Factors Influencing Expression of luxCDABE and nah Genes in Pseudomonas putida RB1353(NAH7, pUTK9) in Dynamic Systems

Bioluminescent reporter organisms have been successfully exploited as analytical tools for in situ determination of bioavailable levels of contaminants in static environmental samples. Continued characterization and development of such reporter systems is needed to extend the application of these bioreporters to in situ monitoring of degradation in dynamic environmental systems. In this study, the naphthalene-degrading, lux bioreporter bacterium Pseudomonas putida RB1353 was used to evaluate the relative influences of cell growth stage, cell density, substrate concentration, oxygen tension, and background carbon substrates on both the magnitude of the light response and the rate of salicylate disappearance. The effect of these variables on the lag time required to obtain maximum luminescence and degradation was also monitored. Strong correlations were observed between the first three factors and both the magnitude and induction time of luminescence and degradation rate. The maximum luminescence response to nonspecific background carbon substrates (soil extract broth or Luria broth) was 50% lower than that generated in response to 1 mg of sodium salicylate liter⁻¹. Oxygen tension was evaluated over the range of 0.5 to 40 mg liter⁻¹, with parallel inhibition to luminescence and degradation rate (20 mg of sodium salicylate liter⁻¹) observed at 1.5 mg liter⁻¹ and below and no effect observed above 5 mg liter⁻¹. Oxygen tensions from 2 to 4 mg liter⁻¹ influenced the magnitude of luminescence but not the salicylate degradation rate. The results suggest that factors causing parallel shifts in the magnitude of both luminescence and degradation rate were influencing regulation of the nah operon promoters. For factors that cause nonparallel shifts, other regulatory mechanisms are explored. This study demonstrates that lux reporter bacteria can be used to monitor both substrate concentration and metabolic response in dynamic systems. However, each lux reporter system and application will require characterization and calibration.     

Effect of temperature,pH, and initial cell number on luxCDABE and nah gene expression during naphthalene and salicylate catabolism in the bioreporter organism Pseudomonas putida RB1353

One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses. For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory. Therefore, this study was conducted to determine what additional factors influence the luminescent response. Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P. putida RB1353 and the maximum degradation rate (V(max)) during salicylate and naphthalene catabolism. Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V(max) during salicylate and naphthalene catabolism, respectively. Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V(max) during salicylate and naphthalene catabolism. Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude. The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1 degrees C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude. Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products. For example, even though lux gene expression was occurring at approximately 35 degrees C, the luciferase enzyme was inactive. Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.     

Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353

One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses. For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory. Therefore, this study was conducted to determine what additional factors influence the luminescent response. Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P. putida RB1353 and the maximum degradation rate (V_max) during salicylate and naphthalene catabolism. Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V_max during salicylate and naphthalene catabolism, respectively. Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V_max during salicylate and naphthalene catabolism. Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude. The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1°C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude. Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products. For example, even though lux gene expression was occurring at ~35°C, the luciferase enzyme was inactive. Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.     

Kinetics of methyl t-butyl ether cometabolism at low concentrations by pure cultures of butane-degrading bacteria

Butane-oxidizing Arthrobacter (ATCC 27778) bacteria were shown to degrade low concentrations of methyl t-butyl ether (MTBE; range, 100 to 800 microg/liter) with an apparent half-saturation concentration (K(s)) of 2.14 mg/liter and a maximum substrate utilization rate (k(c)) of 0.43 mg/mg of total suspended solids per day. Arthrobacter bacteria demonstrated MTBE degradation activity when grown on butane but not when grown on glucose, butanol, or tryptose phosphate broth. The presence of butane, tert-butyl alcohol, or acetylene had a negative impact on the MTBE degradation rate. Neither Methylosinus trichosporium OB3b nor Streptomyces griseus was able to cometabolize MTBE.     

