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An autonomously bioluminescent Saccharomyces cerevisiae BLYAhS bioreporter was developed in this study for the simple and rapid detection of dioxin-like compounds (DLCs) and aryl hydrocarbon receptor (AhR) agonists. This recombinant yeast reporter was based on a synthetic bacterial luciferase reporter gene cassette (lux) that can produce the luciferase as well as the enzymes capable of self-synthesizing the requisite substrates for bioluminescent production from endogenous cellular metabolites. As a result, bioluminescent signal production is generated continuously and autonomously without cell lysis or exogenous reagent addition. By linking the expression of the autobioluminescent lux reporter cassette to AhR activation via the use of a dioxin-responsive promoter, the S. cerevisiae BLYAhS bioreporter emitted a bioluminescent signal in response to DLC exposure in a dose-responsive manner. The model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), could be detected within 4 h with a half maximal effective concentration (EC 50 ) of ~ 8.1 nM and a lower detection limit of 500 pM. The autobioluminescent response of BLYAhS to other AhR agonists, including 2,3,7,8-tetrachlorodibenzofuran (TCDF), polychlorinated bisphenyl congener 126 (PCB-126) and 169 (PCB-169), 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), benzo[a]pyrene (BaP), and β-naphthoflavone (bNF), were also characterized in this study. The non-destructive and reagent-free nature of the BLYAhS reporter assay facilitated near-continuous, automated signal acquisition without additional hands-on effort and cost, providing a simple and cost-effective method for rapid DLC detection.

Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A(2) (PLA(2)) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA(2) in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA(2) inhibited both (3)H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA(2). Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA(2).

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants that can, under proper conditions, be degraded by microorganisms. The responses of a riverine sedimentary microbial community to PAH contamination were examined using an integrated biochemical assay that yielded data on PAH concentration, total microbial biomass, and microbial community structure and were interpreted using perturbation theory and the subsidy-stress gradient. Microbial mineralization of naphthalene, anthracene, fluorene, and phenanthrene was observed 24 h after their addition to all sediments sampled and ranged from 0.9 to 16.3% in ambient sediments and from 14.8 to 35.8% in contaminated sediments. Total microbial biomass, determined by phospholipid phosphate, increased in response to intermediate PAH concentration and decreased at sites with the highest PAH concentration (p < 0.05) during seven out of nine (78%) seasonal sampling periods. The two sampling periods that were not statistically different followed periods of high water and cold temperatures. Phospholipid fatty acid analysis of microbial community structure analysis indicated that increases in the relative abundance of gram-negative aerobes and heterotrophic eukaryotes were responsible, in part, for these observed increases in total microbial biomass. These findings (increased degradation rates, increased biomass at intermediate PAH concentrations, and altered community structure) indicate that a component of the microbial community responded to PAH as a usable input and are consistent with the predictions of perturbation theory and a subsidy-stress gradient.

Previous studies demonstrated that Alcaligenes eutrophus A5 transforms 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) to 4-chlorobenzoate via a meta-ring fission product. The initial reactions could be catalyzed by either monooxygenase or dioxygenase enzymes. In the present study, a transient intermediate that accumulated during the transformation of DDT by the biphenyl-grown cells was identified as 1,1,1-trichloro-2-(4-chlorophenyl-2,3-dihydro-4,6-cyclohexadiene)-2-(4'- chlorophenyl)ethane (DDT-2,3-dihydrodiol) on the basis of mass spectral analysis after n-butylboronic acid derivatization. The dihydrodiol undergoes a characteristic acid-catalyzed dehydration to produce phenols. 1H-NMR indicated a cis-relative stereochemistry. The results indicate that the biphenyl dioxygenase from A. eutrophus A5 catalyzes the dihydroxylation of DDT at the unsubstituted carbons on the aromatic ring to produce DDT-2,3-dihydrodiol.

The distribution of alginate genes encoding biosynthesis of alginate was examined for bacterial isolates associated with corrosive biofilms recovered from source water, cooling lines, and reactor surfaces of a nuclear power plant. A total of 120 diverse Gram-positive and -negative isolates were obtained. Using DNA:DNA hybridization, 11 isolates were shown to contain sequences homologous to structural (algD, algG, alg-76) and/or regulatory (albB) alginate biosynthetic genes derived from an alginate-producing cystic fibrosis isolate of Pseudomonas aeruginosa (FRD1). Identification of isolates was accomplished by fatty acids methyl esters (FAME) analysis and the Biolog identification system. Nine of the twelve isolates were identified as various Pseudomonas spp., and two additional Gram-negative isolates were tentatively identified as Aeromonas veronii and Stenotrophomonas maltophilia. The remaining isolate was identified as a Gram-positive Bacillus pumilus. The results of the investigation extend current knowledge on the distribution of alginate biosynthetic genes in environmental isolates and permits the development of a more environmentally realistic model system to investigate the role of exopolymer production in biofilm formation and biocorrosion processes.

