Effects of Molybdate and Selenite on Formate and Nitrate Metabolism in Escherichia coli

The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.     

Genetic Map Position of the pdxH Gene in Escherichia coli

The gene for pyridoxine phosphate oxidase, pdxH, is located 1.2 min beyond aroD, proximal to trp.     

Manganese-Resistant Mutants of Escherichia coli: Physiological and Genetic Studies

Manganese is growth inhibitory for Escherichia coli. The manganese concentration required for inhibition is dependent upon the magnesium concentration of the medium. Mutants have been isolated which are partially resistant to manganese inhibition in both liquid and solid media. From conjugation experiments, the genetic locus for manganese-resistance, mng, appears to be between 34 and 37 min on the E. coli genetic map. Experiments with radioactive (28)Mg lead to the tentative conclusion that the mng mutants are altered in the inhibition constant for manganese as a competitive inhibitor for the mangnesium accumulation system. Once high manganese enters the cells, it displaces internal magnesium and leads to a net cellular loss and hence growth inhibition. The mng mutants are somewhat less subject to manganese-induced magnesium loss under comparable conditions than are manganese-sensitive wild-type cells.     

Identification of High γ-Aminobutyric Acid Producing Marine Yeast Strains by Physiological and Biochemical Characteristics and Gene Sequence Analyses

Four marine yeasts isolated from the Pacific Ocean off Japan (Siki No. 4, Siki No. 15, Hach No. 6, and Inub No. 11), which showed high γ-aminobutyric acid (GABA) producing abilities, were identified and classified by physiological and biochemical characteristics and gene sequence analyses. Analysis of biochemical data suggested that while Siki No. 15 was identical to Candida, the remaining three isolates belonged to the genus Pichia. However, these data were insufficient to resolve their identity at the species level. Subsequently, analysis of the 5.8S rRNA genes and the two internal transcribed spacer regions (ITS) sequences revealed that Siki No. 15 belongs to Pichia guilliermondii, while the remaining three isolates corresponded to Pichia anomala. Since Siki No. 4 showed slightly different biochemical properties than the other two isolates, which were otherwise identical, we sought to investigate the sequences of the intergenic spacer region 1 (IGS1). We observed few nucleotide changes, suggesting that the Hach No. 6 and Inub No. 11 isolates belong to different but new strains for which we propose the names P. anomola MR-1 and MR-2 respectively.     

Identification,Source, and Metabolism of N-Ethylglutamate in Escherichia coli

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-²H₄]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.During work on developing methods for the analysis of the amino acids generated by recombinant archaeal mutases, I developed procedures for the recovery and analysis of the free amino acids present in cell extracts of Escherichia coli. When these methods were applied to analysis of E. coli grown on LB broth, I always found a large amount of an unknown amino acid. Here I report on the identification of this amino acid as N-ethylglutamate (NEG). NEG has never been reported as a natural product. I demonstrate that NEG is readily taken up by E. coli and can serve as the sole source of nitrogen when the cells are grown on M9 glucose medium.     

Adenine Deaminase Activity of the yicP Gene Product of Escherichia coli

During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli chromosome, suggesting that, like Bacillus subtilis, E. coli has an adenine deaminase. As the yicP gene of E. coli shares about 35% identity with the B. subtilis adenine deaminase gene (ade), we cloned yicP from the E. coli genome and developed a strain that overexpressed its product. The enzyme was purified from a cell extract of E. coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase [EC 3.5.4.2] activity. It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa. The enzyme required manganese ions as a cofactor, and adenine was its only substrate. Its optimum pH was 6.5-7.0 and its optimum temperature was 60°C. The apparent K_m for adenine was 0.8 mM.     

Genetic and Physiological Analysis of Glutamate Decarboxylase in Escherichia coli K-12

No correlation was found between glutamate decarboxylase (GAD) activity and the ability of Escherichia coli K-12 strains to grow on glutamate. A gene, gad, determining GAD activity maps near gltC, which controls glutamate permease.     

Characterization of the Dextranase Gene (dex) of Streptococcus mutans and Its Recombinant Product in an Escherichia coli Host

The gene (dex), which encodes the Streptococcus mutans dextranase (Dex), was cloned in Escherichia coli. The E. coli host harboring a recombinant plasmid (pSD2) containing an 8-kb BamHI insert produced a Dex protein of 133 kDa as well as smaller enzymes of 118, 104, and 88 kDa. The Dex produced by the recombinant E. coli was apparently located in the cytoplasmic fraction, not in the periplasmic nor the extracellular fractions. Subcloning and deletion analysis of pSD2 showed that the structural gene of Dex was encoded by a 4-kb BamHI-SalI fragment. The fragment also contained the dex promoter which was effective in the E. coli cell.     

