Engineering a bioluminescent bioreporter from an environmentally sourced mercury-resistant Enterobacter cloacae strain for the detection of bioavailable mercury.

AIM: Escherichia coli is the conventional choice as the host strain for whole-cell bioreporter construction due to its well-understood genetics and well-established cloning protocols. However, for real-world environmental biosensing applications, it is often beneficial to use a bacterial strain derived directly from the environment under study to better ensure chemical target specificity and optimal response time. The aim of this study was to develop a whole-cell bioreporter for detection of bioavailable mercury by replacing E. coli with a wild-type bacterial host derived from a soil environment. MATERIALS AND RESULTS: In this study, an Enterobacter cloacae strain isolated from soil derived from a municipal and electronic waste dumping site was engineered to serve as a bioluminescent bioreporter for mercury toxicity by linking its merR-like gene and promoter sequence to a reorganized luxABCDE gene cassette from Photorhabdus luminescens. This bioreporter, designated as E. cloacae DWH4lux , detected mercury (HgCl2 ) at a minimum concentration of 0·2 µg l-1 with a linear response profile being maintained between a range of 0·4-1600 µg l-1 (R2  = 0·9604) with a peak bioluminescent response occurring within 1 h after exposure. No significant synergistic or antagonistic influences were observed on the bioluminescent response by other contaminating metal elements. Enterobacter cloacae DWH4lux was also demonstrated to detect mercury effectively in artificially contaminated water sample with linear correlation (R2  = 0·9623). CONCLUSIONS: The results indicated that E. cloacae DWH4lux could detect mercury in quantities below the US Environmental Protection Agency's permitted limit values (2 µg l-1 ). Hence, it is concluded that E. cloacae DWH4lux has the potential to serve as an effective whole-cell bioreporter for the environmental monitoring of mercury contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new insight into the recruitment of mercury-tolerant bacterial hosts derived from environmental samples over the conventional lab-based E. coli host for the construction of mercury bioreporters. With improved response time and selectivity, the environmentally sourced bacteria can serve as an alternative host choice to improve biosensing technology in the near future.

Response of the bioluminescent bioreporter Pseudomonas fluorescens HK44 to analogs of naphthalene and salicylic acid.

Pseudomonasfluorescens HK44 is a lux-based bioluminescent bioreporter capable of selective luminescence in the presence of naphthalene and/or salicylic acid intermediate of its metabolism. We attempted to induce bioluminescence (BL) in this strain with 72 compounds, viz. substituted naphthalenes, naphthalene-like compounds (e.g., quinoline), substituted salicylic acids, salicylic acid-like compounds (e.g., 2-anthranilic acid), oligocyclic aromates, and intermediates of naphthalene metabolism to better discriminate response specificity. From them, 42 induced BL significantly lower as compared to naphthalene, three (viz. isoquinoline, o-cresol, and salicylamide) induced BL significantly greater than naphthalene, and 27 yielded no bioluminescent response whatsoever. Strain HK44 is therefore not prone to extensive false-positive signaling and can serve as a fairly specific indicator organism for naphthalene bioavailability. At elevated concentrations, 41 compounds inhibited BL. Thus, the inclusion of constitutive bioreporter controls as indicators of sample toxicity is vital to successful biosensing application.

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents.

AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.

Bioluminescent bioreporter integrated-circuit sensing of microbial volatile organic compounds.

A bioluminescent bioreporter for the detection of the microbial volatile organic compound p-cymene was constructed as a model sensor for the detection of metabolic by-products indicative of microbial growth. The bioreporter, designated Pseudomonas putida UT93, contains a Vibrio fischeri luxCDABE gene fused to a p-cymene/p-cumate-inducible promoter derived from the P. putida F1 cym operon. Exposure of strain UT93 to 0.02-850 ppm p-cymene produced self-generated bioluminescence in less than 1.5 h. Signals in response to specific volatile organic compounds (VOCs) such as m- and p-xylene and styrene, also occurred, but at two-fold lower bioluminescent levels. The bioreporter was interfaced with an integrated-circuit microluminometer to create a miniaturized hybrid sensor for remote monitoring of p-cymene signatures. This bioluminescent bioreporter integrated-circuit device was capable of detecting fungal presence within approximately 3.5 h of initial exposure to a culture of p-cymene-producing Penicillium roqueforti.

