Hydrocarbon-degrading potential of microbial communities from Arctic plants.

AIMS: To explore rhizospheric microbial communities from Arctic native plant species evaluating their bacterial hydrocarbon-degrading capacities. METHODS AND RESULTS: Eriophorum scheuchzeri, Potentilla cf. rubricaulis, Oxyria digyna, Salix arctica and Puccinellia angustata plant species were collected at Eureka (Canadian high Arctic) along with their rhizospheric soil samples. Their bacterial community fingerprints (16S rRNA gene, DGGE) were distinctive encompassing members from the phyla: Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria. Isolated diesel-degrading bacteria belonged to the phyla Actinobacteria and Proteobacteria. Strains of Mycobacterium, Nocardia, Rhodococcus, Intrasporangiaceae, Leifsoni and Arthrobacter possessed alkB and Pseudomonas possessed both ndoB and xylE gene sequences. Two Rhodococcus strains mineralized [1-(14) C] hexadecane at 5 and -5°C. From the rhizosphere of P. angustata, larger numbers of hydrocarbon-degrading bacteria were isolated than from other plant rhizosphere samples and all three genes alkB (from Actinobacteria), ndoB and xylE (from Pseudomonas) were detected by PCR. CONCLUSIONS: (i) Arctic plants have unique rhizospheric bacterial communities. (ii) P. angustata has potential for phytoremediation research at high Arctic soils. (iii) Isolated bacteria mineralized hydrocarbons at ambient low temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first rhizospheric exploration examining the phytoremediation potential of five Arctic plants and evaluating their microbial hydrocarbon-degrading capacities.

Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry.

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.

Cloning and expression of the polychlorinated biphenyl-degradation gene cluster from Arthrobacter M5 and comparison to analogous genes from gram-negative bacteria.

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.

Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns.

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.

Distribution and Biogeochemical Importance of Bacterial Populations in a Thick Clay-Rich Aquitard System.

To investigate the distribution of microbial biomass, populations and activities within a clay-rich, low hydraulic conductivity (10-11 to 10-12 m s-1) aquitard complex, cores were aseptically obtained from a series of overlying clayey deposits; a fractured till, unfractured till (20-30 ka BP), a disturbed interfacial zone (20-30 ka BP), and a Cretaceous clay aquitard (71-72 Ma BP). The results of confocal microscopy studies, culture methods, molecular approaches, and extractive fatty acid analyses all indicated low bacterial numbers that were non-homogeneously distributed within the sediments. Various primers for catabolic genes were used to amplify extracted DNA. Results indicated the presence of eubacterial 23S rDNA, and the narH gene for nitrate reductase and ribulose-1,5-biphosphate carboxylase (RuBP carboxylase). Although there was no evidence of limitation by electron acceptors or donors, sulfate-reducing bacteria were not detected below the fractured till zone, using PCR, enrichment, or culture techniques. Denaturing gradient gel electrophoresis (DGGE) analyses indicated differences in community composition and abundance between the various geologic units. Results of FAME analyses of sediments yielded detectable extractable fatty acids throughout the aquitard complex. Bacterial activities were demonstrated by measuring mineralization of (14C) glucose. Porewater chemistry and stable isotope data were in keeping with an environment in which extremely slow growing, low populations of bacteria exert little impact. The observations also support the contention that in low permeability sediments bacteria may survive for geologic time periods.

Strategies for augmenting the pentachlorophenol degradation potential of UASB anaerobic granules.

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.

Methanogen community composition and rates of methane consumption in Canadian High Arctic permafrost soils.

Increasing permafrost thaw, driven by climate change, has the potential to result in organic carbon stores being mineralized into carbon dioxide (CO2) and methane (CH4) through microbial activity. This study examines the effect of increasing temperature on community structure and metabolic activity of methanogens from the Canadian High Arctic, in an attempt to predict how warming will affect microbially controlled CH4 soil flux. In situ CO2 and CH4 flux, measured in 2010 and 2011 from ice-wedge polygons, indicate that these soil formations are a net source of CO2 emissions, but a CH4 sink. Permafrost and active layer soil samples were collected at the same sites and incubated under anaerobic conditions at warmer temperatures, with and without substrate amendment. Gas flux was measured regularly and indicated an increase in CH4 flux after extended incubation. Pyrosequencing was used to examine the effects of an extended thaw cycle on methanogen diversity and the results indicate that in situ methanogen diversity, based on the relative abundance of the 16S ribosomal ribonucleic acid (rRNA) gene associated with known methanogens, is higher in the permafrost than in the active layer. Methanogen diversity was also shown to increase in both the active layer and permafrost soil after an extended thaw. This study provides evidence that although High Arctic ice-wedge polygons are currently a sink for CH4, higher arctic temperatures and anaerobic conditions, a possible result of climate change, could result in this soil becoming a source for CH4 gas flux.

