Detection of organic compounds with whole-cell bioluminescent bioassays.

Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices.

Integrated metagenomics and metatranscriptomics analyses of root-associated soil from transgenic switchgrass.

The benefits of using transgenic switchgrass with decreased levels of caffeic acid 3-O-methyltransferase (COMT) as biomass feedstock have been clearly demonstrated. However, its effect on the soil microbial community has not been assessed. Here we report metagenomic and metatranscriptomic analyses of root-associated soil from COMT switchgrass compared with nontransgenic counterparts.

Comparison of fecal indicators with pathogenic bacteria and rotavirus in groundwater.

Groundwater is routinely analyzed for fecal indicators but direct comparisons of fecal indicators to the presence of bacterial and viral pathogens are rare. This study was conducted in rural Bangladesh where the human population density is high, sanitation is poor, and groundwater pumped from shallow tubewells is often contaminated with fecal bacteria. Five indicator microorganisms (E. coli, total coliform, F+RNA coliphage, Bacteroides and human-associated Bacteroides) and various environmental parameters were compared to the direct detection of waterborne pathogens by quantitative PCR in groundwater pumped from 50 tubewells. Rotavirus was detected in groundwater filtrate from the largest proportion of tubewells (40%), followed by Shigella (10%), Vibrio (10%), and pathogenic E. coli (8%). Spearman rank correlations and sensitivity-specificity calculations indicate that some, but not all, combinations of indicators and environmental parameters can predict the presence of pathogens. Culture-dependent fecal indicator bacteria measured on a single date did not predict total bacterial pathogens, but annually averaged monthly measurements of culturable E. coli did improve prediction for total bacterial pathogens. A qPCR-based E. coli assay was the best indicator for the bacterial pathogens. F+RNA coliphage were neither correlated nor sufficiently sensitive towards rotavirus, but were predictive of bacterial pathogens. Since groundwater cannot be excluded as a significant source of diarrheal disease in Bangladesh and neighboring countries with similar characteristics, the need to develop more effective methods for screening tubewells with respect to microbial contamination is necessary.

The evolution of the bacterial luciferase gene cassette (lux) as a real-time bioreporter.

The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.

Pathogen detection using engineered bacteriophages.

Bacteriophages, or phages, are bacterial viruses that can infect a broad or narrow range of host organisms. Knowing the host range of a phage allows it to be exploited in targeting various pathogens. Applying phages for the identification of microorganisms related to food and waterborne pathogens and pathogens of clinical significance to humans and animals has a long history, and there has to some extent been a recent revival in these applications as phages have become more extensively integrated into novel detection, identification, and monitoring technologies. Biotechnological and genetic engineering strategies applied to phages are responsible for some of these new methods, but even natural unmodified phages are widely applicable when paired with appropriate innovative detector platforms. This review highlights the use of phages as pathogen detector interfaces to provide the reader with an up-to-date inventory of phage-based biodetection strategies.

Draft genome sequence of the polycyclic aromatic hydrocarbon-degrading, genetically engineered bioluminescent bioreporter Pseudomonas fluorescens HK44.

Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of ∼6.1 Mb of sequence indicates that 30% of the traits are unique and distributed over five genomic islands, a prophage, and two plasmids.

Bacteriophage reporter technology for sensing and detecting microbial targets.

Bacteriophages (phages) are bacterial viruses evolutionarily tuned to very specifically recognize, infect, and propagate within only a unique pool of host cells. Knowledge of these phage host ranges permits one to devise diagnostic tests based on phage-host recognition profiles. For decades, fundamental phage typing assays have been used to identify bacterial pathogens on the basis of the ability of phages to kill, or lyse, the unique species, strain, or serovar to which they are naturally targeted. Over time, and with a better understanding of phage-host kinetics and the realization that there exists a phage specific for nearly any bacterial pathogen of clinical, foodborne, or waterborne consequence, a variety of improved, rapid, sensitive, and easy-to-use phage-mediated detection assays have been developed. These assays exploit every stage of the phage recognition and infection cycle to yield a wide variety of pathogen monitoring, detection, and enumeration formats that are steadily advancing toward new biosensor integrations and advanced sensing technologies.