Biochemical Genetics of the Cryptic Gene System for Cellobiose Utilization in Escherichia coli K12

The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in microbial evolution. Wild-type E. coli K12 do not utilize the beta-glucoside sugars, arbutin, salicin and cellobiose. A Cel+ (cellobiose utilizing) mutant which grows on cellobiose, arbutin, and salicin was isolated previously from wild-type E. coli K12. Biochemical assays indicate that a cel structural gene (celT) specifies a single transport protein that is a beta-glucoside specific enzyme of the phosphoenolpyruvate-dependent phosphotransferase system. The transport protein phosphorylates beta-glucosides at the expense of phosphoenolpyruvate. A single phosphoglucosidase, specified by celH, hydrolyzes phosphorylated cellobiose, arbutin, and salicin. The genes of the cel system are expressed constitutively in the Cel+ mutant, whereas they are not expressed at a detectable level in the wild-type strain. The transport and hydrolase genes are simultaneously silenced or simultaneously expressed and thus constitute an operon. Cel+ strains which fail to utilize one or more beta-glucosides express the transport system at a lower level than do Cel+ strains which grow on all three beta-glucosides. Other strains inducibly express a gene which specifies transport of arbutin but not the other beta-glucosides. The arbutin transport gene, arbT, maps outside of the cel locus.     

cre基因在大肠杆菌中的表达及表达蛋白活性的检测

Cre重组酶来自噬菌体P1,可以识别特异的loxP位点的DNA序列,并进行专一性的剪切和拼接,利用PCR技术将cre基因克隆至原核表达载体pET-29a,在大肠杆菌BL21（DE3）得到了高效表达,采用DEAE－52柱层析的方法对表达蛋白进行了纯化,体外生物学活性检测表明,表达蛋白对含有同向loxP位点的质粒有切割活性。     

补料速度对工程菌生长和产物表达的影响研究

通过研究各培养阶段补料速度对温敏启动子控制的rIL-2工程菌E.coliK802(pLY-4)培养密度和rIL-2表达的影响,发现在各培养阶段控制不同的补料速度有利于提高菌密度和rIL-2表达,缩短培养周期。确定了rIL-2工程菌高密度培养方案,三批重复实验,平均菌密度为58OD_(600);菌干重18.0g/L,rIL-2表达水平为42.4％。     

Biochemical and Genetic Analyses of the Role of Yeast Casein Kinase 2 in Salt Tolerance

Saccharomyces cerevisiae cells lacking the regulatory subunit of casein kinase 2 (CK-2), encoded by the gene CKB1, display a phenotype of hypersensitivity to Na(+) and Li(+) cations. The sensitivity of a strain lacking ckb1 is higher than that of a calcineurin mutant and similar to that of a strain lacking HAL3, the regulatory subunit of the Ppz1 protein phosphatase. Genetic analysis indicated that Ckb1 participates in regulatory pathways different from that of Ppz1 or calcineurin. Deletion of CKB1 increased the salt sensitivity of a strain lacking Ena1 ATPase, the major determinant for sodium efflux, suggesting that the function of the kinase is not mediated by Ena1. Consistently, ckb1 mutants did not show an altered cation efflux. The function of Ckb1 was independent of the TRK system, which is responsible for discrimination of potassium and sodium entry, and in the absence of the kinase regulatory subunit, the influx of sodium was essentially normal. Therefore, the salt sensitivity of a ckb1 mutant cannot be attributed to defects in the fluxes of sodium. In fact, in these cells, both the intracellular content and the cytoplasm/vacuole ratio for sodium were similar to those features of wild-type cells. The possible causes for the salt sensitivity phenotype of casein kinase mutants are discussed in the light of these findings.     

