John Sanseverino

Rapid, Sensitive Bioluminescent Reporter Technology for Naphthalene Exposure and Biodegradation

A bioluminescent reporter plasmid for naphthalene catabolism (pUTK21) was developed by transposon (Tn4431) insertion of the lux gene cassette from Vibrio fischeri into a naphthalene catabolic plasmid in Pseudomonas fluorescens. The insertion site of the lux transposon was the nahG gene encoding for salicylate hydroxylase. Luciferasemediated light production from P. fluorescens strains harboring this plasmid was induced on exposure to naphthalene or the regulatory inducer metabolite, salicylate. In continuous culture, light induction was rapid (15 minutes) and was highly responsive to dynamic changes in naphthalene exposure. Strains harboring pUTK21 were responsive to aromatic hydrocarbon contamination in Manufactured Gas Plant soils and produced sufficient light to serve as biosensors of naphthalene exposure and reporters of naphthalene biodegradative activity. The robust and sensitive nature of the bioluminescent reporter technology suggests that new sensing methods can be developed for on-line process monitoring and control in complex environmental matrices.

Use of Saccharomyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds

ABSTRACT An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17β-estradiol over the concentration range of 1.2 × 10−8 through 5.6 × 10−12 M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) × 10−10 and (2.4 ± 1.0) × 10−10 M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 × 10−11 to 2.8 × 10−9 M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.

Analyses of Polycyclic Aromatic Hydrocarbon Degrading Bacteria Isolated from Contaminated Soils

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria isolated from PAH-contaminated soils were analyzed genotypically and phenotypically for their capacity for metabolism of naphthalene and other PAH substrates. The methods used for the analyses were DNA hybridization using NAH7-derived gene probes, PAH spray plate assays, 14C-PAH mineralization assays, and dioxygenase activity assays. The results of the analyses showed a dominant number of PAH-degrading bacteria with a NAH7-like genotype. The results support the continued use of the nahA probe for contaminated soils to monitor the genetic potential of indigenous microorganisms to degrade PAHs. However, the finding of non-it nahA-hybridizing PAH-degrading bacteria show the limitation of NAH7-derived gene probes. Fifteen percent (13/89) of PAH-degrading bacteria isolated were not detected with the nahA gene probe. Four isolates (designated A5PH1, A8AN3, B1PH2, and B10AN1) did not hybridize with any of the NAH7-derived gene probes ( nahA, nahG, nahH, and nahR) used in this study. Considering the numerous unculturable microorganisms in nature and their potential genotypes, NAH7-derived gene probes may underestimate the microbial potential to catabolize PAHs. This necessitates development of new gene probes for enumeration and isolation of PAH-degrading bacteria to better understand the in situ microbial potential to degrade PAHs.

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

Background The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Methodology/Principal Findings Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. Conclusions/Significance The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

Screening of Potentially Hormonally Active Chemicals Using Bioluminescent Yeast Bioreporters

Saccharomyces cerevisiae bioluminescent bioreporter assays were developed previously to assess a chemicals estrogenic or androgenic disrupting potential. S. cerevisiae BLYES, S. cerevisiae BLYAS, S. cerevisiae BLYR, were used to assess their reproducibility and utility in screening 68, 69, and 71 chemicals for estrogenic, androgenic, and toxic effects, respectively. EC(50) values were 6.3 +/- 2.4 x 10(-10)M (n = 18) and 1.1 +/- 0.5 x 10(-8)M (n = 13) for BLYES and BLYAS, using 17beta-estradiol and 5alpha-dihydrotestosterone over concentration ranges of 2.5 x 10(-12) through 1.0 x 10(-6)M, respectively. Based on analysis of replicate standard curves and comparison to background controls, a set of quantitative rules have been formulated to interpret data and determine if a chemical is potentially hormonally active, toxic, both, or neither. The results demonstrated that these assays are applicable for Tier I chemical screening in Environmental Protection Agencys Endocrine Disruptor Screening and Testing Program as well as for monitoring endocrine-disrupting activity of unknown chemicals in water.

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.

Saccharomyces cerevisiae BLYAS, a New Bioluminescent Bioreporter for Detection of Androgenic Compounds

ABSTRACT A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp), yielded a bioluminescent bioreporter responsive to androgenic chemicals. Using dihydrotestosterone (DHT) as a standard, the response time and the 50% effective concentration values were 3 to 4 h and (9.7 ± 4.6) × 10−9 M, respectively. The lower limit of detection in response to DHT was 2.5 × 10−9 M, and in response to testosterone it was 2.5 × 10−10 M. This strain is suitable for high-throughput screening of chemicals with potential for remote environmental monitoring systems because of the assay speed, sensitivity, and self-containment.

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.

Complete genome sequence of Thauera aminoaromatica strain MZ1T

Thauera aminoaromatica strain MZ1T, an isolate belonging to genus Thauera, of the family Rhodocyclaceae and the class the Betaproteobacteria, has been characterized for its ability to produce abundant exopolysaccharide and degrade various aromatic compounds with nitrate as an electron acceptor. These properties, if fully understood at the genome-sequence level, can aid in environmental processing of organic matter in anaerobic cycles by short-circuiting a central anaerobic metabolite, acetate, from microbiological conversion to methane, a critical greenhouse gas. Strain MZ1T is the first strain from the genus Thauera with a completely sequenced genome. The 4,496,212 bp chromosome and 78,374 bp plasmid contain 4,071 protein-coding and 71 RNA genes, and were sequenced as part of the DOE Community Sequencing Program CSP_776774.

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].