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The study was conducted to assess the impact of nutrients, dissolved oxygen (DO) concentration and nickel on the formation of river biofilm, its structure, its role and the composition of the community. However, the outcome indicated that the groups were a part of the bacteria developing either from the freshwater and marine environments or from the agricultural soils or from the waste matter of the industries.

Research has been conducted on biodegradation of petroleum hydrocarbons in Alpine soils. The authors have investigated the prevalence of genotypes involved in n-alkane, aromatic hydrocarbon and polycyclic aromatic hydrocarbon degradation using PCR hybridization analyses of the soil microbial community DNA.

Determining the toxicity of contaminants to soil organisms under field conditions is hampered by site-specific and temporal factors that modulate contaminant availability. Assessing the pollution-induced community tolerance (PICT) of indigenous microbial communities integrates these complex environmental factors. The purpose of this study wasto determine if the PICT response was proportional to 2,4,6-trinitrotoluene (TNT) concentrations in soil, if changes detected by PICT were also evident in soil microbial community composition, and if the PICT response correlated with phytotoxicity assays. Microorganisms extracted from TNT-contaminated field soils were mixed with a solution containing six different concentrations of TNT and inoculated into Biolog ECO plates. The utilization rate of substrates was determined overa 7-d period. Denaturing gradient gel electrophoretic analysis of a portion of the gene encoding l6S rDNA described the structure of the soil microbial community. Phytoindicators (Poa compressa and P. palustris L.) of TNT pollution were identified and used to assess TNT phytotoxicity in soil samples. The TNT (in Biolog wells) greatly inhibited microbial communities from locations with low in situ TNT exposure.The inhibition of microbial use of L-asparagine, L-phenylalanine, and D-glucosaminic acid by TNT (in Biolog wells) increased as TNT concentration in soil decreased. Locations differing in ECO-PICT response also differed in their microbial community composition and TNT phytotoxicity. Decreased phytotoxicity of field soils corresponded to decreases in PICT. The results from this study indicated that ECO-PICT is an effective assay to rapidly detect TNT exposure and toxicity in soil microbial communities. [ABSTRACT FROM AUTHOR]

The influence of moisture on the survival, movement and degradation activity of a 2,4-D degrading bacterium, Burkholderia cepacia strain BRI6001L, genetically engineered to contain bioluminescent and lactose utilization genes, was studied in unsaturated soil columns. The distance traveled by BRI6001L was dependent on the clay content of the soil, higher clay contents being responsible for higher filtration coefficients. Long term survival, in excess of one year, was attributed to strain BRI6001L's ability to survive dry conditions. Changes in the 2,4-D biodegradation rate showed a better correlation with the BRI6001L population density than with the total viable bacterial population. At moisture levels between field capacity and 40% moisture (-33 kPa to -100 kPa) 2,4-D degradation was attributed mainly to BRI6001L. At moisture levels between 6 and 15%, 2,4-D disappearance was attributed to the indigenous microbial population, with no degradation occurring at moisture levels below 6%. Returning the moisture to above 40%led to an increase of 4 orders of magnitude in the BRI6001L population density and to a 10-fold increase in the 2,4-D degradation rate. The ability to monitor a specific microbial population using reporter genes has demonstrated the importance of controlling moisture levels for maximizing biodegradation rates in unsaturated soil environments. [ABSTRACT FROM AUTHOR]

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E.faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential. [ABSTRACT FROM AUTHOR]

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001, supplemented with carbon (glucose), nitrogen (NH4Cl), phosphorus (KH2PO4), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. Both nirS and nirK denitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N2, suggesting that emission of N2O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, and nirS and nirK genes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence of nirS and nirK genes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P ≤ 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane. [ABSTRACT FROM AUTHOR]

Fluorescent microspheres were applied in a novel fashion during subsurface drilling of permafrost and ground ice in the Canadian High Arctic to monitor the exogenous microbiological contamination of core samples obtained during the drilling process. Prior to each drill run, a concentrated fluorescent microsphere (0.5-µm diameter) solution was applied to the interior surfaces of the drill bit, core catcher, and core tube and allowed to dry. Macroscopic examination in the field demonstrated reliable transfer of the microspheres to core samples, while detailed microscopic examination revealed penetration levels of less than 1 cm from the core exterior. To monitor for microbial contamination during downstream processing of the permafrost and ground ice cores, a Pseudomonas strain expressing the green fluorescent protein (GFP) was painted on the core exterior prior to processing. Contamination of the processed core interiors with the GFP-expressing strain was not detected by culturing the samples or by PCR to detect the gfp marker gene. These methodologies were quick, were easy to apply, and should help to monitor the exogenous microbiological contamination of pristine permafrost and ground ice samples for downstream culture-dependent and culture-independent microbial analyses. [ABSTRACT FROM AUTHOR]