The persistence and interaction between newly isolated strains ofPseudomonas aeruginosa and resident bacteriophages indigenous to a freshwater environment was monitored over 45 days in lake water microcosms. The interaction between susceptible and resistant bacteria with pure phage (UT1) particles or a mixed phage population (M1) was investigated by following temporal changes in host density, phage-to-bacteria ratio (PBR), and the appearance of apparent prophage carriers within the host population. Decay rates of the phage (UT1) ranged from 0.054 hour(-1) in natural water to 0.027 hour(-1) in filtered lake water. About 45% of sensitive bacteria incubated with phase UT1 were pseudolysogenic within 12 hours of incubation in natural lake water. This process was delayed until 72 hours in the steile lake water control, suggesting that host-phage interaction is promoted in the presence of a viable natural microbial community. Phage UT1 appeared to stabilize the density of host bacteria in lake water at a level of 10(4) colony-forming units (cfu) ml(-1). Bacterial coexistence with the mixed phage (M1) population resulted in an oscillating equilibrium with the PBR stabilizing at about 3. The presence of extraneous homoimmune phages appeared to be detrimental to the stability of the pseudolysogens, which were maintained at a lower population density than prophage-free cells in lake water containing the mixed phage (M1) population.

The environmental and ecological significance of catabolic plasmids and their host strains are discussed in the context of their potential application for environmental biotechnology. Included is a comprehensive list of naturally occurring discrete catabolic plasmids isolated from either natural habitats or selective enrichment studies. General properties, such as plasmid maintenance, stability and transfer, are discussed together with the techniques for plasmid detection and monitoring in the environment. The issues concerning the construction of catabolic strains with new or broader substrate ranges and the uses of monocultures or consortia for in situ treatment are addressed.

Numerical taxonomic analysis of a freshwater bacterial guild demonstrated that the bacteria capable of growth on phenanthrene and polychlorinated biphenyl media were representative of the taxa obtained from low nutrient oligotrophic media. The diversity of heterotrophic bacteria and members of new taxa recovered from the guild followed a poisson distribution relative to the number of isolation media used. Moderately high nutrient, yeast extract peptone and glucose agar was found to be the most selective isolation medium relative to the total number of taxa recovered whereas low nutrient, lake water agar was the least selective medium used. Carbon source utilization patterns of the isolated taxa indicated that taxa within the guild had broad niche ranges and could potentially occupy many niches within a dynamic environment. The structure of the bacterial guild was dominated by mesophilic oligotrophs.The results of this investigation demonstrate that potential biodegradative populations are representative of the diverse taxa found in uncontaminated freshwater environments.

Strain ABS10, a Gram-negative, pleomorphic bacterium isolated from a pristine aquifer in Ada, Oklahoma, was studied as a candidate for the introduction and expression of plasmid DNA in a native ground water isolate. This organism was originally typed as anArthrobacter sp. due to its morphological phase change and Gram-variable reaction upon Gram staining. The fatty acid methyl ester profile of ABS10 revealed a high similarity withPseudomonas putida. DNA-DNA hybridization showed 81% homology between ABS10 andP. putida. 16S rRNA sequence analysis showed ABS 10 to be a member of the Gamma division of the purple photosynthetic bacteria. The organism has been designatedPseudomonas geomorphus due to its isolation from a subterranean sample and the morphological phase change from rods in young cultures to cocci in older cultures. The broad host range plasmid RP4 was introduced into ABS10 and stably maintained, indicating that RP4 may serve as a vehicle for the introduction of catabolic genes into this organism.

Comparative numerical taxonomic analyses, using fatty acid methyl ester (FAME) profiles and phenetic characteristics, were conducted to examine bacterial guild structure in freshwater sediments. Both approaches were used to examine a subset of 60 OTUs obtained from a previously well characterized microbial community in sediments of a shallow fast flowing stream. For both classifications, greater than 80% of the OTUs were recovered in 11 and 12 major groups for FAME and phenetics approaches. However, there was not complete correspondence for the groupings of the 2 classifications, with most FAME groups being distributed among the phenetic groups and 2 phenetic groups not providing usable characteristics for FAME analysis. The results did demonstrate significant taxonomic variation in bacteria capable of occupying the same or a similar fundamental niche.