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Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications. Nurmohammad Shaikh, 2003.As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E . coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1 . Between these events, sorbitol-fermenting E . coli O157:H- presumably diverged from this clade . We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx . Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E . coli O157:H7 strains that lack stx1 (stx1-negative strains) . Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region . A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer . An stx2 bacteriophage usually occupies wrbA in stx1+/stx2+ E . coli O157:H7, but wrbA is unexpectedly unoccupied in most stx1-negative/stx2+ E . coli O157:H7 strains, the presumed progenitors of stx1+/stx2+ E . coli O157:H7 . Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E . coli O157:H7 strains tested; bile salts usually attenuate excision . These data demonstrate the unexpected diversity of the chromosomal architecture of E . coli O157:H7 (with novel truncated bacteriophages and multiple stx2 bacteriophage insertion sites), suggest that stx1 acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined . The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea. Verena Siewers, 2004.The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction . Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps . We present here evidence that this "direct" pathway is indeed the only one used by an ABA-overproducing strain of B . cinerea . Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway . Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions . Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis:  Bcaba1 mutants contained no residual ABA . Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene .
Characterization and Regulation of the gbuA Gene, Encoding Guanidinobutyrase in the Arginine Dehydrogenase Pathway of Pseudomonas aeruginosa PAO1. Yuji Nakada, 2002.The arginine dehydrogenase (or oxidase) pathway catabolically converts arginine to succinate via 2-ketoglutarate and 4-guanidinobutyrate (4-GB) with the concomitant formation of CO2 and urea . Guanidinobutyrase (GBase; EC 3.5.3.7) catalyzes the conversion of 4-guanidinobutyrate to 4-aminobutyrate and urea in this pathway . We investigated the structure and regulation of the gene for GBase (designated gbuA) of Pseudomonas aeruginosa PAO1 and characterized the gbuA product . The gbuA and the adjacent gbuR genes were cloned by functional complementation of a gbuA9005 mutant of strain PAO1 defective in 4-GB utilization . The deduced amino acid sequence of GbuA (319 amino acids; Mr 34,695) assigned GBase to the arginase/agmatinase family of C-N hydrolases . Purified GbuA was a homotetramer of 140 kDa that catalyzed the specific hydrolysis of 4-GB with Km and Kcat values of 49 mM and 1,012 s-1, respectively . The divergent gbuR gene, which shared the intergenic promoter region of 206 bp with gbuA, encoded a putative regulatory protein (297 amino acids; Mr 33,385) homologous to the LysR family of proteins . Insertional inactivation of gbuR by a gentamicin resistance cassette caused a defect in 4-GB utilization . GBase and gbuA'::'lacZ fusion assays demonstrated that this gbuR mutation abolishes the inducible expression of gbuA by exogenous 4-GB, indicating that GbuR participates in the regulation of this gene . Northern blotting located an inducible promoter for gbuA in the intergenic region, and primer extension localized the transcription start site of this promoter at 40 bp upstream from the initiation codon of gbuA . The gbuRA genes at the genomic map position of 1547000 are unlinked to the 2-ketoarginine utilization gene kauB at 5983000, indicative of at least two separate genetic units involved in the arginine dehydrogenase pathway . Temperature Sensing by the dsrA Promoter. F. Repoila, 2003.Synthesis of the small regulatory RNA DsrA is under temperature control . The minimal dsrA promoter of 36 bp contains sufficient information to ensure such regulation . In vivo, we have analyzed the critical elements responsible for the temperature control of dsrA by using a collection of chimeric promoters combining various elements of the dsrA promoter and the lacUV5 promoter, which does not respond to temperature . Our results favor an RNA polymerase-DNA interaction model instead of a trans-acting factor for temperature regulation . While all of the elements of the dsrA promoter contribute to temperature-sensitive expression, the sequence of the -10 box and the spacer region are the essential elements for the thermal response of the dsrA promoter . The proper context for these promoter elements, including at least one of the flanking elements, the -35 region or the start site region, is also required . Point mutations demonstrate that the sequence of the -10 box imposes constraints on the length and the sequence of the spacer and/or its AT richness, even at low temperature . These results show a complex interdependence of different regions in the promoter for temperature regulation .
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