The effect of contamination with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on an indigenous soil bacterial community was examined in two uncontaminated loam soil columns possessing native grasses. One column was spiked twice with RDX crystals for a total RDX load of 1000 mg (kg soil)(-1). The reduced metabolite of RDX degradation, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine, was observed in the column leachate, suggesting anaerobic degradation of RDX. Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA from both contaminated and uncontaminated columns produced identical banding patterns which were stable over the course of the experimental period. The bacterial diversity remained high in the contaminated column, as determined by restriction fragment length polymorphism and rarefaction analyses of random 16S rDNA clones. These combined results suggested that long-term exposure to 1000 mg RDX (kg soil)(-1) did not produce an observable effect on bacterial diversity or the numerically dominant members of the indigenous soil bacterial community.

The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4). In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a rubredoxin reductase (rubB). The alkB3 and alkB4 homologs were found as separate genes which were not part of alk gene clusters. Functional heterologous expression of some of the rhodococcal alk genes (alkB2, rubA2, and rubA4 [NRRL B-16531]; alkB2 and rubB [Q15]) was achieved in Escherichia coli and Pseudomonas expression systems. Pseudomonas recombinants containing rhodococcal alkB2 were able to mineralize and grow on C(12) to C(16) n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to the Pseudomonas putida GPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus.

Abstract The prevalence of four alkane monooxygenase genotypes (Pseudomonas putida GPo1, Pp alkB; Rhodococcus sp. strain Q15, Rh alkB1 and Rh alkB2; and Acinetobacter sp. strain ADP-1, Ac alkM) in hydrocarbon-contaminated and pristine soils from the Arctic and Antarctica were determined by both culture-independent (PCR hybridization analyses) and culture-dependent (colony hybridization analyses) molecular methods, using oligonucleotide primers and DNA probes specific for each of the alk genotypes. PCR hybridization of total soil community DNA detected the rhodococcal alkB genotypes in most of the contaminated (Rh alkB1, 18/20 soils; Rh alkB2, 13/20) and many pristine soils (Rh alkB1, 9/10 soils; Rh alkB2, 7/10), while Pp alkB was generally detected in the contaminated soils (15/20) but less often in pristine soils (5/10). Ac alkM was rarely detected in the soils (1/30). The colony hybridization technique was used to determine the prevalence of each of the alk genes and determine their relative abundance in culturable cold-adapted (5 degrees C) and mesophilic populations (37 degrees C) from eight of the polar soils. The cold-adapted populations, in general, possessed relatively higher percentages of the Rh alkB genotypes (Rh alkB1, 1.9% (0.55); Rh alkB2, 2.47% (0.89)), followed by the Pp alkB (1.13% (0.50)), and then the Ac alkM (0.53% (0.36)). The Rh alkB1 genotype was clearly more prevalent in culturable cold-adapted bacteria (1.9% (0.55)) than in culturable mesophiles (0.41 (0.55)), suggesting that cold-adapted bacteria are the predominant organisms possessing this genotype. Overall, these results indicated that (i) Acinetobacter spp. are not predominant members of polar alkane degradative microbial communities, (ii) Pseudomonas spp. may become enriched in polar soils following contamination events, and (iii) Rhodococcus spp. may be the predominant alkane-degradative bacteria in both pristine and contaminated polar soils.

Anaerobic degradation of pentachlorophenol (PCP) is an example of a process that may benefit from enrichment or bioaugmentation. In one approach, enrichment acceleration was attempted by applying an on-line control-based selective stress strategy to a native anaerobic upflow sludge bed (UASB) system; this strategy linked PCP loading rate to methane production. As a result, the reactor biomass potential for PCP complete dechlorination reached a rate of 4 mg g(-1) volatile suspended solid (VSS) day(-1) within a period of 120 days. In another approach, a pure culture, Desulfitobacterium frappieri PCP-1, a strictly anaerobic Gram-positive bacterium, was used to augment the granular biomass of the UASB reactor. This also resulted in a specific degradation rate of 4 mg PCPg(-1) VSS day(-1); however, this potential was attained within 56 days. Fluorescent in situ hybridization (FISH) showed that the PCP-1 strain was able to rapidly attach to the granule and densely colonize the outer biofilm layer.