Salicylate biodegradation by various algal-bacterial consortia under photosynthetic oxygenation

Four green microalgae (Chlorella sorokiniana, Chlorella vulgaris, Scenedesmus obliquus and Selenastrum capricornutum), a wild Bolivian microalga strain and two cyanobacteria (Anabaena catenula and Microcystis aeruginosa) were compared for tolerance to salicylate, O2 production capacity and ability to support salicylate degradation by a Ralstonia basilensis strain in symbiotic microcosms with the microalgae. Microcystis aeruginosa had the highest tolerance to salicylate at 500 mg l(-1) and 1500 mg l(-1) but only produced 0.7 mg O2 l(-1) h(-1) in the absence of pollutant. Chlorella sorokiniana resisted salicylate at 1500 mg l(-1) with the highest O2 production in the absence of salicylate (26 mg l(-1) h(-1)) closely followed by the Bolivian microalga (23 mg l(-1) h(-1)) and Chlorella vulgaris (21 mg l(-1) h(-1)). Selenastrum capricornutum and Anabaena catenula were completely inhibited by salicylate at 500 mg l(-1). When inoculated with Ralstonia sp. and supplied with salicylate, Chlorella sorokiniana had the highest removal rate (19 mg l(-1) h(-1)), followed by the wild Bolivian strain (18 mg l(-1) h(-1)) and Chlorella vulgaris (14 mg l(-1) h(-1)).     

Chromosomally located gene fusions constructed in Acinetobacter sp. ADP1 for the detection of salicylate

Acinetobacter sp. ADP1 is a common soil-associated bacterium with high natural competency, allowing it to efficiently integrate foreign DNA fragments into its chromosome. This property was exploited to engineer salicylate-inducible luxCDABE and green fluorescent protein (GFP) variants of Acinetobacter sp. ADP1. Specifically, Acinetobacter sp. ADPWH_lux displayed the higher sensitivity when comparing the two variants (minimum detection c. 0.5-1 microM salicylate) and a faster turnover of the lux marker gene, making it suitable for whole-cell luminescence assays of salicylate concentration. In contrast, the longer maturation and turnover times of the GFP protein make the Acinetobacter sp. ADPWH_gfp variant more suited to applications involving whole-cell imaging of the presence of salicylate. The sensitivity of the luxCDABE variant was demonstrated by assaying salicylate production in naphthalene-degrading cultures. Assays using ADPWH_lux specifically mapped the kinetics of salicylate production from naphthalene and were similar to that observed by high-performance liquid chromatography (HPLC) data. However, ADPWH_lux exhibited the higher sensitivity, when compared with HPLC, for detecting salicylate production during the first 24 h of naphthalene metabolism. These data demonstrate that the engineered Acinetobacter variants have significant potential for salicylate detection strategies in laboratory and field studies, especially in scenarios where genetic stability of the construct is required for in situ monitoring.     

Development of bespoke bioluminescent reporters with the potential for in situ deployment within a phenolic-remediating wastewater treatment system

A suite of ecologically relevant, site-specific bioreporters was constructed by transposon mutagenesis of microorganisms isolated from a polluted phenolic-remediating wastewater treatment system. Four Pseudomonad species were engineered to carry a stable chromosomal copy of the lux operon (luxCDABE) derived from Photorhabdus luminescens. These recombinant reporter microorganisms were tested for bioluminescence response to relevant phenol concentrations in the laboratory and to phenolic-containing effluents generated by an industrial wastewater treatment plant. The reporters displayed proportional responses of bioluminescence decay with increasing phenol concentrations up to 800 mg l(-1) of phenol. When deployed against samples from the treatment system, they showed superior operational range and sensing capabilities to that observed for industry standard microorganisms such as Vibrio fischeri. Specifically, the engineered strains accurately predicted toxicity shifts in all the treatment compartments under study (with phenolic concentrations ranging from approximately 10 to 600 mg l(-1)) with a low coefficient of variation of replicate determinations (between 1.16% and 8.32%). This work highlights the utility of genetic modification of native microorganisms from sites of interest to provide robust and ecologically relevant organism-based reagents for toxicity monitoring with the potential for in situ deployment.     

Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans

This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n-decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.     

