Thiosulphate improves yield of hydrogen production from glucose by the immobilized formate hydrogenlyase system of Escherichia coli.

Escherichia coli cells with formate hydrogenlyase activity that were immobilized in agar beads produced hydrogen from glucose at the approximate yield of 0.6 mole of hydrogen per mole. Succinate or thiosulphate added to glucose increased hydrogen production two-fold. Thiosulphate did not increase the rate of hydrogen production but reduced the consumption of glucose for the same amount of hydrogen produced compared to control. Oxaloacetate and traces of pyruvate instead of succinate accumulated at the end when thiosulphate was present.     

Synthesis and lysis of formate by immobilized cells of Escherichia coli

Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH(2) (initial) value of the immobilized cell, and subsequently cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized cells. The pH optimum range, K(m), and specific activity of the immobilized cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g cell-h, respectively. The cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.     

A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12

Abstract Five different c -type cytochromes have been detected during anaerobic growth of various Escherichia coli strains in different media. None of these cytochromes was detectable in aerobically-grown cultures. Only a single, 43 kDa cytochrome was synthesized in response to the presence of trimethylamine-N-oxide: synthesis of this cytochrome was unaffected by the presence of nitrate or nitrite, was repressed by oxygen, but was dependent upon a funtional tor operon located at minute 22 (coordinate 1070 kb) on the E. coli chromosome. The other four cytochromes, masses 16, 18, 24 and 50 kDa, were induced by nitrite coordinately with formate-dependent nitrite reductase activity, but repressed by oxygen and nitrate. As only the 18 kDa and 50 kDa cytochromes are encoded by the nrf operon located at minute 92 (coordinate 4366 kb), there must be other loci, possibly essential for formate-dependent nitrite reduction, encoding the 16 kDa and 24 kDa cytochromes. No other c -type cytochrome was detected under any growth condition tested.     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H₂) when grown on glucose. H₂ formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H₂ was found to be dependent on Hyd-4 and the F₀F₁-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate H₂ production dependent on pH was investigated. In both types of cells, H₂ production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F₀F₁-ATPase. The results indicate that Hyd-3 has a major role in H₂ production at acidic pH independently on the F₀F₁-ATPase.     

Induction of cadmium tolerance in Escherichia coli K-12

Abstract Cadmium ions are bacteriocidal, resulting in exponential killing that starts immediately after exposure. We have shown that pretreatment with sublethal concentrations of cadmium induces tolerance. Protection against cadmium killing can also be obtained by preincubation at elevated temperatures, known to induce the heat-shock response. However, in contrast to pretreatment at elevated temperatures, exposure to sublethal cadmium concentrations does not induce thermotolerance.     

The EnvZ protein of Salmonella typhimurium LT-2 and Escherichia coli K-12 is located in the cytoplasmic membrane

Abstract The osmoregulated expression of the porin proteins OmpC and OmpF in S. typhimurium and E. coli is dependent on the regulatory proteins OmpR and EnvZ. The function of the EnvZ protein is not clear. In order to establish the cellular location of EnvZ two different methods of buoyant sucrose density centrifugation was employed. The presence of EnvZ in the different fractions was visualised by immunoblotting. It was conclusively shown that the EnvZ protein is located in the cytoplasmic membrane fraction. The result is in agreement with the available sequence data which shows that the EnvZ polypeptide contains two long hydrophobic stretches.     

大肠杆菌K-12转醛醇酶基因talB的克隆及在运动发酵单胞菌CP4中的表达

研究了E.coliK-12转醛醇酶基因(talB)在自身启动子和在Z.mobilisCP4eno基因启动子的启动下在E.coliDH5α和Z.mobilisCP4中的表达情况。首先克隆了E.coliK-12talB基因,并连接到穿梭载体pZB1上构建成pZB1-talB;然后利用PCR重叠延伸技术将E.coliK-12talB自身的启动子换成Z.mobilisCP4eno的启动子,构建得到pZB1-Peno-talB。将这两个质粒分别转化E.coliDH5α和Z.mobilisCP4。对转化子粗酶液进行的转醛醇酶酶活力测定结果表明,E.coli talB自身启动子和Z.mobilis eno启动子能以基本相同的效率启动talB基因在E.coli和Z.mobilis中的表达。     