Tropic1808基因的原核表达及其表达产物的生物活性

Tropic180 8基因是新近获得的一个鼠源性的cDNA .Tropic180 8基因开放阅读框架片段通过PCR方法从质粒中扩增后 ,重组入表达载体pET 2 1a中 ,转化大肠杆菌BL2 1(DE3 ) .用IPTG诱导目的蛋白的表达 ,SDS PAGE并凝胶图象分析确定目的蛋白表达水平占细菌总蛋白的 14%以上 .表达蛋白在N端融合有 16个氨基酸 ,将表达蛋白电转移至PVDF膜 .氨基酸序列分析表明 ,其N端第 17～ 2 5位氨基酸序列与Tropic180 8基因编码序列一致 .利用融合部分含T7·Tag ,通过亲和层析纯化表达蛋白 ,经Westernblot检测为目的蛋白 ,加入到无血清培养的新生SD大鼠背根神经节 (DRG)中 ,观察到表达蛋白对DRG具有促进存活和促进突起生长的作用 .     

Cloning and Identification of the hemG Gene Encoding Protoporphyrinogen Oxidase (PPO) of Escherichia coli K-12

Cells of the VSR751 strain, which was previously isolated asa photoresistant revertant of the visA-deleted (hemH-deleted)strain of Escherichia coli K-12, accumulated uroporphyrin (uro),coproporphyrin (copro) and protoporphyrin IX (proto), but didnot accumulate as much protoporphyrin as cells of the parentalstrain (hemH-deleted). Therefore, we concluded that strain VSR751must be defective in protoporphyrinogen oxidase (PPO), the productof the hemG gene. By complementation analysis using VSR751,we isolated and identified this gene. The hemG gene is locatedat 86 mim on the E. coli chromosome, just upstream of the rrnAoperon, and is transcribed clockwise in the same direction asthe rrnA operon. This gene encodes a 181-amino acid proteinwith a calculated molecular mass of about 21 kDa. Sequence analysisrevealed the presence of flavodoxin motif, suggesting that acofactor of this enzyme is flavin mononucleotide, which is consistentwith the previous report that the mammalian PPO had the flavincofactor.     

Identification of a spermidine excretion protein complex (MdtJI) in Escherichia coli

A spermidine excretion protein in Escherichia coli was looked for among 33 putative drug exporters thus far identified. Cell toxicity and inhibition of growth due to overaccumulation of spermidine were examined in an E. coli strain deficient in spermidine acetyltransferase, an enzyme that metabolizes spermidine. Toxicity and inhibition of cell growth by spermidine were recovered in cells transformed with pUCmdtJI or pMWmdtJI, encoding MdtJ and MdtI, which belong to the small multidrug resistance family of drug exporters. Both mdtJ and mdtI are necessary for recovery from the toxicity of overaccumulated spermidine. It was also found that the level of mdtJI mRNA was increased by spermidine. The spermidine content in cells cultured in the presence of 2 mM spermidine was decreased, and excretion of spermidine from cells was enhanced by MdtJI, indicating that the MdtJI complex can catalyze excretion of spermidine from cells. It was found that Tyr⁴, Trp⁵, Glu¹⁵, Tyr⁴⁵, Tyr⁶¹, and Glu⁸² in MdtJ and Glu⁵, Glu¹⁹, Asp⁶⁰, Trp⁶⁸, and Trp⁸¹ in MdtI are involved in the excretion activity of MdtJI.     

Identification and Characterization of a Two-Component Sensor-Kinase and Response-Regulator System (DcuS-DcuR) Controlling Gene Expression in Response to C4-Dicarboxylates in Escherichia coli

The dcuB gene of Escherichia coli encodes an anaerobic C₄-dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition, dcuB expression is strongly induced by C₄-dicarboxylates, suggesting the presence of a novel C₄-dicarboxylate-responsive regulator in E. coli. This paper describes the isolation of a Tn10 mutant in which the 160-fold induction of dcuB expression by C₄-dicarboxylates is absent. The corresponding Tn10 mutation resides in the yjdH gene, which is adjacent to the yjdG gene and close to the dcuB gene at ~93.5 min in the E. coli chromosome. The yjdHG genes (redesignated dcuSR) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the dcuSR operon restored the C₄-dicarboxylate inducibility of dcuB expression in the dcuS mutant to levels exceeding those of the dcuS⁺ strain by approximately 1.8-fold. The dcuS mutation affected the expression of other genes with roles in C₄-dicarboxylate transport or metabolism. Expression of the fumarate reductase (frdABCD) operon and the aerobic C₄-dicarboxylate transporter (dctA) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C₄-dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the dcuS mutant was normal. However, under aerobic conditions with C₄-dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a dctA mutant. Studies employing a dcuA dcuB dcuC triple mutant unable to transport C₄-dicarboxylates anaerobically revealed that C₄-dicarboxylate transport is not required for C₄-dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a ~140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C₄-dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of Klebsiella pneumoniae. DcuS is not closely related to the C₄-dicarboxylate-sensing DctS or DctB protein of Rhodobacter capsulatus or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.     

