Bruce M. Applegate

Physiological considerations of environmental applications of lux reporter fusions

The performance of the bioluminescent reporter bacterium Pseudomonas fluorescens HK44, which contains a nahGluxCDABE fusion for naphthalene catabolism and bioavailability was investigated for environmental samples and mixed contaminants. For aqueous extracts from fuel hydrocarbon-contaminated soils, a reproducible bioluminescence response was obtained, which coincided with, but overestimated the presence of naphthalene. Therefore, the strain’s bioluminescence response to mixed contaminants was investigated further. The strain showed a linear correlation between bioluminescence and the amount of JP-4 jet fuel present in an aqueous solution, representing a mixture of compounds including naphthalene. However, some non-inducing organic solvents such as toluene, p-xylene and acetone caused a significant bioluminescence increase as well. The analysis of nah-lux mRNA from cells exposed to toluene revealed that the bioluminescence response was not due to increased nahG-luxCDABE gene expression, whereas increased lux mRNA levels were found with exposure to naphthalene or JP-4 jet fuel. While different mixture combinations of solvents resulted in either additive or intermediate effects, the combination of naphthalene and solvent resulted in a synergistic effect on the bioluminescence response. The addition of n-decanal, a substrate for luciferase, showed that the cells were aldehyde-limited. If aldehyde was added, only the presence of naphthalene caused a significantly increased bioluminescence response over the control. The solvent effects were dependent on the physiological status of the reporter culture and were present in growing, but not in resting cell cultures. It was postulated that the increase in bioluminescence after exposure to solvents was due to changed fatty acid synthesis patterns affecting the aldehyde supply for the bioluminescence reaction. Exposure to toluene resulted in altered membrane fatty acid composition and release of fatty acids from the cells. Exposure to n-alkanes resulted in minor changes in the bioluminescence response, whereas, exposure to heavy metals or cyanide resulted in significant reductions in the overall bioluminescence. These results demonstrate the utility of bioluminescent reporter bacteria for environmental applications, as well as the need for adequate experimental controls in interpreting environmental sensing data.

Molecular diagnostics of polycyclic aromatic hydrocarbon biodegradation in Manufactured Gas Plant soils

Traditional methods for quantifying specific catabolic bacterial populations underestimate the true population count due to the limitations of the necessary laboratory cultivation methods. Likewise,in situ activity is also difficult to assess in the laboratory without altering the sample environment. To circumvent these problems and achieve a truein situ bacterial population count and activity measurement, new methods based on molecular biological analysis of bacterial nucleic acids were applied to soils heavily contaminated with polycyclic aromatic hydrocarbons (PAH). In addition, a naphthalene-lux reporter system was used to determine bioavailability of naphthalene within these soils. DNA extracted from seven PAH-contaminated soils and hybridized with thenahA gene probe indicated that the naphthalene degradative genes were present in all samples in the range of 0.06 to 0.95 ng/100 µl DNA extract which was calculated to represent 3.2×106 to 1.1×1010 cells/g soil (assuming one copy of these genes per cell).14C-naphthalene mineralization was observed in all contaminated soils with14CO2 mineralization rates ranging from 3.2×10−5 to 304,920.0×10−5 µg g soil−1h−1. Phenanthrene, anthracene, and benzo(a)pyrene were mineralized also in several soils. Messenger RNA transcripts ofnahA were isolated and quantified from 4 soils. Only one soil tested, soil B, was inducible with salicylate above thein situ nahA gene transcript level. Two of the soils, C and G, were already fully inducedin situ. The naphthalene mineralization rate correlated positively with the amount ofnahA gene transcripts present (r=0.99). Naphthalene was bioavailable in soils A, D, E, G, and N as determined by a bioluminescent response from the naphthalene-lux reporter system. Taken together, these data provided information on what the naphthalene-degrading bacterial population was experiencingin situ and what approaches would be necessary to increase activity.

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.

Response of Soil Microorganisms to As-Produced and Functionalized Single-Wall Carbon Nanotubes (SWNTs)

The use of single-wall carbon nanotubes (SWNTs) in manufacturing and biomedical applications is increasing at a rapid rate; however data on the effects of a potential environmental release of the materials remain sparse. In this study, soils with either low or high organic matter contents as well as pure cultures of E. coli are challenged with either raw as-produced SWNTs (AP-SWNTs) or SWNTs functionalized with either polyethyleneglycol (PEG-SWNTs) or m-polyaminobenzene sulfonic acid (PABS-SWNTs). To mimic chronic exposure, the soil systems were challenged weekly for six weeks; microbial activities and community structures for both the prokaryote and eukaryote community were evaluated. Results show that repeated applications of AP-SWNTs can affect microbial community structures and induce minor changes in soil metabolic activity in the low organic matter systems. Toxicity of the three types of SWNTs was also assessed in liquid cultures using a bioluminescent E. coli-O157:H7 strain. Although decreases in light were detected in all treated samples, low light recovery following glucose addition in AP-SWNTs treatment and light absorption property of SWNTs particles suggest that AP-SWNTs suppressed metabolic activity of the E. coli, whereas the two functionalized SWNTs are less toxic. The metals released from the raw forms of SWNTs would not play a role in the effects seen in soil or the pure culture. We suggest that sorption to soil organic matter plays a controlling role in the soil microbiological responses to these nanomaterials.