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

Genomic DNA from nine individual bacteria, isolated from a diclofop-methyl-degrading biofilm consortium, was extracted for genetic characterization. The degradation of diclofop-methyl produces metabolites that are known intermediates or substrates for bacteria that degrade a variety of chlorinated aromatic compounds. Accordingly, oligonucleotide primers were designed from specific catabolic genes for chlorinated organic degradation pathways, and tested by PCR to determine if these genes are involved in diclofop-methyl degradation. DNA homology between the PCR products and the known catabolic genes investigated by Southern hybridization analysis and by sequencing, suggested that novel catabolic genes are functioning in the isolates. Specific fluorescent oligonucleotides were designed for two of the isolates, following 16S rDNA sequencing and identification of each of the isolates. These probes were successfully used for fluorescent in situ hybridization (FISH) studies of the two isolates in the biofilm consortium.

We examined physiological adaptations which allow the psychrotroph Rhodococcus sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5 degrees C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5 degrees C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [beta-D-Gal-(1-3)-D-GlcNAc and alpha-L-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5 degrees C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5 degrees C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.

The acetonitrile-sonication extraction method (US EPA SW-846 Method 8330) and aquatic-based toxicity tests were used on laboratory and field samples, to characterize the ecotoxicity of soils contaminated with energetic substances. Spiked soil studies indicated that 2,4, 6-trinitrotoluene (TNT)-dependent soil toxicity could be measured in organic extracts and aqueous leachates using the 15-min Microtox (Vibrio fischeri, IC50=0.27 to 0.94 mg TNT/liter incubation medium) and 96-h Selenastrum capricornutum growth inhibition (IC50=0.62 to 1. 14 mg/liter) toxicity tests. Analyses of leachates of composite soil samples [containing TNT and some TNT metabolites, 1,3,5-trinitro-1,3, 5-triazacyclohexane (RDX), and 1,3,5,7-tetranitro-1,3,5, 7-tetrazacyclooctane (HMX)] from an explosives manufacturing facility, indicated toxicities similar to those found in the TNT-spiked soil studies and pure TNT in solution, and suggested that TNT was the major toxicant. Using TNT as a model toxicant in soils having different moisture contents (20% vs dry) and textures (sandy vs clayey-sandy) but similar organic matter content (3-4%), multi-factorial analyses of Microtox test data revealed that these soil factors significantly influenced the TNT extractability from soil and subsequent toxicity measurements. Taken together, data indicate that the modified Method 8330 may be used in conjunction with ecotoxicity tests to reflect the toxic potential of soils contaminated with energetic substances., (Copyright 1999 Academic Press.)

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS-, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.

The psychorotrophic Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5 degrees C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C10 to C21 alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 degrees C. Mineralization of hexadecane at 5 degrees C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis the A gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 degrees C.

Chloroaliphatics are major components of bleached kraft mill effluents. Gene probes and oligonucleotide primers were developed to monitor kraft pulp mill effluent treatment systems for the presence of key genes (dehalogenases) responsible for the dehalogenation of chloroaliphatic organics. The primers were used for polymerase chain reaction (PCR) analysis of genomic DNA extracted from dehalogenating bacterial isolates and from total community DNA extracted from water and sediments of mill effluent treatment system. PCR amplification with oligonucleotide primers designed from dhlB, encoding the haloacid dehalogenase from Xanthobacter autotrophicus, revealed the presence of dehalogenase genes in both aerated lagoons and stabilization basins. Similarly, positive results were obtained with mmoX primers designed from the soluble methane monooxygenase gene of Methylococcus capsulatus Bath. The haloacetate dehalogenase encoding gene (dehH2) from Moraxella sp. was typically not detected in mill effluent treatment systems unless the biomass was selectively enriched. DNA sequence analysis of several PCR fragaments revealed significant similarity to known dehalogenase amd methane monooxygenase genes. The results indicated a broad distribution of known dehalogenation genes and bacteria with chloroorganic-degrading potential in the mill effluent treatment systems.

Three hydrocarbon-degrading psychrotrophic bacteria were isolated from petroleum-contaminated Arctic soils and characterized. Two of the strains, identified as Pseudomonas spp., degraded C5 to C12 n-alkanes, toluene, and naphthalene at both 5 and 25 degrees C and possessed both the alk catabolic pathway for alkane biodegradation and the nah catabolic pathway for polynuclear aromatic hydrocarbon biodegradation. One of these strains contained both a plasmid slightly smaller than the P. oleovorans OCT plasmid, which hybridized to an alkB gene probe, and a NAH plasmid similar to NAH7, demonstrating that both catabolic pathways, located on separate plasmids, can naturally coexist in the same bacterium.