Protein localization in Escherichia coli K-12: an analysis of ompC-lacZ gene fusions

Abstract Two conditionally expressed lacZU131 gene fusions were constructed in vivo to the ompC gene which encodes a major outer membrane protein in Escherichia coli . The resulting hybrid molecules contained approximately 25% and 50% of the mature OmpC protein fused to the LacZ. Export analysis showed that under nonoverproducing conditions essentially all synthesized OmpC-LacZ hybrid protein was effectively processed in vivo unless the signal peptide cleavage was inhibited by ethanol addition. Also, the hybrid proteins were highly accessible to solid phase iodination of whole cells under conditions where cytoplasmic proteins remained unlabelled. Thus, hybrids containing large portions of the OmpC protein were clearly recognized by the cellular export machinery, and probably all synthesized hybrid protein was partially translocated through the cytoplasmic membrane.     

大肠杆菌酸性磷酸酶基因克隆表达及应用

将大肠杆菌K-12的酸性磷酸酶(AphA)完整基因和去信号肽基因分别克隆到pET-28a(+)栽体上,并转化入大肠杆菌BL21( DE3)中.经诱导检测,重组菌均能表达出高活性的可溶性酶蛋白,去信号肽表达更稳定.对重组菌的活性研究表明,相对于野生菌,重组菌酶活力得到大幅度提高,同时,以pNPP、肌苷为底物进行磷酸转移催化反应,在pH4.0-6.0、反应温度37℃条件下,约有30％的肌苷可转化为IMP,但随着反应的进行所形成的IMP又被该酶降解,向反应液中加入EDTA即可明显抑制酶的水解活性,减缓IMP的降解速率.     

Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli

Through complementation of a trans-acting regulatory mutation a gene has been cloned whose product is required for the formate induction of the anaerobic expression of the formate hydrogenlyase structural genes. By restriction analysis, and from the size of the encoded protein, the gene could be identified as being equivalent to fhlA described by Sankar et al. (1988). The nucleotide sequence of the fhlA gene was determined and it was shown to code for a protein with a calculated Mr of 78,467. Analysis of the derived amino acid sequence showed that the carboxy-terminal domain of FHLA shares considerable sequence similarity with NIFA and NTRC, which are the 'regulators' of two-component regulatory systems. Carboxy-terminal truncation of, and introduction of amino-terminal deletions in, the fhlA gene delivered inactive gene products. When overexpressed, FHLA mediates activation of expression of the formate dehydrogenase and hydrogenase structural genes in the presence of formate also under aerobic growth conditions. FHLA appears to bind to the upstream regulatory sequence (URS) in the 5' flanking region of the fdhF gene since activation of fdhF expression was dependent on the presence of the URS.     

formation for phenylethylamine oxidase expressed in Escherichia coli K-12

Abstract Arthrobacter globiformis amine oxidase produced by Escherichia coli cells grown in copper-depleted media was reported to undergo activation due to formation of its topaquinone cofactor in a copper-dependent autocatalytic reaction. Likewise, a mutated E. coli amine oxidase located in the cytoplasm was reported to form topaquinone autocatalytically in an EDTA-sensitive reaction. Here we show unequivocally that formation of an amine oxidase lacking topaquinone is primarily a consequence of the location of the enzyme in the cytoplasm rather than the level of copper in the growth medium. For E. coli , insertion of copper into apoamine oxidase and subsequent topaquinone formation occur after export of the apoenzyme into the periplasm.     

SUMO基因的内在原核启动子活性及其在大肠杆菌蛋白表达系统中的应用

SUMO融合系统已成为目前大肠杆菌重组蛋白生产的重要手段,但在载体构建效率和蛋白可溶性等方面仍有待改进。本研究在PCR克隆酿酒酵母SUMO基因Smt3(Sm) 时意外发现Sm具有组成型原核启动子活性;而且经软莓BPROM程序预测发现大多数物种SUMO基因编码区都具有依赖s70的原核启动子。进一步通过整合Sm启动子和Sm 3￠末端StuⅠ位点特性以及引入His标签和超酸增溶标签,构建了基于Sm’-LacZα融合基因的一系列通用克隆表达载体,并通过蓝白斑筛选和SDS-PAGE分析进行了多个靶蛋白基因的克隆和表