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Characterization of the Second LysR-Type Regulator in the Biphenyl-Catabolic Gene Cluster of Pseudomonas pseudoalcaligenes KF707. Takahito Watanabe, 2003.Pseudomonas pseudoalcaligenes KF707 possesses a biphenyl-catabolic (bph) gene cluster consisting of bphR1A1A2-(orf3)-bphA3A4BCX0X1X2X3D . The bphR1 (formerly orf0) gene product, which belongs to the GntR family, is a positive regulator for itself and bphX0X1X2X3D . Further analysis in this study revealed that a second regulator belonging to the LysR family (designated bphR2) is involved in the regulation of the bph genes in KF707 . The bphR2 gene was not located near the bph gene cluster, and its product (BphR2) exhibited a high level of similarity to NahR (the naphthalene- and salicylate-catabolic regulator belonging to the LysR family) in plasmid NAH7 of Pseudomonas putida . A strain containing a disrupted bphR2 gene failed to grow on biphenyl as a sole source of carbon, and the BphD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) activity was significantly reduced compared to that of wild-type strain KF707 . Furthermore, the same strain exhibited extremely low transcription of bphR1, bphA1, bphC, bphX0, and bphD . However, when the bphR2 gene was provided in trans to the bphR2-disrupted strain, the transcription level of these genes was restored . These results indicate that bphR2 regulates the bph genes positively as a second regulator together with BphR1 . Structural and Topographical Studies of the Type IV Bundle-Forming Pilus Assembly Complex of Enteropathogenic Escherichia coli. Jaiweon Hwang, 2003.The type IV bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for virulence in orally challenged human volunteers and for the localized adherence and autoaggregation in vitro phenotypes . BFP filament biogenesis and function are encoded by the 14-gene bfp operon . The BFP assembly complex, containing a BfpB-His6 fusion protein, was chemically cross-linked in situ, and the complex was then purified from BFP-expressing EPEC by a combination of nickel- and BfpB antibody-based affinity chromatography . Characterization of the isolated complex by immunoblotting using BFP protein-specific antibodies showed that at least 10 of the 14 proteins specified by the bfp operon physically interact to form an oligomeric complex . Proteins localized to the outer membrane, inner membrane, and periplasm are within this complex, thus demonstrating that the complex spans the periplasmic space . A combination of immunofluorescence and immuno-gold thin-section transmission electron microscopy studies localized this complex to one pole of the cell . Isolation and Characterization of a Generalized Transducing Phage for Pseudomonas aeruginosa Strains PAO1 and PA14. Jonathan M. Budzik, 2004.A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of Pseudomonas aeruginosa . A clear-plaque variant of this bacteriophage was isolated . DMS3 is capable of mediating generalized transduction within and between P . aeruginosa strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of P . aeruginosa . Production of Plant-Specific Flavanones by Escherichia coli Containing an Artificial Gene Cluster. Eui Il Hwang, 2003.In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically . Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS) . For the purpose of production of flavanones in Escherichia coli, three sets of an artificial gene cluster which contained three genes of heterologous origins—PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata—were constructed . The constructions of the three sets were done as follows: (i) PAL, 4CL, and CHS were placed in that order under the control of the T7 promoter (PT7) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single PT7 in front of PAL, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both PT7 and the RBS . These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid . E . coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid . Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones . This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin .
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