Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment.

Microorganisms maintain the biosphere by catalyzing biogeochemical processes, including biodegradation of organic chemical pollutants. Yet seldom have the responsible agents and their respective genes been identified. Here we used field-based stable isotopic probing (SIP) to discover a group of bacteria responsible for in situ metabolism of an environmental pollutant, naphthalene. We released 13C-labeled naphthalene in a contaminated study site to trace the flow of pollutant carbon into the naturally occurring microbial community. Using GC/MS, molecular biology, and classical microbiological techniques we documented 13CO2 evolution (2.3% of the dose in 8 h), created a library of 16S rRNA gene clones from 13C labeled sediment DNA, identified a taxonomic cluster (92 of 95 clones) from the microbial community involved in metabolism of the added naphthalene, and isolated a previously undescribed bacterium (strain CJ2) from site sediment whose 16S rRNA gene matched that of the dominant member (48%) of the clone library. Strain CJ2 is a beta proteobacterium closely related to Polaromonas vacuolata. Moreover, strain CJ2 hosts the sequence of a naphthalene dioxygenase gene, prevalent in site sediment, detected before only in environmental DNA. This investigative strategy may have general application for elucidating the bases of many biogeochemical processes, hence for advancing knowledge and management of ecological and industrial systems that rely on microorganisms.

A comparitive study of ammonia-oxidizing bacteria in lab-scale industrial wastewater treatment reactors.

The diversity and community structure of the beta-proteobacterial ammonia oxidising bacteria (AOB) in a range of different lab-scale industrial wastewater treatment reactors were compared. Three of the reactors treat waste from mixed domestic and industrial sources whereas the other reactor treats waste solely of industrial origin. PCR with AOB selective primers was combined with denaturing gradient ge electrophoresis to allow comparative analysis of the dominant AOB populations and the phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing or direct sequencing of bands excised from DGGE gels. Different AOB were found within and between different reactors. All AOB sequences identified were grouped within the genus Nitrosomonas. Within the lab-scale reactors there appeared to be selection for a low diversity of AOB and predominance of a single AOB population. Furthermore, the industrial input in both effluents apparently selected for salt tolerant AOB, most closely related to Nitrosococcus mobilis and Nitrosomonas halophila.

Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA.

Our goal was to develop a field soil biodegradation assay using (13)C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived (13)C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of (13)C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for (13)CO(2) respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of (13)CO(2) emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF(6), that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired (13)CO(2). Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of (13)CO(2) released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of (13)C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with (13)C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, and Alcaligenes spp. for phenol; Pseudomonas, Acinetobacter, and Variovorax spp. for naphthalene; and Acinetobacter, Enterobacter, Stenotrophomonas, and Pantoea spp. for caffeine.

A comparison of autotrophic ammonia-oxidizing bacteria in full- and laboratory-scale wastewater treatment reactors.

Lab-scale reactors are commonly used to simulate full-scale plants as they permit the effects of defined experimental perturbations to be evaluated. Ideally, lab- and full-scale reactors should possess similar microbial populations. To determine this we compared the diversity of the beta-proteobacterial autotrophic ammonia-oxidising bacteria (AOB) in a full-scale and lab-scale biological aerated filter (BAF) using PCR with AOB selective primers combined with denaturing gradient gel electrophoresis (DGGE). PCR amplified 16S rRNA gene fragments from the nitrification unit of the lab-and full-scale BAF were subjected to cloning and sequencing to determine the phylogenetic affiliation of the AOB. A high degree of comparability between the lab-and full-scale BAF was observed with respect to AOB populations. However minor differences were apparent. The importance of these minor constituents in the overall performance of the reactor is unknown. Nonetheless the lab-scale reactor in this study did appear to reflect the dominant AOB community within the full-scale equivalent.

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

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 (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees 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 alpha/beta 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.

Biodegradation of Glycerol Trinitrate and Pentaerythritol Tetranitrate by Agrobacterium radiobacter.

Bacteria capable of metabolizing highly explosive and vasodilatory glycerol trinitrate (GTN) were isolated under aerobic and nitrogen-limiting conditions from soil, river water, and activated sewage sludge. One of these strains (from sewage sludge) chosen for further study was identified as Agrobacterium radiobacter subgroup B. A combination of high-pressure liquid chromatography and nuclear magnetic resonance analyses of the culture medium during the growth of A. radiobacter on basal salts-glycerol-GTN medium showed the sequential conversion of GTN to glycerol dinitrates and glycerol mononitrates. Isomeric glycerol 1,2-dinitrate and glycerol 1,3-dinitrate were produced simultaneously and concomitantly with the disappearance of GTN, with significant regioselectivity for the production of the 1,3-dinitrate. Dinitrates were further degraded to glycerol 1- and 2-mononitrates, but mononitrates were not biodegraded. Cells were also capable of metabolizing pentaerythritol tetranitrate, probably to its trinitrate and dinitrate analogs. Extracts of broth-grown cells contained an enzyme which in the presence of added NADH converted GTN stoichiometrically to nitrite and the mixture of glycerol dinitrates. The specific activity of this enzyme was increased 160-fold by growth on GTN as the sole source of nitrogen.

Comparison of pathways for biodegradation of monomethyl sulphate in Agrobacterium and Hyphomicrobium species.

Different mechanisms have been proposed previously for the biodegradation of monomethyl sulphate (MMS) in Agrobacterium sp. and Hyphomicrobium sp. Sulphate liberation from MMS in Agrobacterium sp. M3C was previously shown to be O2-dependent, whereas in several Hyphomicrobium spp. the initiating step has been considered hitherto to be hydrolytic and catalysed by methyl sulphatase. In the present study, Agrobacterium and Hyphomicrobium strains were compared for their ability to oxidize MMS and its potential metabolites in the oxygen electrode. MMS-grown Agrobacterium sp. M3C and Hyphomicrobium sp. MS223 oxidized MMS with consumption of 0.5 mol O2 per mol of substrate, but they were unable to oxidize methanol. By repeatedly challenging MMS-grown Hypomicrobium with MMS in the electrode chamber, all the O2 in the electrode became exhausted, at which point SO4(2-) liberation stopped although excess MMS was available. SO4(2-) release resumed immediately when O2 was re-admitted to the electrode chamber. Thus liberation of SO4(2-) from MMS in the oxygen electrode was dependent on the continuing availability of O2. Hyphomicrobium sp. MS223 therefore closely resembled Agrobacterium sp. M3C in its obligatory requirement for O2 in MMS degradation. Unlike Agrobacterium sp. M3C, Hyphomicrobium sp. MS223 was able to grow on methanol and methanol-grown cells oxidized methanol (0.5 mol O2 per mol of substrate) but not MMS. Cyclopropanol, an inhibitor of methanol dehydrogenase, abolished oxidation of methanol by methanol-grown Hyphomicrobium sp. MS223 but did not affect oxidation of MMS by MMS-grown cells. Thus Hyphomicrobium sp. MS223 expresses enzymes for oxidation of methanol when needed for growth on this compound, but not when grown on MMS.(ABSTRACT TRUNCATED AT 250 WORDS)