The cloned sulfur oxidation (desulfurization) genes (sox) for dibenzothiophene (DBT) from the prototype Rhodococcus sp. strain IGTS8 were used in Southern hybridization and PCR experiments to establish the DNA relatedness in six new rhodococcal isolates which are capable of utilizing DBT as a sole sulfur source for growth. The ability of these strains to desulfurize appears to be an exclusive property of a 4-kb gene locus on a large plasmid of ca. 150 kb in IGTS8 and ca. 100 kb in the other strains. Besides a difference in plasmid profile, IGTS8 is distinguishable from the other strains in at least the copy number of the insertion sequence IS1166, which is associated with the sox genes.

Polyvinylpolypyrrolidone spin columns were used to rapidly purify crude soil DNA extracts from humic materials for polymerase chain reaction (PCR) analysis. The PCR detection limit for the tfdC gene, encoding chlorocatechol dioxygenase from the 2,4-dichlorophenoxyacetic acid degradation pathway, was 10(1)-10(2) cells/g soil in inoculated soils. The procedure could be applied to the amplification of biodegradative genes from indigenous microbial populations from a wide variety of soil types, and the entire analysis could be performed within 8 h.

Bioremediation of polluted temperate and cold temperature environments may require the activity of psychrotrophic bacteria, because their low temperature growth range parallels the ambient temperatures encountered in these environments. In the present study, 135 psychrotrophic microorganisms isolated from a variety of ecosystems in Canada were examined for their ability to mineralize 14C-labelled toluene, naphthalene, dodecane, hexadecane, 2-chlorobiphenyl, and pentachlorophenol. A number of the psychrotrophic strains mineralized toluene, naphthalene, dodecane, and hexadecane. None of the psychrotrophs were capable of mineralizing 2-chlorobiphenyl or pentachlorophenol. Those strains demonstrating mineralization activity were subsequently screened by the polymerase chain reaction (PCR) and Southern hybridization of PCR products for the presence of catabolic genes (alkB, ndoB, todCl, and xylE) involved in known bacterial biodegradative pathways for these compounds. Some of the psychrotrophs able to mineralize toluene and naphthalene possessed catabolic genes that hybridized to xylE or todCl, and ndoB, respectively. The alkB PCR fragments obtained from the strains that mineralized dodecane and hexadecane did not hybridize to an alkB gene probe derived from Pseudomonas oleovorans. Psychrotrophic strain Q15, identified as a Rhodococcus sp., also mineralized the C28 n-paraffin octacosane. A gene probe constructed from the "alkB" PCR fragment from strain Q15 did hybridize with the alkB PCR fragments from most of the psychrotrophic alkane biodegraders, indicating that the alkB primers may be amplifying another gene(s), perhaps with low homology to P. oleovorans alkB, which may be involved in the biodegradation of both short chain (dodecane) and longer chain alkanes (hexadecane, octacosane). All of the psychrotrophic biodegradative isolates examined were capable of mineralization activity at both 23 and 5 degrees C, indicating their potential for low temperature bioremediation of petroleum hydrocarbon contaminated sites.

The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading various isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K(infm) and V(infmax) values of 1.4 (mu)M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting the presence of two uptake systems for benzoic acid with distinct K(infm) (0.72 and 5.3 (mu)M) and V(infmax) (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3(prm1), 4(prm1)-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.

A significant proportion of the naturally occurring hydrocarbon-degrading populations within Alaskan sediments affected by the Exxon Valdez oil spill had both the xylE and alkB genes and could convert hexadecane and naphthalene to carbon dioxide; a greater proportion of the population had xylE than had alkB, reflecting the composition of the residual oil at the time of sampling; nearly equal populations with xylE alone, alkB alone, and xylE + alkB genes together were found after exposure to fresh crude oil; populations with xylE lacking alkB increased after enrichment on naphthalene. Thus, the genotypes of hydrocarbon-degrading populations reflected the composition of the hydrocarbons to which they were exposed.

Eighty regional strains of Vibrio isolated from the seasonally cold waters of coastal Newfoundland, and a number of Vibrio reference cultures, were studied. The regional strains had been isolated from the brown macroalga Alaria esculenta and the giant scallop Placopecten magellanicus and were known to grow at 4 degrees C. The strains were grouped according to their arginine-dihydrolase reactions and examined by numerical analysis. According to phenotypic properties the arginine-dihydrolase positive strains closely resembled Vibrio splendidus biovar I. Most clusters of the arginine-dihydrolase negative strains appeared to be unique but the closest phenotypic resemblance among some strains was with Vibrio ordalii. Some strains were examined using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique for fingerprinting and it was shown that the regional strains were significantly different from either V. splendidus biovar I or V. ordalii. Generally, the strains from seaweed clustered separately from those that were from scallops. Strains in some clusters, especially those from the seaweed, were able to utilize most of the compounds that were tested as sole sources of carbon and energy.