A luxCDABE-based bioluminescent bioreporter for the detection of phenol.

A bioluminescent reporter strain, Acinetobacter sp. DF4-8, was constructed for the detection of phenol by inserting a mopR-like promoter upstream of the Vibrio fischeri bioluminescent luxCDABE gene cassette in a modified mini-Tn5 construct. When introduced into the chromosome of Acinetobacter sp. DF4, the bioreporter produced a sensitive bioluminescent response to phenol at concentrations ranging from 2.5 to 100 ppm. This response was linear (R(2)=0.986) in the range from 20 to 90 ppm. A significant bioluminescent response was also recorded when strain DF4-8 was incubated with slurries from aged, phenol-contaminated soil.

Metabolism of DHEA by cytochromes P450 in rat and human liver microsomal fractions.

Administration of dehydroepiandrosterone (DHEA) to rodents produces many unique biological responses, some of which may be due to metabolism of DHEA to more biologically active products. In the current study, DHEA metabolism was studied using human and rat liver microsomal fractions. In both species, DHEA was extensively metabolized to multiple products; formation of these products was potently inhibited in both species by miconazole, demonstrating a principal role for cytochrome P450. In the rat, use of P450 form-selective inhibitors suggested the participation of P4501A and 3A forms in DHEA metabolism. Human liver samples displayed interindividual differences in that one of five subjects metabolized DHEA to a much greater extent than the others. This difference correlated with the level of P4503A activity present in the human liver samples. For one subject, troleandomycin inhibited hepatic microsomal metabolism of DHEA by 78%, compared to 81% inhibition by miconazole, suggesting the importance of P4503A in these reactions. Form-selective inhibitors of P4502D6 and P4502E1 had a modest inhibitory effect, suggesting that these forms may also contribute to metabolism of DHEA in humans. Metabolites identified by LC-MS in both species included 16alpha-hydroxy-DHEA, 7alpha-hydroxy-DHEA, and 7-oxo-DHEA. While 16alpha-hydroxy-DHEA appeared to be the major metabolite produced in rat, the major metabolite produced in humans was a mono-hydroxylated DHEA species, whose position of hydroxylation is unknown.

Molecular regulation of genes encoding xenobiotic-metabolizing enzymes: mechanisms involving endogenous factors.

It is widely recognized that xenobiotic-metabolizing enzymes play a fundamental role in the basic processes of carcinogenesis and toxicity on one hand, and chemoprevention and drug efficacy on the other. Realization that different factors can profoundly affect the expression of these enzymes at the genome level has resulted in an enhanced appreciation of the importance these genes play in our modern industrialized age. There continues to be rapid proliferation of studies addressing the molecular regulation of these genes. The discovery of common signal transduction pathways and transcription factors that dictate tissue and developmental-specific expression, as well as variation in expression within a given tissue, suggest that there may be significant interaction among these various regulatory systems. This report is a summary of a symposium that was part of the Structure, Function and Regulation of Cytochromes P450 and Xenobiotic Metabolizing Enzymes satellite meeting of the 2000 joint meeting of the American Society for Biochemistry and Molecular Biology, the American Society for Pharmacology and Experimental Therapeutics, the French Pharmacological Society, and the Pharmacological Society of Canada held in Boston, Massachusetts. This symposium brought together several speakers who addressed specific receptor-mediated signal transduction pathways involved in the regulation of xenobiotic-metabolizing enzymes, as well as other molecular mechanisms whereby endogenous factors are involved in controlling tissue- and developmental-specific expression.

An integrated CMOS microluminometer for low-level luminescence sensing in the bioluminescent bioreporter integrated circuit.

We report an integrated CMOS microluminometer for the detection of low-level bioluminescence in whole cell biosensing applications. This microluminometer is the microelectronic portion of the bioluminescent bioreporter integrated circuit (BBIC). This device uses the n-well/p-substrate junction of a standard bulk CMOS IC process to form the integrated photodetector. This photodetector uses a distributed electrode configuration that minimizes detector noise. Signal processing is accomplished with a current-to-frequency converter circuit that forms the causal portion of the matched filter for dc luminescence in wide-band white noise. Measurements show that luminescence can be detected from as few as 4 x 10(5) cells/ml.

