆碱酯酶的活性;并对靶标基因烟碱型乙酰胆碱受体（nAChR）β1亚基基因进行了突变检测。【结果】北京海淀、河南许昌和河北邯郸的棉蚜对吡虫啉敏感;河北廊坊、新疆阿克苏、山东德州及新疆奎屯地区的棉蚜对吡虫啉的抗性倍数(resistance ratio, RR)分别为22.6, 26.3,53.5和61.1倍,为中等水平抗性。酶活力对比研究发现,阿克苏和奎屯地区的棉蚜多功能氧化酶的比活力分别是敏感种群(北京种群)的1.7和1.8倍,羧酸酯酶的比活力分别是敏感种群的1.6和1.7倍,谷胱甘肽S-转移酶的比活力均是敏感种群的1.5倍,但是乙酰胆碱酯酶比活力在棉蚜种群间差异不显著。靶标基因突变检测表明,河北廊坊、新疆阿克苏、山东德州及新疆奎屯棉蚜种群nAChR β1亚基均存在与吡虫啉抗性相关的精氨酸到苏氨酸（R81T）突变。【结论】结果提示,多功能氧化酶、羧酸酯酶和谷胱甘肽S-转移酶活力升高以及nAChR β1亚基R81T突变与棉蚜对吡虫啉的抗性形成相关。     

酿酒酵母Mbp1基因缺失突变株耐受性研究

为了研究Mbp1基因对工业酿酒酵母(Saccharomyces cerevisiae)耐受性的影响,在敲除野生型工业酿酒酵母MF1015菌株Mbp1基因的基础上,分析Mbp1基因缺失菌株(突变菌株)与野生型在乙醇耐受性、耐热性、细胞壁完整性、呼吸强度、海藻糖含量、过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、乙醇脱氢酶(ADH)活性等方面的差异。结果表明:在含刚果红或SDS的平板上突变菌株的菌落比野生型小、菌体存活率比野生型低;突变菌株的乙醇耐受性和耐热性均下降,在乙醇浓度为9%的液体培养基中,突变菌株的最大OD600约为3.15,而野生型最大OD600值约为3.48;在30℃和37℃时突变菌株和野生型的生长趋势基本一致,而在40℃时,突变菌株最大OD600约为3.0,而野生型约为4.5;用9%乙醇处理后,突变菌株胞内海藻糖含量、过氧化氢酶、过氧化物酶、超氧化物歧化酶、乙醇脱氢酶酶活分别比野生型低49%、80%、24%、37%、73%,而未经乙醇处理的突变菌株和野生型酶活无明显差别。推测Mbp1基因有助于工业酿酒酵母适应外部不良环境。     

Effect of glyphosate toxicity on growth, pigment and alkaline phosphatase activity in cyanobacterium Anabaena doliolum: a role of inorganic phosphate in glyphosate tolerance

Response of glyphosate toxicity on photoautotrophic cyanobacterium A. doliolum and its mutant strain was investigated. Chlorophyll a content of both the wild type and mutant strain in the presence of glyphosate (N-phosphonomethyl glycine) initially showed an increasing trend when supplemented with Pi and a declining tendency under the Pi-starved condition. The results suggested that both the wild type and mutant strains were more sensitive to glyphosate in the absence of phosphate. Alkaline phosphatase activity of wild type strain in the presence of Pi, enhanced in response to addition of glyphosate (40 microg/ml), but the activity remained unaltered by addition of glyphosate in the Pi-starved cells, whereas the alkaline phosphatase activity in the mutant strain under both Pi-starved as well as unstarved conditions was stimulated (approximately 5.4 and 3.1-fold, respectively) by addition of glyphosate. The results on alkaline phosphatase activity indicated a glyphosate-induced depletion in the phosphate content of the cells, particularly in the mutant strain, as evident from the stimulated activity of alkaline phosphatase. It is suggested that enzyme activity in the Pi-starved wild type cells may not be influenced any further by glyphosate, as cellular phosphate reserve might not be available for further depletion.     

Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C(317)-->T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.     

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase.

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-lon. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30 degrees C and 5% of that at 40 degrees C. At 42 degrees C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30 degrees C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30 degrees C and almost the same in both strains at 42 degrees C. The mutant had no obvious phenotype regarding its growth properties.     

Isolation and Characterization of a Mutant of Arthrobacter sp. Strain GLP-1 Which Utilizes the Herbicide Glyphosate as Its Sole Source of Phosphorus and Nitrogen

Arthrobacter sp. strain GLP-1, grown on glucose as a carbon source, utilizes the herbicide glyphosate [N-(phosphonomethyl)glycine] as its sole source of phosphorus as well as its sole source of nitrogen. The mutant strain GLP-1/Nit-1 utilizes glyphosate as its sole source of nitrogen as well. In strain GLP-1, P_i was a potent competitive inhibitor of glyphosate uptake (K_i, 24 μM), while the affinity of P_i for the uptake system of strain GLP-1/Nit-1 was reduced by 2 orders of magnitude (K_i, 2.3 mM). It is concluded that the inability of strain GLP-1 to utilize glyphosate as a source of nitrogen is due to the stringent control of glyphosate uptake by excess phosphate released during the degradation of the herbicide.     

