The global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. Of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids.

Sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), formed by the hydrolysis of the plant sulfolipid, is a major component of the biological sulfur cycle. However, pathways for its catabolism are poorly delineated. We examined the hypothesis that mineralization of sulfoquinovose to inorganic sulfate is initiated by reactions of the glycolytic and/or Entner-Doudoroff pathways in bacteria. Metabolites of [U-13 C]sulfoquinovose were identified by 13 C-nuclear magnetic resonance (NMR) in strains of Klebsiella and Agrobacterium previously isolated for their ability to utilize sulfoquinovose as a sole source of carbon and energy for growth, and cell extracts were analyzed for enzymes diagnostic for the respective pathways. Klebsiella sp. strain ABR11 grew rapidly on sulfoquinovose, with major accumulations of sulfopropandiol (2,3-dihydroxypropanesulfonate) but no detectable release of sulfate. Later, when sulfoquinovose was exhausted and growth was very slow, sulfopropandiol disappeared and inorganic sulfate and small amounts of sulfolactate (2-hydroxy-3-sulfopropionate) were formed. In Agrobacterium sp. strain ABR2, growth and sulfoquinovose disappearance were again coincident, though slower than that in Klebsiella sp. Release of sulfate was still late but was faster than that in Klebsiella sp., and no metabolites were detected by 13 C-NMR. Extracts of both strains grown on sulfoquinovose contained phosphofructokinase activities that remained unchanged when fructose 6-phosphate was replaced in the assay mixture with either glucose 6-phosphate or sulfoquinovose. The results were consistent with the operation of the Embden-Meyerhoff-Parnas (glycolysis) pathway for catabolism of sulfoquinovose. Extracts of Klebsiella but not Agrobacterium also contained an NAD ؉ -dependent sulfoquinovose dehydrogenase activity, indicating that the Entner-Doudoroff pathway might also contribute to catabolism of sulfoquinovose.

Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (K m ‫؍‬ 23 M) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35°C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the ␣/␤ barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.Nitrate esters such as glycerol trinitrate (GTN) and pentaerythritol tetranitrate (PETN) are widely used both as explosives and as vasodilators in the treatment of angina. During their long history of exploitation in these applications (24, 34), there has been ample opportunity during production, storage, and use for significant contamination of land sites and water courses to occur, so that site remediation of explosive residues is now an urgent issue worldwide. Moreover, the current acceleration in demilitarization of ordnance and rocket formulations will produce further waste material, raising new environmental concerns. The environmental issues, together with the rarity of naturally occurring analogs (14), make the discovery of microorganisms which can degrade such compounds and thus influence their environmental fate of particular interest. Earlier interest in the elimination of nitrate ester explosives in wastewater treatment plants (35,36) and in their metabolism in fungal organisms (6,7,29,30) led to the first report (21) of denitration of GTN in pure bacterial cultures of Bacillus thuringiensis plus B. cereus and Enterobacter agglomerans, apparently occurring via a hydrolytic pathway, although formation of nitrate was not demonstrated. In contrast, White et al. showed unequivocally that assimilation of nitrogen from GTN in pure cultures of a Pseudomonas sp. (37) and Agrobacterium radiobacter (38) occurred via nitrite (not nitrate) with the concomitant formation of mainly glycerol 1,3-dinitrate (1,3-GDN) and small amounts of the corresponding 1,2-isomer. Cells were able to denitrate both of the dinitrates to mononitrates but not beyond, and they also converted PETN to its tri-and dinitrates. The enzyme responsible was identif...

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Nonylphenol ethoxylate (NPEOx) homologs present in commercial mixtures were rapidly adsorbed to and desorbed from native river sediment. Adsorption isotherms, established using high‐performance liquid chromatography analysis to monitor individual homologs NPEO3 to NPEO13 simultaneously, were linear for each component adsorbing to native sediment, organic‐free sediment, and kaolinite, usually with small positive intercepts on the isotherms indicating an additional low‐capacity, high‐affinity binding site. The adsorption partition coefficients (Kd) for the native sediment decreased progressively from 1,460 L/kg for NPEO3 to 450 L/kg for NPEO10, then increased again slightly for higher homologs. In contrast, Kd values for organic‐free sediment (range 230–590 L/kg) or kaolinite (190–490 L/kg) increased steadily from NPEO3 to NPEO13. Adsorptions to silica and alumina were very weak, but to sewage sludge all components adsorbed strongly (Kd values 12,000–33,000, with a maximum at NPEO7). The adsorption to sewage sludge was related to the low‐capacity, high‐affinity sites observed for native sediment. Dependence of Kd values on ethoxylate chain length was analyzed, in terms of both possible adsorption mechanisms, and the environmental fate and impact of NPEOxs as endocrine disruptors.

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The P2 primary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (200-250-fold) by column chromatography on DEAE-cellulose, Sephadex G-100 and butyl-agarose. The intact protein is a dimer with a mol. wt. of 160 000. Activity towards primary alkyl sulphate esters was maximal at pH 8.3, varied little in the range pH 7.8-8.7, but decreased sharply at higher pH. For a homologous series of primary alkyl sulphate substrates (C6-C12), logKm decreased linearly with increasing chain length, corresponding to a contribution to the free energy of association between enzyme and substrate of -2.5kJ/mol for each additional CH2 group in the alkyl chain. logKi for the competitive inhibition by secondary alkyl 2-sulphate esters followed a similar pattern (-2.4kJ/mol for each additional CH2 group) except that only n-1 carbon atoms effectively participate in hydrophobic bonding, implying that the C-1 methyl group is not involved. logKi values for inhibition primary alkanesulphonates also depended linearly on chain length but with a diminished gradient, indicating a free-energy increment of -1.2kJ/mol per additional CH2 group. The collective results showed the presence of a hydrophobic site on the enzyme capable of accomodating an alkyl chain of considerable length. Cationic structures (in the form of arginine, lysine or histidine), whose presence might be expected for binding the anionic sulphate group, were not detectable at the active site.