Influence of nutrient inputs, hexadecane, and temporal variations on denitrification and community composition of river biofilms.

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 < or = 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.

Utilization of fluorescent microspheres and a green fluorescent protein-marked strain for assessment of microbiological contamination of permafrost and ground ice core samples from the Canadian High Arctic.

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-microm 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.

Molecular analysis and development of 16S rRNA oligonucleotide probes to characterize a diclofop-methyl-degrading biofilm consortium.

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.

Influence of environmental factors on 2,4-dichlorophenoxyacetic acid degradation by Pseudomonas cepacia isolated from peat.

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.

Assessment of the biodegradation potential of psychrotrophic microorganisms.

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 influence of moisture on microbial transport, survival and 2,4-D biodegradation with a genetically marked Burkholderia cepacia in unsaturated soil columns.

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.

Biodegradation of petroleum hydrocarbons by psychrotrophic Pseudomonas strains possessing both alkane (alk) and naphthalene (nah) catabolic pathways.

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.

Frequency of genes in aromatic and aliphatic hydrocarbon biodegradation pathways within bacterial populations from Alaskan sediments.

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.

Uptake of Benzoic Acid and Chloro-Substituted Benzoic Acids by Alcaligenes denitrificans BRI 3010 and BRI 6011.

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.

Enhanced selection of an anaerobic pentachlorophenol-degrading consortium.

A rapid enrichment approach based on a pentachlorophenol (PCP) feeding strategy which linked the PCP loading rate to methane production was applied to an upflow anaerobic sludge bed reactor inoculated with anaerobic sludge. Due to this strategy, over a 140-day experimental period the PCP volumetric load increased from 2 to 65 mg L(R)(-1) day(-1) with a near zero effluent concentration of PCP. Dechlorination dynamics featured sequential appearance of 3,4,5-chlorophenol, 3,5-chloro- phenol, and 3-chlorophenol in the reactor effluent. Profiling of the reactor population by denaturing gradient gel electrophoresis (DGGE) revealed a correlation between the appearance of dechlorination intermediates and bands on the DGGE profile. Nucleotide sequencing of newly detected 16S rDNA fragments suggested the proliferation of Clostridium and Syntrophobacter/Syntrophomonas spp. in the reactor during PCP degradation. Published by John Wiley & Sons, Inc.

Phenanthrene mineralization by Pseudomonas sp. UG14.

A phenanthrene-mineralizing Pseudomonas sp., designated UG14, was isolated from creosote-contaminated soil. It contained two plasmids, of approximately 77 kb and 76 kb, the smaller of which contained DNA sequences that hybridized with probes specific for ndoB and xylE, genes involved in catabolism of aromatic hydrocarbons. At initial phenanthrene concentrations of 10, 50, 200 and 1000 mg/l broth, 27%, 19%, 7.7% and 3.3%, respectively, of the [9-(14)C]phenanthrene was recovered as (14)CO2 after 36 days' incubation at 30°C. Most (14)C-label was converted to a water-soluble metabolite tentatively identified as 1-hydroxy-2-naphthoic acid. Rhamnolipid biosurfactants produced by P. aeruginosa UG2 enhanced mineralization of 50 mg phenanthrene/l by Pseudomonas sp. UG14. With the biosurfactant at 0, 25 and 250 mg rhamnose equivalents/l, 6.5%, 8.2% and 9.8%, respectively, of the phenanthrene was mineralized after 35 days.

Numerical analysis and the application of random amplified polymorphic DNA polymerase chain reaction to the differentiation of Vibrio strains from a seasonally cold ocean.

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.

Survival of lux-lac-marked biosurfactant-producing Pseudomonas aeruginosa UG2L in soil monitored by nonselective plating and PCR.

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.