Two reporter systems, lacZY and luxAB, were stably integrated into the chromosome of Pseudomonas aeruginosa UG2, a biosurfactant-producing strain. Growth and rhamnolipid production of the UG2 wild-type and reporter gene-bearing UG2L strains were similar in liquid culture. A spontaneous rifampin-resistant detecting UG2Lr, allowed antibiotic selection. Phenotypic characteristics were compared for usefulness in detecting UG2Lr colonies: morphology, fluorescent pigment production, light emission (lux), X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) cleavage (lac), and rifampin resistance. Survival patterns of UG2, UG2L, and UG2Lr strains were similar in sandy loam soil microcosms over 12 12 weeks. The lac marker was not suitable for monitoring P. aeruginosa UG2Lr in soil since 20 to 42% of cultured, aerobic, heterotrophic soil microorganisms formed blue, lactose-positive colonies. The lux genes provided a stable and unequivocal reporter system, as effective as conventional antibiotic plating, for tracking microorganisms nonselectively at 10(3) CFU/g of soil. Three months after inoculation into oil-contaminated and uncontaminated soil microcosms, UG2Lr cells were recovered at 10(7) and 10(4) cells per g (dry weight) of soil, respectively. Detection by PCR amplification of part of the luxA gene confirmed a decrease in UG2Lr cell numbers in uncontaminated soil. In combination, antibiotic resistance, bioluminescence, and PCR analyses provided sensitive detection and quantitative enumeration of P. aeruginosa UG2Lr in soil.

Arthrobacter M5 was characterized genetically to determine if the catabolic pathway (controlled by the bph genes), responsible for polychlorinated biphenyl (PCB) biodegradation in this Gram-positive strain, was similar to the pathways characterized from various Gram-negative bacteria. Arthrobacter M5 was originally isolated as a contaminant from a culture of the PCB degrader, Acinetobacter sp. strain P6. A bph-specific oligodeoxyribonucleotide (oligo) gene probe (bphC2) was designed by aligning the published sequences of two bphC genes (encoding 2,3-dihydroxybiphenyl dioxygenase) and synthesizing a 29-nucleotide (nt) fragment from a conserved region of the gene. The bphC2 oligo was used as a probe to identify a 10-kb HindIII fragment of total DNA from Arthrobacter M5 and subsequently to isolate Escherichia coli clones possessing bphC. The PCB-degradation genes were expressed in E. coli, but expression was increased by subcloning in Pseudomonas aeruginosa. The nt and amino acid sequences of the region corresponding to the Arthrobacter M5 bphC gene showed a very high degree of homology with the published sequences of bphC genes from Gram-negative bacteria.

Xanthobacter autotrophicus GJ10 is a bacterium that can degrade short-chain halogenated aliphatic compounds such as 1,2-dichloroethane. A 200-kb plasmid, pXAU1, was isolated from this strain and shown to contain the dhlA gene, which codes for haloalkane dehalogenase, the first enzyme in the degradation pathway of 1,2-dichloroethane by GJ10. Loss of pXAU1 resulted in loss of haloalkane dehalogenase activity, significantly decreased chloroacetaldehyde dehydrogenase activity, and loss of resistance to mercuric chloride but did not affect the activity level of haloalkanoate dehalogenase, the second dehalogenase in the degradation of 1,2-dichloroethane.

A Pseudomonas cepacia, designated strain BRI6001, was isolated from peat by enrichment culture using 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. BRI6001 grew at up to 13 mM 2,4-D, and degraded 1 mM 2,4-D at an average starting population density as low as 1.5 cells/ml. Degradation was optimal at acidic pH, but could also be inhibited at low pH, associated with chloride release from the substrate, and the limited buffering capacity of the growth medium. The only metabolite detected during growth on 2,4-D was 2,4-dichlorophenol (2,4-DCP), and degradation of the aromatic nucleus was by intradiol cleavage. Growth lag times prior to the on-set of degradation, and the total time required for degradation, were linearly related to the starting population density and the initial 2,4-D concentration. BRI6001, grown on 2,4-D, oxidized a variety of structurally similar chlorinated aromatic compounds accompanied by stoichiometric chloride release.