Molecular Subtyping and Genetic Analysis of the Enterohemolysin Gene (ehxA) from Shiga Toxin-Producing Escherichia coli and Atypical Enteropathogenic E. coli

Analyses of the distribution of virulence factors among different Escherichia coli pathotypes, including Shiga toxin-producing E. coli (STEC), may provide some insight into the mechanisms by which different E. coli strains cause disease and the evolution of distinct E. coli types. The aim of this study was to examine the DNA sequence of the gene for enterohemolysin, a plasmid-encoded toxin that readily causes the hemolysis of washed sheep erythrocytes, and to assess the distribution of enterohemolysin subtypes among E. coli isolates from various human and animal sources. The 2,997-bp ehxA gene was amplified from 227 (63.8%) of 356 stx- and/or eae-positive E. coli strains isolated from cattle and sheep and from 24 (96.0%) of 25 STEC strains isolated from humans with diarrheal disease. By using PCR and restriction fragment length polymorphism (RFLP) analysis of ehxA, six distinct PCR-RFLP types (A to F) were observed, with strains of subtypes A and C constituting 91.6% of all the ehxA-positive strains. Subtype A was associated mainly with ovine strains with stx only (P < 0.001), and subtype C was associated with bovine eae-positive strains (P < 0.001). Eleven ehxA alleles were fully sequenced, and the phylogenetic analysis indicated the presence of three closely related (>95.0%) ehxA sequence groups, one including eae-positive strains (subtypes B, C, E, and F) and the other two including mainly eae-negative STEC strains (subtypes A and D). In addition to being widespread among STEC strains, stx-negative, eae-positive strains (atypical enteropathogenic E. coli strains) isolated from cattle and sheep have similar ehxA subtypes and hemolytic activities.     

大肠杆菌rpoH基因的克隆,表达及其产物的纯化

本文用ＰＣＲ方法获得大肠杆菌热休克蛋白转录因子σ３２的编码基因ｒｐｏＨ,并克隆在含有ｔａｃ启动子的表达载体ｐＵＨＥ中,经ＩＰＴＧ诱导,在大肠杆菌中表达了Ｃ端融合有６个寡聚组氨酸的σ３２。表达产物经金属螯合层析一步纯化,达到ＳＤＳ－ＰＡＧＥ银染一条带纯度,氨基酸组成分析及Ｎ端序列分析结果与文献报道一致。３５Ｓ细胞内参入实验表明：即使在较低的温度下,表达产物σ３２（Ｈｉｓ）６也能导致热休克蛋白如ＧｒｏＥｌ、ＤｎａＫ、Ｈｔｐ的大量合成．     

Transkingdom Genetic Transfer from Escherichia coli to Saccharomyces cerevisiae as a Simple Gene Introduction Tool

Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP and IncQ groups. We also investigated forms of transfer of these vectors from Escherichia coli into Saccharomyces cerevisiae to develop TKC as a simple gene introduction method. Both types of vectors were transferred precisely, conserving the origin of transfer (oriT) sequences, but IncP-based vectors appeared to be more efficient than an IncQ-based vector. Interestingly, unlike in agrobacterial T-DNA (transfer DNA) transfer, the efficiency of TKC transfer was similar between a wild-type yeast strain and DNA repair mutants defective in homologous recombination (rad51Δ and rad52Δ) or nonhomologous end joining (rad50Δ, yku70Δ, and lig4Δ). Lastly, a shuttle vector with two repeats of IncP-type oriT (oriT^P) sequences flanking a marker gene was constructed. TKC transfer of this vector resulted in precise excision of both the oriT^P loci as well as the marker gene, albeit at a low frequency of 17% of all transconjugants. This feature would be attractive in biotechnological applications of TKC. Taken together, these results strongly suggest that in contrast to agrobacterial T-DNA transfer, the circularization of vector single-stranded DNA occurs either before or after transfer but requires a factor(s) from the donor. TKC is a simple method of gene transfer with possible applications in yeast genetics and biotechnology.