Bioluminescent most-probable-number monitoring of a genetically engineered bacterium during a long-term contained field release.

Pseudomonas fluorescens HK44 is a lux-based bioluminescent bioreporter capable of emitting light upon exposure to naphthalene, salicylate, and other substituted analogs. The bacterium was inoculated into intermediate-scale field lysimeters and population dynamics were monitored with time. Two methods were used to enumerate cell numbers in soil: a standard selective plating technique with colony hybridization verification and a modified lux-based most-probable-number (lux-MPN) assay based on the detection of bioluminescence. The lux-MPN assay was developed and evaluated as a possible supplement or replacement for the labor-intensive and time-consuming selective plating assay. Comparisons between selective plate counts and lux-MPN population estimates showed similar trends over the 2-year study, except that lux-MPN estimates were consistently less than selective plate counts. Verification of P. fluorescens HK44 genotype through colony hybridization techniques revealed that selective plating was actually overestimating HK44 populations and that lux-MPN values were more closely approximating true HK44 cell densities, except within the first few weeks after inoculation, when lux-MPN estimates underrepresented population densities. Thus, utilizing bioluminescence as a population monitoring tool for lux-based microorganisms was shown to be more effective and precise than standard selective plating techniques, and provided an accurate ecological analysis of P. fluorescens HK44 population dynamics over an extended period.

Field applications of genetically engineered microorganisms for bioremediation processes.

Genetically engineered microorganisms (GEMs) have shown potential for bioremediation applications in soil, groundwater, and activated sludge environments, exhibiting enhanced degradative capabilities encompassing a wide range of chemical contaminants. However, the vast majority of studies pertaining to genetically engineered microbial bioremediation are supported by laboratory-based experimental data. In general, relatively few examples of GEM applications in environmental ecosystems exist. Unfortunately, the only manner in which to fully address the competence of GEMs in bioremediation efforts is through long-term field release studies. It is therefore essential that field studies be performed to acquire the requisite information for determining the overall effectiveness and risks associated with GEM introduction into natural ecosystems.

Methionine S-oxidation in human and rabbit liver microsomes: evidence for a high-affinity methionine S-oxidase activity that is distinct from flavin-containing monooxygenase 3.

Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3, which has a Km value for methionine S-oxidation of approximately 4 mM, and exhibits high selectivity for formation of the d-diastereoisomer of methionine sulfoxide. The current studies provide evidence for an additional methionine S-oxidase activity in liver microsomes. Human and rabbit liver microsomes exhibited a biphasic response to methionine at concentrations ranging from 0.05 to 10 mM, as indicated by both Eadie-Hofstee plots and nonlinear regression. The low-affinity component of the biphasic response had Km values of approximately 3 and 5 mM for humans and rabbits, respectively, as well as high diastereoselectivity for methionine sulfoxide formation. The low-affinity activity in rabbit liver microsomes was inhibited by methimazole, S-allyl-l-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had Km values of approximately 0.07 and 0.04 mM for humans and rabbits, respectively, as well as lower diastereoselectivity for methionine sulfoxide formation. Further characterization of the high-affinity activity in rabbit liver microsomes indicated lack of involvement of cytochrome P450s or reactive oxygen species. The high-affinity activity was inhibited 25% by potassium cyanide and greater than 50% by methimazole and S-allyl-l-cysteine. Mild heat treatment produced 85% inhibition of the low-affinity activity, but only 30% inhibition of the high-affinity activity. Both high- and low-affinity activities were decreased by 85% in flavin-depleted microsomes. Because these results suggested the additional S-oxidase activity has characteristics of an FMO, recombinant human FMO4 was evaluated as a potential catalyst of this activity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl-l-cysteine, with similar diastereoselectivity to the high-affinity microsomal S-oxidase; however, the Km values for both reactions appeared to be greater than 10 mM. In summary, these studies provide evidence for two microsomal methionine S-oxidase activities. FMO3 is the predominant catalyst at millimolar concentrations of methionine. However, at micromolar methionine concentrations, there is an additional S-oxidase activity that is distinct from FMO3.

Species and sex differences in expression of flavin-containing monooxygenase form 3 in liver and kidney microsomes.