Influence of nanophase titania topography on bacterial attachment and metabolism

Surfaces with nanophase compared to conventional (or nanometer smooth) topographies are known to have different properties of area, charge, and reactivity. Previously published research indicates that the attachment of certain bacteria (such as Pseudomonas fluorescens 5RL) is higher on surfaces with nanophase compared to conventional topographies, however, their effect on bacterial metabolism is unclear. Results presented here show that the adhesion of Pseudomonas fluorescens 5RL and Pseudomonas putida TVA8 was higher on nanophase than conventional titania. Importantly, in terms of metabolism, bacteria attached to the nanophase surfaces had higher bioluminescence rates than on the conventional surfaces under all nutrient conditions. Thus, the results from this study show greater select bacterial metabolism on nanometer than conventional topographies, critical results with strong consequences for the design of improved biosensors for bacteria detection.

Structure-activity relationships of antibacterial and biocompatible copolymers.

The development of polymers that are both bactericidal and biocompatible would have many applications and are currently of research interest. Following the development of strongly bactericidal copolymers of 4-vinylpyridine and poly(ethylene glycol) methyl ether methacrylate, biocompatibility assays have been completed on these materials to measure their potential biocompatibility. In this article, a new methodology for measuring protein interaction was developed for water-soluble polymers by coupling proteins to surfaces and then measuring the adsorption of copolymers onto these surfaces. Ellipsometry was then used to measure the thickness of adsorbed polymers as a measurement of biocompatibility. These results were then compared and correlated with the results of other biocompatibility assays previously conducted on these polymers, affording a greater understanding of the biocompatibility of the copolymers as well as improving the understanding of the effect of hydrophilic and hydrophobic groups that is vital for the development of these materials.

Combined lipid/DNA extraction method for environmental samples

Abstract Previously, separate methods have been developed for the extraction and purification of lipids and DNA from soils and sediments. This paper describes a new method for the isolation of both lipids and DNA from the same environmental sample. This combined method is based on the Bligh and Dyer lipid extraction technique. Upon phase separation, lipids partition into the organic phase and DNA partitions into the aqueous phase. DNA extraction and recovery from the solid phase is necessary under certain conditions. Preliminary experiments performed with 32P-labeled DNA in the absence of soil showed that greater than 98% of the total DNA was present in the aqueous phase after the modified Bligh and Dyer extraction. Analysis of the DNA by polyacrylamide gel electrophoresis and autoradiography demonstrated that no degradation of DNA occurred during the lipid extraction procedure. Lipid extraction of lyophilized cells showed that DNA was released from Pseudomonas putida and Bacillus subtilis cells corresponding to 26±5 and 14±4% of the theoretical DNA yield, respectively. The combined lipid/DNA extraction method was applied to both lyophilized cells and wet cells added to soil. Analysis by DNA:DNA hybridization showed that approx. 40–50% of the DNA from cells added to soil was recovered after lipid extraction relative to samples treated only with conventional DNA extraction. Estimation of cell number per gram soil based on either lipid or DNA analysis showed good agreement with actual numbers added based on plate counts of the inocula. DNA extracts from samples which had been lipid-extracted also had lower amounts of humic material. Although some DNA was not recovered after lipid extraction, that which was recovered was of sufficiently high quality for hybridization analysis. This method shows utility for the co-recovery of both lipids and DNA from a single sample; this is particularly useful when a small sample size is all that is available or procurable.

Nano/Micro and Spectroscopic Approaches to Food Pathogen Detection

Despite continuing research efforts, timely and simple pathogen detection with a high degree of sensitivity and specificity remains an elusive goal. Given the recent explosion of sensor technologies, significant strides have been made in addressing the various nuances of this important global challenge that affects not only the food industry but also human health. In this review, we provide a summary of the various ongoing efforts in pathogen detection and sample preparation in areas related to Fourier transform infrared and Raman spectroscopy, light scattering, phage display, micro/nanodevices, and nanoparticle biosensors. We also discuss the advantages and potential limitations of the detection methods and suggest next steps for further consideration.

Chlorine dioxide oxidation of dihydronicotinamide adenine dinucleotide (NADH).

The oxidation of dihydronicotinamide adenine dinucleotide (NADH) by chlorine dioxide in phosphate buffered solutions (pH 6-8) is very rapid with a second-order rate constant of 3.9 x 10(6) M(-1) s(-1) at 24.6 degrees C. The overall reaction stoichiometry is 2ClO2(*) per NADH. In contrast to many oxidants where NADH reacts by hydride transfer, the proposed mechanism is a rate-limiting transfer of an electron from NADH to ClO2(*). Subsequent sequential fast reactions with H(+) transfer to H2O and transfer of an electron to a second ClO2(*) give 2ClO2(-), H3O(+), and NAD(+) as products. The electrode potential of 0.936 V for the ClO2(*)/ClO2(-) couple is so large that even 0.1 M of added ClO2(-) (a 10(3) excess over the initial ClO2(*) concentration) fails to suppress the reaction rate.

Biodegradation genes as marker genes in microbial ecosystems

For the detection of specific biodegradative microorganisms in the environment, DNA hybridization has been extensively used and throughly reviewed [1, 4, 10]. More often, DNA hybridization can be applied in colony hybridization where the cells from any soil sample are washed off, cultured on nutrient containing agar plates, transferred to nylon membranes, lysed, their DNA fixed to the nylon membranes and subsequently hybridized with appropriate catabolic gene probe(s) [11]. DNA hybridization can also be used with directly extracted DNA from soil or any other source. Colony hybridization first requires culturing of cells while direct DNA extraction is done by in situ lysis of cells in the soil. Therefore, direct extraction of DNA overcomes the primary disadvantage in colony hybridization (culturability) as only a small fraction of the actual bacterial population in the soil is culturable. The DNA obtained through direct extraction from soil is usually blotted on nylon membrane and hybridized with appropriate catabolic gene probe(s) as in colony hybridization [7].