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G S Sayler

Publications and source records attributed to G S Sayler.

At least 19 recordsLinked to original sources
PubMed2002

Lipid analysis of the response of a sedimentary microbial community to polycyclic aromatic hydrocarbons.

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

Biomass↗
PubMed2002

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.

Acinetobacter↗
PubMed2001

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.

Biosensing Techniques↗
PubMed2001

Quantification of toluene dioxygenase induction and kinetic modeling of TCE cometabolism by Pseudomonas putida TVA8.

As measured by the toluene-induced bioluminescent response of Pseudomonas putida TVA8 in batch experiments, toluene dioxygenase (Tod) enzyme activities are dependent on toluene concentration between 0 and 30 mg/L. To provide a measure of the Tod activity for use in Michaelis-Menten competitive-inhibition kinetics, a correlation between toluene concentration and induced Tod activity as measured by an induced bioluminescent response of P. putida TVA8 is presented as a nondimensional Tod activity parameter. A packed-bed, radial-flow bioreactor (RFB) using the bioreporter P. putida TVA8A serves as the model system for studying the effect of the enzyme activity parameter on model predictions of vapor-phase toluene oxidation and trichloroethylene (TCE) cometabolism. Mass balances were performed on a differential section of the RFB to describe the radial transport of vapor-phase toluene and TCE through a bulk gas phase and the concomitant biological reaction in a stationary biofilm phase. The finite-element Galerkin weak-statement formulation with first-order basis functions was used to find the optimum solution to the highly nonlinear, coupled equations. For this RFB system with toluene concentrations less than 1 mg/L in the bulk gas phase, the Tod activity parameter enables accurate predictions of steady-state TCE degradation rate (0.27 microg TCE/min).

Kinetics↗
PubMed2001

Observations on the preferential biodegradation of selected components of polyaromatic hydrocarbon mixtures.

The capacity of the naphthalene degrading enzyme (NAH) system of Pseudomonas fluorescens 5R and a number of other NAH system bacterial isolates to degrade mixtures of polyaromatic hydrocarbons (PAHs) and heterocyclic compounds were examined. It was found that all the examined organisms displayed similar patterns of preferential compound degradation when presented with the same mixture. Using strains that possess portions of the NAH system, this preferential degradation was localized to the activity of naphthalene dioxygenase. Comparisons of the first-order rates of compound degradation with the structures of the mixture components indicated that increased deviation from the base structure of naphthalene led to slower disappearance. Structural features that were found to decrease the rate of compound degradation include an increase in the number of methyl substituents and an increase in the size of a substituent.

Biodegradation, Environmental↗
PubMed2001

Whole-cell biocomputing.

The ability to manipulate systems on the molecular scale naturally leads to speculation about the rational design of molecular-scale machines. Cells might be the ultimate molecular-scale machines and our ability to engineer them is relatively advanced when compared with our ability to control the synthesis and direct the assembly of man-made materials. Indeed, engineered whole cells deployed in biosensors can be considered one of the practical successes of molecular-scale devices. However, these devices explore only a small portion of cellular functionality. Individual cells or self-organized groups of cells perform extremely complex functions that include sensing, communication, navigation, cooperation and even fabrication of synthetic nanoscopic materials. In natural systems, these capabilities are controlled by complex genetic regulatory circuits, which are only partially understood and not readily accessible for use in engineered systems. Here, we focus on efforts to mimic the functionality of man-made information-processing systems within whole cells.

Computational Biology↗
PubMed2001

Gene expression monitoring in soils by mRNA analysis and gene lux fusions.

Two methods recently developed to monitor the gene expression of microbial communities in soil are the extraction and detection of messenger RNA from soil microorganisms and the construction and use of lux-based bioreporter strains. The goal of these approaches is to assess microbial activity in natural and impacted soil environments.

Artificial Gene Fusion↗
PubMed2001

Screening for ribosomal-based false positives following prokaryotic mRNA differential display.

Differential display (DD) and the closely related RNA arbitrarily primed PCR (RAP-PCR) have become the molecular tools of choice for identifying and isolating differentially expressed genes in both eukaryotic and prokaryotic systems. However, one of the current drawbacks of both techniques is the high number of false positives generated. In prokaryotic applications, the many false positive typically generated by DD are subsequently identified as rRNAs because of their greater abundance compared to mRNAs. To circumvent this problem, full-length 16S and 23S rDNA probes, derived from Pseudomonas putida G7 and Pseudomonas aeruginosa FRD1, respectively, were used as a prescreening approach to discriminate between those bands, which appear to be differentially expressed mRNAs, but in fact are rRNAs, following prokaryotic mRNA DD.