Toxicological assessment of selective pesticides towards plant growth promoting activities of phosphate solubilizing Pseudomonas aeruginosa

The study was designed to assess the effect of selected pesticides (metribuzin, glyphosate, imidacloprid, thiamethoxam, hexaconazole, metalaxyl and kitazin) at the recommended and higher rates on plant growth promoting activities of Pseudomonas aeruginosa strain PS1 isolated from mustard (Brassica compestris) rhizosphere. The strain PS1 was specifically chosen owing to its substantial tolerance against pesticides, phosphate solubilization and considerable production of indole acetic acid, siderophores, exo-polysaccharides, HCN and ammonia. Plant growth promoting traits of the strain PS1 decreased consistently as the concentrations of each pesticide was increased from the recommended dose to the higher ones. Generally, the maximum toxicity to plant growth promoting traits was displayed by pesticides at three times the recommended field rate. However, the effect on the plant growth promoting activities of the P. aeruginosa strain PS1 at the recommended dose of each pesticide was less hazardous. This study revealed an additional aspect of the toxicological mechanisms of the pesticides through which they suppress the plant growth.     

Correlation Between Reduced Nicotinamide Adenine Dinucleotide Phosphate Levels and Morphological Changes in Neurospora crassa

A colonial mutant of Neurospora crassa, previously shown to be altered in the structure of glucose-6-P dehydrogenase [a reduced nicotinamide adenine dinucleotide phosphate (NADPH) producing reaction], contained only 40% as much NADPH in extracts as did the wild type. A partial revertant strain, when grown at 23 C, had the same total NADPH content as the wild type, but, at 34 C, had lower levels of NADPH as well as a colonial morphology. A revertant with complete wild-type morphology had wild-type levels of NADPH. Two different colonial mutants, which have also been reported to be altered in NADPH-generating reactions, were found to have a lower content of NADPH, whereas other colonial mutants had wild-type levels. The wild-type strain, when grown under conditions in which it contained a lower total content of NADPH, had a morphology similar to that of a colonial mutant. The evidence indicates that lowered NADPH content leads to a dramatic alteration in the morphology of Neurospora, but not necessarily vice versa. The possible pleiotropic effects of the NADPH deficiency are discussed.     

Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions. The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter. The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower. In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM. A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter. We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.     

Pathogenic potential of a collagenase gene from Aeromonas veronii

The role of collagenase as a mechanism of bacterial pathogenicity in some pathogenic bacteria has been reported. The information on the role of collagenase in Aeromonas spp. pathogenesis is scant. In the present study, a mutant Aeromonas veronii RY001 that is deficient in the putative collagenase gene acg was constructed and compared with the wild-type strain for virulence factors. Bacterial cells and cell-free extracellular products of the mutant had significantly less collagenolytic activity, but there were not significant differences in caseinolytic, gelatinolytic, and elastolytic activities. Adhesion and invasion abilities of the mutant strain on epithelioma papillosum of carp cells was only 56% of that of the wild-type strain, and the cytotoxicity of the mutant strain to epithelioma papillosum of carp cells was only 42% of that of the wild-type strain. The LD50 values of the wild-type strain were determined as 1.6 x 10(6) and 3.5 x 10(5) cfu in goldfish and mice, respectively, whereas the mutant RY001 strain showed slightly higher values (i.e., 2.8 x 10(6) and 1.4 x 10(6) cfu in goldfish and mice, respectively). These results indicated the involvement of the collagenase gene in the pathogenesis of A. veronii.     

Replacement of arginine 246 by histidine in the beta subunit of Escherichia coli H+-ATPase resulted in loss of multi-site ATPase activity

A mutant strain KF43 of Escherichia coli defective in the beta subunit of H+-translocating ATPase (F0F1) was examined. In this mutant, replacement of Arg246 by His was identified by DNA sequencing of the mutant gene and confirmed by tryptic peptide mapping. The mutant F1-ATPase was defective in multi-site hydrolysis of ATP but was active in uni-site hydrolysis. Studies on the kinetics of uni-site hydrolysis indicated that the k1 (rate of ATP binding) was similar to that of the wild-type, but the k-1 (rate of release of ATP) could not be measured. The mutant enzyme had a k3 (rate of release of inorganic phosphate) about 15-fold higher than that of the wild-type and showed 3 orders of magnitude lower promotion from uni- to multi-site catalysis. These results suggest that Arg246 or the region in its vicinity is important in multi-site hydrolysis of ATP and is also related to the binding of inorganic phosphate. Reconstitution experiments using isolated subunits suggested that hybrid enzymes (alpha beta gamma complexes) carrying both the mutant and wild-type beta subunits were inactive in multi-site hydrolysis of ATP, supporting the notion that three intact beta subunits are required for activity of the F1 molecule.     

Disruption of the acetate kinase (<Emphasis Type="Italic">ack</Emphasis>) gene of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.