Flavin-containing monooxygenase (FMO) 3 is the predominant FMO isoform in adult human liver; however, little is known about its expression in common laboratory species. Studies have shown FMO3 levels to be sex-dependent in mouse liver, but not in human liver. The current study was undertaken to determine the expression of FMO3 in liver and kidney microsomes from multiple species, and to determine whether the sex dependence seen in mouse liver extends to other species and/or tissues. FMO3 had previously been shown to be the major FMO involved in methionine S-oxidation in rat and rabbit liver microsomes. In this study, species differences in FMO3 levels were assessed in liver microsomes from humans, rats, dogs, mice, and rabbits, and in kidney microsomes from rats, dogs, mice, and rabbits, by comparing methionine S-oxidase activities. Species differences were noted in male liver microsomes, with rabbits having 3-fold higher methionine S-oxidase activity than mice and dogs and 1.5-fold higher activity than humans and rats. Species differences were also noted in male and female kidney microsomes, with rats exhibiting 2- to 6-fold higher methionine S-oxidase activity than the other species. Sex differences in FMO3 levels were assessed using methionine S-oxidase activity and immunoblotting, and were noted only in liver microsomes from mice and dogs, with females having higher levels than males. Results also show that FMO3 orthologs from multiple species are catalytically similar with regard to methionine, S-allyl-L-cysteine, and S-(1,2-dichlorovinyl)-L-cysteine S-oxidations.

Oxidation of cysteine S-conjugates by rabbit liver microsomes and cDNA-expressed flavin-containing mono-oxygenases: studies with S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2,2-trichlorovinyl)-L-cysteine, S-allyl-L-cysteine, and S-benzyl-L-cysteine.

Rabbit liver microsomes catalyzed the highly stereoselective, NADPH- and time-dependent S-oxidation of S-benzyl-L-cysteine (SBC), S-allyl-L-cysteine (SAC), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) to their respective sulfoxides. Methimazole, a flavin-containing mono-oxygenase (FMO) substrate, inhibited S-oxidation of all four conjugates. The cytochrome P450 inhibitor 1-benzylimidazole did not affect SAC, SBC, or DCVC S-oxidation but inhibited the S-oxidation of TCVC. Solubilization of microsomes also inhibited TCVC activity, whereas SBC, SAC, and DCVC activities were not affected. Because these results suggested that FMOs were the major catalysts of SBC, SAC, and DCVC sulfoxidations, the four conjugates were evaluated as substrates for cDNA-expressed rabbit FMO isoforms FMO1, FMO2, FMO3, and FMO5. At equimolar concentrations (10 mM), FMO1 S-oxidized SBC and SAC, but no sulfoxides were detected with DCVC or TCVC. FMO3 S-oxidized all four conjugates. Km values determined with FMO3 were comparable to Km values from rabbit liver microsomes. S-Oxidation by FMO2 was detected only with SAC, and no sulfoxides were detected in incubations with FMO5. These results show that FMO isoforms can catalyze cysteine conjugate S-oxidation and that the specific isoform involved depends on the structure of the cysteine conjugate. The cysteine conjugates with more nucleophilic sulfur atoms, SAC and SBC, were much better FMO substrates than those having the less nucleophilic sulfur atoms DCVC and TCVC. The sulfoxides of TCVC and DCVC were reactive toward GSH, whereas the sulfoxides of SBC and SAC were not reactive. These results provide evidence for different chemical reactivities of these sulfoxides.

Characterization of the methionine S-oxidase activity of rat liver and kidney microsomes: immunochemical and kinetic evidence for FMO3 being the major catalyst.