Mass transfer studies using cloned-luminous strain of Xanthomonas campestris

Measurements of mass transfer in a highly viscous pseudoplastic broth, which is typical to Xanthomonas campestris fermentations, are difficult to obtain by conventional methods and little data is available. A novel research method that uses bioluminescence for mass transfer studies has been developed. A plasmid carrying the luminescence operon of marine luminous bacteria is introduced into an industrial bacteria, X. campestris. Besides producing the polysaccharide xanthangum, the bioluminescent X. campestris emits measurable light. Monitoring the luminescence is a simple, noncontaminating nondestructive and very sensitive indicator of the metabolic activity of the culture during fermentation. Energy drain due to bioluminescence is very low; growth rate and polysaccharide production rate are close to those of the wild-type strain.Oxygen and substrate mass transfer are determined by inducing step or periodic fluctuations in their concentration and measuring the resultant luminescence response. Oxygen mass transfer coefficients show linear dependence on Reynolds number and an exponential dependence on the average shear rate. Viscosity effect is small at high viscosities but increases rapidly below 10 Pa-s. The influence of oxygen uptake rate is studied.Mass transfer of the limiting component (ammonium ions) is analyzed under pulsating feed conditions. The luminescence declines, following a feed pulse, due to energy investment in active transport of ammonium ions through the cell membrane, it regenerates then to its baseline. The relation between mass transfer and luminescence fluctuation is elucidated.     

Persistence and metabolic activity of Escherichia coli O157:H7 in farm animal faeces

Ruminants, and to a lesser extent monogastric farm animals, are known to be natural reservoirs of Escherichia coli O157:H7, and contact with contaminated faeces has been linked to human infection. This study used a nontoxigenic, chromosomally marked, lux reporter strain to compare the persistence and activity (bioluminescence) of E. coli O157:H7 over 21 days in the faecal liquor of five farm animals: horse, sheep, cow, pig and piglet. Samples were inoculated with the lux E. coli O157:H7 (7.82 log CFU mL(-1)) and stored at 20 +/- 1 degrees C. The organism was recovered from all samples throughout the experimental period, although lower numbers were recovered from horse faecal liquor relative to all other types (P<0.001). The organisms' activity declined in all samples over time and no luminescence could be detected in any sample 21 days postinoculation. However, activity did increase greatly within pig and piglet faeces during initial stages of monitoring and overall luminescence was greater in piglet samples compared with all other samples (P<0.001). This is the first study to demonstrate how both the persistence and metabolic activity of E. coli O157:H7 notably varies within a range of ruminant and nonruminant animal faeces. Further research is needed to elucidate the factors that govern differential persistence and metabolic activity of E. coli O157:H7 within such matrices.     

Photosynthetically oxygenated salicylate biodegradation in a continuous stirred tank photobioreactor

A consortium consisting of a Chlorella sorokiniana strain and a Ralstonia basilensis strain was able to carry out sodium salicylate biodegradation in a continuous stirred tank reactor (CSTR) using exclusively photosynthetic oxygenation. Salicylate biodegradation depended on algal activity, which itself was a function of microalgal concentration, light intensity, and temperature. Biomass recirculation improved the photobioreactor performance by up to 44% but the results showed the existence of an optimal biomass concentration above which dark respiration started to occur and the process efficiency started to decline. The salicylate removal efficiency increased by a factor of 3 when illumination was increased from 50-300 microE/m2.s. In addition, the removal rate of sodium salicylate was shown to be temperature-dependent, increasing from 14 to 27 mg/l.h when the temperature was raised from 26.5 to 31.5 degrees C. Under optimized conditions (300 microE/m2.s, 30 degrees C, 1 g sodium salicylate/l in the feed and biomass recirculation) sodium salicylate was removed at a maximum constant rate of 87 mg/l.h, corresponding to an estimated oxygenation capacity of 77 mg O2/l.h (based on a BOD value of 0.88 g O2/g sodium salicylate for the tested bacterium), which is in the range of the oxygen transfer capacity of large-scale mechanical surface aerators. Thus, although higher degradation rates were attained in the control reactor, the photobioreactor is a cost-efficient process which reduces the cost of aeration and prevents volatilization problems associated with the degradation of toxic volatile organic compounds under aerobic conditions.     

