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New Method of Denitrification Analysis of Bradyrhizobium Field Isolates by Gas Chromatographic Determination of 15N-Labeled N2. Reiko Sameshima-Saito, 2004.To evaluate the denitrification abilities of many Bradyrhizobium field isolates, we developed a new 15N-labeled N2 detection methodology, which is free from interference from atmospheric N2 contamination . 30N2 (15N15N) and 29N2 (15N14N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N2 gas having natural abundance of 15N (0.366 atom%) as a carrier gas . The detection limit was 0.04% 30N2, and the linearity extended at least to 40% 30N2 . When Bradyrhizobium japonicum USDA110 was grown in cultures anaerobically with 15NO3–, denitrification product (30N2) was detected stoichiometrically . A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method . The denitrification abilities were partly correlated with filed sites, Bradyrhizobium species, and the hup genotype . The Naphthalene Catabolic (nag) Genes of Ralstonia sp . Strain U2 Are an Operon That Is Regulated by NagR, a LysR-Type Transcriptional Regulator. Rheinallt M. Jones, 2003.In Ralstonia sp . strain U2, the nag catabolic genes, which encode the enzymes for the pathway that catabolizes naphthalene via the alternative ring cleavage gentisate pathway, are transcribed as an operon under the same promoter . nagR, which encodes a LysR-type transcriptional regulator, is divergently transcribed compared to the nag catabolic genes . A 4-bp frameshift deletion in nagR demonstrated that NagR is required for expression of the nag operon . The transcriptional start of the nag operon was mapped, and a putative -10, -35  70-type promoter binding site was identified . Further upstream, a site proximal to the promoter was identified as a site that has bases which have been found to be conserved in the activator-binding motif of other naphthalene pathways . Transcriptional fusion studies demonstrated that NagR regulates the expression of the nag operon positively in the presence of salicylate and to a lesser extent in the presence of 2-nitrobenzoate . Mutation of the LysR-type activator-binding motif in the nag promoter-proximal region resulted in a loss of inducibility of a lacZ reporter gene transcriptionally fused to nagAa, the first gene of the operon . However, other mutations in the region increased the effectiveness of salicylate as an inducer .
Effects of ISSo2 Insertions in Structural and Regulatory Genes of the Trimethylamine Oxide Reductase of Shewanella oneidensis. Christophe Bordi, 2003.We have isolated three Shewanella oneidensis mutants specifically impaired in trimethylamine oxide (TMAO) respiration . The mutations arose from insertions of an ISSo2 element into torA, torR, and torS, encoding, respectively, the TMAO reductase TorA, the response regulator TorR, and the sensor TorS . Although TorA is not the sole enzyme reducing TMAO in S . oneidensis, growth analysis showed that it is the main respiratory TMAO reductase . Use of a plasmid-borne torE'-lacZ fusion confirmed that the TorS-TorR phosphorelay mediates TMAO induction of the torECAD operon . Transcriptional Cross-Regulation of the Catechol and Protocatechuate Branches of the ß-Ketoadipate Pathway Contributes to Carbon Source-Dependent Expression of the Acinetobacter sp . Strain ADP1 pobA Gene. Patricia C. Brzostowicz, 2003.Transcriptional control of carbon source preferences by Acinetobacter sp . strain ADP1 was assessed with a pobA::lacZ fusion during growth on alternative substrates . The pobA-encoded enzyme catalyzes the first step in the degradation of 4-hydroxybenzoate, a compound consumed rapidly as a sole carbon source . If additional aromatic carbon sources are available, 4-hydroxybenzoate consumption is inhibited by unknown mechanisms . As reported here, during growth on aromatic substrates, pobA was not expressed despite the presence of 4-hydroxybenzoate, an inducer that normally causes the PobR regulator to activate pobA transcription . Growth on organic acids such as succinate, fumarate, and acetate allowed higher levels of pobA expression . In each case, pobA expression increased at the end of the exponential growth phase . Complex transcriptional regulation controlled 4-hydroxybenzoate catabolism in multisubstrate environments . Additional studies focused on the wild-type preference for benzoate consumption prior to 4-hydroxybenzoate consumption . These compounds are degraded via the catechol and protocatechuate branches of the ß-ketoadipate pathway, respectively . Here, mutants were characterized that degraded benzoate and 4-hydroxybenzoate concurrently . These mutants lacked the BenM and CatM transcriptional regulators that normally activate genes for benzoate catabolism . A model is presented in which BenM and CatM prevent pobA expression indirectly during growth on benzoate . These regulators may affect pobA expression by lowering the PcaK-mediated uptake of 4-hydroxybenzoate . Consistent with this model, BenM and CatM bound in vitro to an operator-promoter fragment controlling the expression of several pca genes, including pcaK . These studies provide the first direct evidence of transcriptional cross-regulation between the distinct but analogous branches of the ß-ketoadipate pathway .
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