DNA Probes↗
PubMed2000

Kinetic analysis of a tod-lux bacterial reporter for toluene degradation and trichloroethylene cometabolism.

Kinetics of toluene and trichloroethylene (TCE) degradation and bioluminescence from the bioreporter Pseudomonas putida B2 and TVA8 were investigated utilizing batch and continuous culture, respectively. Degradation was modeled using a Michaelis-Menten expression for the competition of two substrates for a single enzyme system, and bioluminescence was modeled assuming a luciferase enzyme saturational dependence on toluene as the inducer and growth substrate. During the batch experiments, bioluminescence increased at approximately 90 namp/min for initial toluene concentrations of 10 to 50 mg/L, but more slowly at higher toluene concentrations, suggesting maximum promoter induction at below 10 mg/L and toxic effects above 50 mg/L toluene. TCE degradation did not occur until toluene depletion, presumably due to competition between toluene and TCE for the toluene dioxygenase enzyme. During continuous culture, bioluminescence transiently increased, then gradually decreased in response to increasing step changes in toluene feed concentration. Bioluminescence in the CSTR appeared to be limited by growth substrate and/or inducer.

Biodegradation, Environmental↗
PubMed2000

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.

Colony Count, Microbial↗
PubMed2000

The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1.

Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r(2)=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19+/-1.5x10(-4) mg indigo (mg protein)(-1) min(-1) was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0+/-0.2x10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions.

Biofilms↗
PubMed2000

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.

Biodegradation, Environmental↗
PubMed2000

A bioluminescent whole-cell reporter for detection of 2, 4-dichlorophenoxyacetic acid and 2,4-dichlorophenol in soil.

A bioreporter was made containing a tfdRP(DII)-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2, 4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 microM to 5.0 mM. This response was linear (R(2) = 0.9825) in the range of 2.0 microM to 1.1 x 10(2) microM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 x 10(2) microM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2000

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis.

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.

Cloning, Molecular↗
PubMed1999

Validation of genetically engineered bioluminescent surfactant resistant bacteria as toxicity assessment tools.

Bacteria are useful organisms for measuring acute and chronic toxicity. The most popular toxicity tests utilize the inhibition of bioluminescence as an indication of toxicity. An extensive toxicity database on pure chemical compounds has been created using the bioluminescent microorganism, Vibrio fischeri. However, the use of the Microtox assay in applications for environmental samples is not always successful, due to the test organism. Because the genes for bioluminescence have been cloned from V. fischeri, environmentally relevant test strains can be readily constructed. In this study, surfactant-resistant bioluminescent bacterial strains were constructed by transferring a broad host range plasmid containing the bioluminescent genes under the regulation of a constitutive promoter into strains from several bacterial genera. Two test strains, Stenotrophomonas 3664 and Alcaligenes eutrophus 2050, were approximately 400 times more resistant to the nonionic surfactant polyoxyethylene 10 lauryl ether than V. fischeri and are useful for toxicity reduction evaluations of remediation processes which use surfactants for solubilization of hydrophobic pollutants. The use of these strains as alternative test organisms in the Microtox assay was evaluated using nonpolar narcosis as the baseline toxicity mechanisms. The two test strains and V. fischeri indicated linear fits of EC50 values with the octanol/water partition (Kow) for five nonpolar narcotic compounds in acute assays (r2>0.9) with a slope of approximately 1. For all three strains, the y-intercept values were approximately the same, indicating that sensitivity did not vary. These results indicate that the nonpolar narcosis baseline toxicity mechanism may be useful as a general tool to validate the functioning of genetically engineered bioluminescent microorganisms.

Alcaligenes↗
PubMed1999

Containment of a genetically engineered microorganism during a field bioremediation application.

A field release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation. Six large lysimeters were filled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene. A genetically engineered bacterial strain, Pseudomonas fluorescens HK44, was sprayed onto the surface of the soil during soil loading. This strain contains a fusion between the lux genes of Vibrio fischeri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene. Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers. Although approximately 10(14) bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low.

Air Microbiology↗