Enabling trace chemical detection equipment utilized in the field to transduce a biodetection assay would be advantageous from a logistics, training, and maintenance standpoint. Described herein is an assay design that uses an unmodified, commercial off-the-shelf (COTS) ion trap mobility spectrometer to analyze an immunomagnetic enzyme-linked immunosorbant assay (ELISA). The assay, which uses undetectable enzymatic substrates and ELISA-generated detectable products, was optimized to quantitatively report the amount of target in the sample. Optimization of this ELISA design retained the assay specificity and detection limit ( approximately 10(3) E. coli per assay) while decreasing the number of user steps and reducing the assay time to 10 min (>9-fold decrease as compared to past studies). Also discussed are previously undescribed, independent substrate/enzyme/product combinations used in the immunomagnetic ELISA. These discoveries allow for the possibility of a quantitative, multiplexed, 10-min assay that is analyzed by the ion trap mobility spectrometer trace chemical detector.

Human health risk assessments involving contaminated soil include dermal absorption as a potential pathway contributing to the total exposure burden. For PCB-contaminated soil, the U.S. Environmental Protection Agency uses a dermal absorption factor of 14%, based on a 1993 study of dermal absorption in rhesus monkeys. The current study examined several parameters that can influence the dermal absorption of lipophilic hydrocarbons, including soil organic content, particle size, skin residence time, and contaminant "aging" in the soil. Four groups of four female rhesus monkeys each were exposed to radiolabeled Aroclor 1260 either intravenously (100% absorption) or dermally with PCB-spiked soil. Groups exposed for 12 or 24 h to PCBs aged in soil exhibited percutaneous absorption values of 3.43 +/- 0.35 and 4.26 +/- 0.52%, respectively, while a group exposed for 24 h to soil freshly spiked with PCBs exhibited a dermal absorption value of 4.07 +/- 0.46%. Evidence strongly suggests that the factor most responsible for modulating the percutaneous absorption of highly lipophilic compounds from soil is its organic content. The base soil used in the current study with Aroclor 1260 had an organic content of 5-6%(< or =2 mm particle fraction), a value typical for U.S. soil. The organic content of the soil applied to the skin was 8.7%(<150 microm particle fraction), a value that contrasts sharply with the soil containing 0.9% organics used in the 1993 study with Aroclors 1242 and 1254 that produced a dermal absorption value of 14% for PCBs.

The polychlorinated biphenyl (PCB) congener specificities and partial BphA sequences of biphenyl dioxygenase were determined for a set of PCB-degrading bacteria. The strains examined were categorized into two groups based on their ability to degrade 17 PCB congeners. Strains that degraded a broad range of PCBs but had relatively weak activity against di-para-substituted PCBs were designated as having an LB400-type specificity. Strains designated as having a KF707-type specificity degraded a much narrower range of PCBs but had strong activity against certain di-para-substituted congeners. BphA protein sequence comparisons between these two types of strains identified four regions (designated I, II, III, and IV) in which specific sequences were consistently associated with either broad or narrow PCB substrate specificity. The dramatic differences in substrate specificity between LB400 and KF707 appear to result primarily from a combination of mutations in regions III and IV. Altering these regions in the LB400 BphA subunit to correspond to those in the KF707 sequence produced a narrow substrate specificity very similar to that of KF707. Some individual mutations within region III alone were found to improve PCB degradative activity, especially for di-para-substituted congeners. However, the greatest improvements in activity resulted from multiple amino acid modifications in region III, suggesting that the effects of these mutations are cooperative. These results demonstrate the ability to significantly improve PCB oxidative activity through sequence modifications of biphenyl dioxygenase.

A 73-day field study of in situ aerobic biodegradation of polychlorinated biphenyls (PCBs) in the Hudson River shows that indigenous aerobic microorganisms can degrade the lightly chlorinated PCBs present in these sediments. Addition of inorganic nutrients, biphenyl, and oxygen enhanced PCB biodegradation, as indicated both by a 37 to 55 percent loss of PCBs and by the production of chlorobenzoates, intermediates in the PCB biodegradation pathway. Repeated inoculation with a purified PCB-degrading bacterium failed to improve biodegradative activity. Biodegradation was also observed under mixed but unamended conditions, which suggests that this process may occur commonly in river sediments, with implications for PCB fate models and risk assessments.

Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners.

A Beijerinckia species, capable of oxidizing phenanthrene, biphenyl and other polycyclic aromatic hydrocarbons, was shown to contain two plasmids that were designated pKGl and pKG2. The molecular masses of plasmids pKG1 and pKG2, as determined by electron microscopy, were approximately 147 X 10(6) and 20.8 X 10(6) daltons, respectively. Growth of the organism on benzoate led to the isolation of strains that had lost the ability to grow with phenanthrene and biphenyl. All of the Phn-, Bph- strains had also lost the smaller plasmid, pKG2. The results presented suggest that plasmid pKG2 is responsible for the synthesis of enzymes involved in the degradation of phenanthrene and biphenyl.

A suppressor of the mutations which inhibit the establishment of lysogeny in Pseudomonas aeruginosa has been found to be encoded by P. aeruginosa plasmids of four separate incompatibility groups. The wide distribution of this phenotype (designated Sly for suppressor of lysogeny establishment deficiency) indicates that these plasmids are able to enhance or replace host functions involved in the stable establishment of extrachromosomal DNA.

DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to LB400 in PCB-degrading capability. These two organisms showed a strong conservation of restriction sites in the region of DNA encoding PCB metabolism. No other sequence similarities were detected in the two genomes. DNA from the other PCB-degrading strains showed no hybridization to the probe, which demonstrated the existence of at least two distinct classes of genes encoding PCB degradation.

Pseudomonas strain LB400 is able to degrade an unusually wide variety of polychlorinated biphenyls (PCBs). A genomic library of LB400 was constructed by using the broad-host-range cosmid pMMB34 and introduced into Escherichia coli. Approximately 1,600 recombinant clones were tested, and 5 that expressed 2,3-dihydroxybiphenyl dioxygenase activity were found. This enzyme is encoded by the bphC gene of the 2,3-dioxygenase pathway for PCB-biphenyl metabolism. Two recombinant plasmids encoding the ability to transform PCBs to chlorobenzoic acids were identified, and one of these, pGEM410, was chosen for further study. The PCB-degrading genes (bphA,-B,-C, and -D) were localized by subcloning experiments to a 12.4-kilobase region of pGEM410. The ability of recombinant strains to degrade PCBs was compared with that of the wild type. In resting-cell assays, PCB degradation by E. coli strain FM4560 (containing a pGEM410 derivative) approached that of LB400 and was significantly greater than degradation by the original recombinant strain. High levels of PCB metabolism by FM4560 did not depend on the growth of the organism on biphenyl, as it did for PCB metabolism by LB400. When cells were grown with succinate as the carbon source, PCB degradation by FM4560 was markedly superior to that by LB400.