Methionine is oxidized to methionine sulfoxide by rat liver and kidney microsomes in an O2- and NADPH-dependent manner. In all microsomal assays, no methionine sulfone was detected. Use of a monoclonal antibody to rat liver cytochrome P-450 reductase, various cytochrome P-450 and peroxidase inhibitors, antioxidants, and competitive flavin-containing monooxygenase (FMO) substrates suggested that methionine sulfoxidation was exclusively mediated by FMOs. At 5 mM methionine, the d-isomer of methionine sulfoxide was preferentially detected over the l-isomer in both liver (ratio, 5:1) and kidney microsomes (ratio, 12:1); however, at 30 to 40 mM methionine concentrations, the diastereomeric ratio was reduced to approximately 3:1 in both tissues. The Vmax/K(m) ratios determined for the liver and kidney microsomes were similar. Because cDNA-expressed rabbit FMO3 and FMO1 were previously shown to preferentially catalyze methionine and S-benzyl-L-cysteine (SBC) sulfoxidations, respectively, these substrates were used to isolate two distinct S-oxidase activities from the same rat liver microsomal preparation. The purified activities have apparent molecular weights of approximately 55 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings that the methionine S-oxidase reacted intensely with antibodies raised against rabbit FMO3 and the SBC S-oxidase reacted intensely with antibodies raised against rabbit FMO1 provide evidence for these activities being associated with FMO3 and FMO1, respectively. The apparent methionine K(m) determined with the purified methionine S-oxidase was 3.4 mM, whereas the apparent methionine K(m) determined with the purified SBC S-oxidase was 48 mM. The methionine sulfoxide d:l diastereomeric ratio obtained with methionine S-oxidase was 15:1, whereas the diastereomeric ratio obtained with SBC S-oxidase was only 2:1. These results provide strong evidence for the expression of both FMO1 and FMO3 in rat liver microsomes and suggest that FMO3 is the major catalyst of methionine sulfoxidation in rat liver and kidney microsomes.

Effects of Suspended Particulates on the Frequency of Transduction among Pseudomonas aeruginosa in a Freshwater Environment.

Transduction has been shown to play a significant role in the transfer of plasmid and chromosomal DNA in aquatic ecosystems. Such ecosystems contain a multitude of environmental factors, any one of which may influence the transduction process. It was the purpose of this study to show how one of these factors, particulate matter, affects the frequency of transduction. In situ transduction rates were measured in lake water microcosms containing either high or low concentrations of particulate matter. The microcosms were incubated in a freshwater lake in central Oklahoma. Transduction frequencies were found to be enhanced as much as 100-fold in the presence of particulates. Our results suggest that aggregations of bacteriophages and bacterial cells are stimulated by the presence of these suspended particulates. This aggregation increases the probability of progeny phages and transducing particles finding and infecting new host cells. Consequently, both phage production and transduction frequencies increase in the presence of particulate matter.

Transduction of a freshwater microbial community by a new Pseudomonas aeruginosa generalized transducing phage, UT1.

A pseudolysogenic, generalized transducing bacteriophage, UT1, isolated from a natural freshwater habitat, is capable of mediating the transfer of both chromosomal and plasmid DNA between strains of Pseudomonas aeruginosa. Several chromosomal alleles from three different P. aeruginosa strains were found to transduce at frequencies from 10(-8) to 10(-10) transductants per PFU at multiplicities of infection (MOI) between 0.1 and 1. Transduction frequencies of certain alleles increased up to 1000-fold as MOIs were decreased to 0.01. UT1 is also capable of transducing plasmid DNA to indigenous populations of microorganisms in natural lake-water environments. Data obtained in this study suggest that environmentally endemic bacteriophages such as UT1 are formidable transducers of naturally occurring microbial communities. It should be possible to develop model systems to test transduction in freshwater environments using components derived exclusively from these environments.

Evidence for phage-mediated gene transfer among Pseudomonas aeruginosa strains on the phylloplane.

As the use of genetically engineered microorganisms for agricultural tasks becomes more frequent, the ability of bacteria to exchange genetic material in the agricultural setting must be assessed. Transduction (bacterial virus-mediated horizontal gene transfer) is a potentially important mechanism of gene transfer in natural environments. This study investigated the potential of plant leaves to act as surfaces on which transduction can take place among microorganisms. Pseudomonas aeruginosa and its generalized transducing bacteriophage F116 were used as a model system. The application of P. aeruginosa lysogens of F116 to plant leaves resulted in genetic exchange among donor and recipient organisms resident on the same plant. Transduction was also observed when these bacterial strains were inoculated onto adjacent plants and contact was made possible through high-density planting.

Imipenem resistance in pseudomonas aeruginosa PAO: mapping of the OprD2 gene.

Carbapenem antibiotics have been shown to penetrate the outer membrane of Pseudomonas aeruginosa through a unique porin protein, OprD2. We mapped the OprD2 gene by conjugation using plasmid FP2 and by transduction using phage F116L. This gene maps between 71 and 75 min on the PAO1 chromosome.