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Thioesterase II of Escherichia coli Plays an Important Role in 3-Hydroxydecanoic Acid Production. Zhong Zheng, 2004.3-Hydroxydecanoic acid (3HD) was produced in Escherichia coli by mobilizing (R)-3-hydroxydecanoyl-acyl carrier protein-coenzyme A transacylase (PhaG, encoded by the phaG gene) . By employing an isogenic tesB (encoding thioesterase II)-negative knockout E . coli strain, CH01, it was found that the expressions of tesB and phaG can up-regulate each other . In addition, 3HD was synthesized from glucose or fructose by recombinant E . coli harboring phaG and tesB . This study supports the hypothesis that the physiological role of thioesterase II in E . coli is to prevent the abnormal accumulation of intracellular acyl-coenzyme A . A Previously Unrecognized Step in Pentachlorophenol Degradation in Sphingobium chlorophenolicum Is Catalyzed by Tetrachlorobenzoquinone Reductase (PcpD). MingHua Dai, 2003.The first step in the pentachlorophenol (PCP) degradation pathway in Sphingobium chlorophenolicum has been believed for more than a decade to be conversion of PCP to tetrachlorohydroquinone . We show here that PCP is actually converted to tetrachlorobenzoquinone, which is subsequently reduced to tetrachlorohydroquinone by PcpD, a protein that had previously been suggested to be a PCP hydroxylase reductase . pcpD is immediately downstream of pcpB, the gene encoding PCP hydroxylase (PCP monooxygenase) . Expression of PcpD is induced in the presence of PCP . A mutant strain lacking functional PcpD has an impaired ability to remove PCP from the medium . In contrast, the mutant strain removes tetrachlorophenol from the medium at the same rate as does the wild-type strain . These data suggest that PcpD catalyzes a step necessary for degradation of PCP, but not for degradation of tetrachlorophenol . Based upon the known mechanisms of flavin monooxygenases such as PCP hydroxylase, hydroxylation of PCP should produce tetrachlorobenzoquinone, while hydroxylation of tetrachlorophenol should produce tetrachlorohydroquinone . Thus, we proposed and verified experimentally that PcpD is a tetrachlorobenzoquinone reductase that catalyzes the NADPH-dependent reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone . Involvement of the C-Terminal Extension of the  Polypeptide and of the PucC Protein in LH2 Complex Biosynthesis in Rubrivivax gelatinosus.Anne-Soisig Steunou, 2004.The facultative phototrophic nonsulfur bacterium Rubrivivax gelatinosus exhibits several differences from other species of purple bacteria in the organization of its photosynthetic genes . In particular, the puc operon contains only the pucB and pucA genes encoding the ß and polypeptides of the light-harvesting 2 (LH2) complex . Downstream of the pucBA operon is the pucC gene in the opposite transcriptional orientation . The transcription of pucBA and pucC has been studied . No pucC transcript was detected either by Northern blotting or by reverse transcription-PCR analysis . The initiation site of pucBA transcription was determined by primer extension, and Northern blot analysis revealed the presence of two transcripts of 0.8 and 0.65 kb . The half-lives of both transcripts are longer in cells grown semiaerobically than in photosynthetically grown cells, and the small transcript is the less stable . It was reported that the
polypeptide, encoded by the pucA gene, presents a C-terminal extension which is not essential for LH2 function in vitro . The biological role of this alanine- and proline-rich C-terminal extension in vivo has been investigated . Two mutants with C-terminal deletions of 13 and 18 residues have been constructed . Both present the two pucBA transcripts, while their phenotypes are, respectively, LH2+ and LH2–, suggesting that a minimal length of the C-terminal extension is required for LH2 biogenesis . Another important factor involved in the LH2 biogenesis is the PucC protein . To gain insight into the function of this protein in R . gelatinosus, we constructed and characterized a PucC mutant . The mutant is devoid of LH2 complex under semiaerobiosis but still produces a small amount of these antennae under photosynthetic growth conditions . This conditional phenotype suggests the involvement of another factor in LH2 biogenesis .
Cloning and Sequencing of the Histidine Decarboxylase Genes of Gram-Negative, Histamine-Producing Bacteria and Their Application in Detection and Identification of These Organisms in Fish. Hajime Takahashi, 2003.The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products . To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed . A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed . A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested . Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography . Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii . In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product . Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product . A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences . Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification . The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed .
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