Screening of Environmental DNA Libraries for the Presence of Genes Conferring Lipolytic Activity on Escherichia coli

Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.     

Construction of Environmental DNA Libraries in Escherichia coli and Screening for the Presence of Genes Conferring Utilization of 4-Hydroxybutyrate

Environmental DNA libraries from three different soil samples were constructed. The average insert size was 5 to 8 kb and the percentage of plasmids with inserts was approximately 80%. The recombinant Escherichia coli strains (approximately 930,000) were screened for 4-hydroxybutyrate utilization. Thirty-six positive E. coli clones were obtained during the initial screen, and five of them contained a recombinant plasmid (pAH1 to pAH5) which conferred a stable 4-hydroxybutyrate-positive phenotype. These E. coli clones were studied further. All five were able to grow with 4-hydroxybutyrate as sole carbon and energy source and exhibited 4-hydroxybutyrate dehydrogenase activity in crude extracts. Sequencing of pAH5 revealed a gene homologous to the gbd gene of Ralstonia eutropha, which encodes a 4-hydroxybutyrate dehydrogenase. Two other genes (orf1 and orf6) conferring utilization of 4-hydroxybutyrate were identified during subcloning and sequencing of the inserts of pAH1 and pAH3. The deduced orf1 gene product showed similarities to members of the DedA family of proteins. The sequence of the deduced orf6 gene product harbors the fingerprint pattern of enoyl-coenzyme A hydratases/isomerases. The other sequenced inserts of the plasmids recovered from the positive clones revealed no significant similarity to any other gene or gene product whose sequence is available in the National Center for Biotechnology Information databases.     

Altered Na+ and Li+ Homeostasis in Saccharomyces cerevisiae Cells Expressing the Bacterial Cation Antiporter NhaA

The bacterial Na⁺(Li⁺)/H⁺ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na⁺, Li⁺) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H⁺ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na⁺, Li⁺) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H⁺ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionality of plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase.     

Residue Aspartate-147 from the Third Transmembrane Region of Na+/H+ Antiporter NhaB of Vibrio alginolyticus Plays a Role in Its Activity

NhaB is a bacterial Na(+)/H(+) antiporter with unique topology. The pH dependence of NhaB from Vibrio alginolyticus differs from that of the Escherichia coli NhaB homolog. Replacement of Asp-147 with Glu made high H(+) concentrations a requirement for the NhaB activity. Replacement of Asp-147 with neutral amino acids inactivated NhaB.     

Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on Escherichia coli

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C₂ to C₄) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.     

Cloning and Expression of the Gene for the Na+-Coupled Serine Transporter from Escherichia coli and Characteristics of the Transporter

We cloned a gene (sstT) for the Na⁺/serine symporter from the chromosome of Escherichia coli by using a low-copy-number vector and sequenced it. According to the deduced amino acid sequence, the transporter (SstT) consists of 414 amino acid residues. Hydropathy analysis suggested that the SstT protein possesses 9, instead of 12, hydrophobic domains.     

Thermostable NADP+-Dependent Medium-Chain Alcohol Dehydrogenase from Acinetobacter sp. Strain M-1: Purification and Characterization and Gene Expression in Escherichia coli

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C₂ to C₁₄ and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.     

ftsE(Ts) Affects Translocation of K+-Pump Proteins into the Cytoplasmic Membrane of Escherichia coli

The ftsE(Ts) mutation of Escherichia coli causes defects in cell division and cell growth. We expressed alkaline phosphatase (PhoA) fusion proteins of KdpA, Kup, and TrkH, all of which proved functional in vivo as K⁺ ion pumps, in the mutant cells. During growth at 41°C, these proteins were progressively lost from the membrane fraction. The reduction in the abundance of these proteins inversely correlated with cell growth, but the preformed proteins in the membrane were stable at 41°C, indicating that the molecules synthesized at the permissive temperature were diluted in a growth-dependent manner at a high temperature. Pulse-chase experiments showed that KdpA-PhoA was synthesized, but the synthesized protein did not translocate into the membrane of the ftsE(Ts) cells at 41°C and degraded very rapidly. The loss of KdpA-PhoA from the membrane fractions of ftsE(Ts) cells was suppressed by a multicopy plasmid carrying the ftsE⁺ gene. While cell growth stopped when the abundance of these proteins decreased 15-fold, the addition of a high concentration of K⁺ ions specifically alleviated the growth defect of ftsE(Ts) cells but not cell division, and the cells elongated more than 100-fold. We conclude that one of the causes of growth cessation in the ftsE(Ts) mutants is a defect in the translocation of K⁺-pump proteins into the cytoplasmic membrane.     

三结构域重组FN多肽表达质粒的构建及其表达产物性质的初步鉴定

为探讨三结构域重组迁连蛋白（ＦＮ）在肿瘤治疗中的作用,构建了两个三结构域重组ＦＮ表达质粒ｐＦ９４－６２和ｐＦ９４－８２,它们分别编码两个重组多肽：ＣＨ６２（ＦＮＰｒｏ１２３９－Ｓｅｒ１５１５经Ｍｅｔ、Ａｌａ １６９０－Ｖａｌ２０４９相连）和ＣＨ８２（从ＣＨ６２中删除了ＨｅｒｐⅡＣ端和ＣｅｌｌⅡ结构域Ｎ端的Ｐｒｏ１９５３－Ｇｌｕ１９７８）。含表达质粒ｐＦ９４－８２的工程菌经３７℃培养,ＣＨ８２得到表     

Suppressor Scanning at Positions 177 and 236 in the Escherichia coli Lactose/H+ Cotransporter and Stereotypical Effects of Acidic Substituents That Suggest a Favored Orientation of Transmembrane Segments Relative to the Lipid Bilayer

Acidic substituents for Ala-177 (helix 6) or Tyr-236 (helix 7) in LacY cause effects on sugar recognition and cosubstrate coupling that are stereotypical of neutral substituents. Thus, helices 6 and 7 are probably oriented to produce little side-chain contact with the low dielectric lipid bilayer at positions 177 and 236.     

Replication-Dependent Recruitment of the β-Subunit of DNA Polymerase III from Cytosolic Spaces to Replication Forks in Escherichia coli

The beta-subunit of DNA polymerase III is located as one or two condensed clusters within the nucleoid-occupied space in exponentially growing cells of Escherichia coli. When chromosome replication is terminated after incubation at nonpermissive temperature in a temperature-sensitive dnaC mutant, the beta-subunit is located in the cytosolic spaces of the cell poles.