Degradation of polycyclic aromatic hydrocarbon compounds under various redox conditions in soil-water systems

This study evaluated the microbial degradation of naphthol, naphthalene, and acenaphthene, under aerobic, anaerobic, and denitrification conditions in soil-water systems. Chemical degradation of naphthol and naphthalene in the presence of a manganese oxide was also studied. Naphthol, naphthalene, and acenaphthene were degraded microbially under aerobic conditions from initial aqueous-phase concentrations of 9, 7, and 1 mg/liter to nondetectable levels in 3, 10, and 10 days, respectively. Under anaerobic conditions naphthol degraded to nondetectable levels in 15 days, whereas naphthalene and acenaphthene showed no significant degradation over periods of 50 and 70 days, respectively. Under denitrification conditions naphthol, naphthalene, and acenaphthene were degraded from initial aqueous-phase concentrations of 8, 7, and 0.4 mg/liter to nondetectable levels in 16, 45, and 40 days, respectively. Acclimation periods of approximately 2 days under aerobic conditions and 2 weeks under denitrification conditions were observed for both naphthalene and acenaphthene. Abiotic degradation of naphthalen and naphthol were evaluated by reaction with manganese oxide, a minor soil constituent. In the presence of a manganese oxide, naphthalene showed no abiotic degradation over a period of 9 weeks, whereas the aqueous naphthol concentration decreased from 9 mg/liter to nondetectable levels in 9 days. The results of this study show that low-molecular-weight, unsubstituted, polycyclic aromatic hydrocarbons are amenable to microbial degradation in soil-water systems under denitrification conditions.     

Vibrio fischeri uses two quorum-sensing systems for the regulation of early and late colonization factors

Vibrio fischeri possesses two quorum-sensing systems, ain and lux, using acyl homoserine lactones as signaling molecules. We have demonstrated previously that the ain system activates luminescence gene expression at lower cell densities than those required for lux system activation and that both systems are essential for persistent colonization of the squid host, Euprymna scolopes. Here, we asked whether the relative contributions of the two systems are also important at different colonization stages. Inactivation of ain, but not lux, quorum-sensing genes delayed initiation of the symbiotic relationship. In addition, our data suggest that lux quorum sensing is not fully active in the early stages of colonization, implying that this system is not required until later in the symbiosis. The V. fischeri luxI mutant does not express detectable light levels in symbiosis yet initiates colonization as well as the wild type, suggesting that ain quorum sensing regulates colonization factors other than luminescence. We used a recently developed V. fischeri microarray to identify genes that are controlled by ain quorum sensing and could be responsible for the initiation defect. We found 30 differentially regulated genes, including the repression of a number of motility genes. Consistent with these data, ain quorum-sensing mutants displayed an altered motility behavior in vitro. Taken together, these data suggest that the sequential activation of these two quorum-sensing systems with increasing cell density allows the specific regulation of early colonization factors (e.g., motility) by ain quorum sensing, whereas late colonization factors (e.g., luminescence) are preferentially regulated by lux quorum sensing.     

Degradation of polycyclic aromatic hydrocarbon compounds under various redox conditions in soil-water systems.

This study evaluated the microbial degradation of naphthol, naphthalene, and acenaphthene, under aerobic, anaerobic, and denitrification conditions in soil-water systems. Chemical degradation of naphthol and naphthalene in the presence of a manganese oxide was also studied. Naphthol, naphthalene, and acenaphthene were degraded microbially under aerobic conditions from initial aqueous-phase concentrations of 9, 7, and 1 mg/liter to nondetectable levels in 3, 10, and 10 days, respectively. Under anaerobic conditions naphthol degraded to nondetectable levels in 15 days, whereas naphthalene and acenaphthene showed no significant degradation over periods of 50 and 70 days, respectively. Under denitrification conditions naphthol, naphthalene, and acenaphthene were degraded from initial aqueous-phase concentrations of 8, 7, and 0.4 mg/liter to nondetectable levels in 16, 45, and 40 days, respectively. Acclimation periods of approximately 2 days under aerobic conditions and 2 weeks under denitrification conditions were observed for both naphthalene and acenaphthene. Abiotic degradation of naphthalen and naphthol were evaluated by reaction with manganese oxide, a minor soil constituent. In the presence of a manganese oxide, naphthalene showed no abiotic degradation over a period of 9 weeks, whereas the aqueous naphthol concentration decreased from 9 mg/liter to nondetectable levels in 9 days. The results of this study show that low-molecular-weight, unsubstituted, polycyclic aromatic hydrocarbons are amenable to microbial degradation in soil-water systems under denitrification conditions.