BackgroundThe 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 FindingsAutonomous 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/SignificanceThe 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.

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 17␤-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 estrogendisrupting chemicals in the environment.

In vivo bioluminescent imaging (BLI) is increasingly being utilized as a method for modern biological research. This process, which involves the noninvasive interrogation of living animals using light emitted from luciferase-expressing bioreporter cells, has been applied to study a wide range of biomolecular functions such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis, cancer treatment, and disease progression. This article will review the various bioreporter/biosensor integrations of BLI and discuss how BLI is being applied towards a new visual understanding of biological processes within the living organism.

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Bacteriophages occur in high numbers in environmental ecosystems and are thus significant mediators of microbial survival and activities. However, interactions between microbial populations and phages in situ have been largely ignored. Current understanding of the process relies on studies performed with well-fed, laboratory-grown host bacteria. The purpose of the experiments reported here was to determine bacteriophage-host interactions under environmentally relevant conditions of nutrient limitation. These studies have revealed the importance of a phenomenon called pseudolysogeny in the maintenance of viral genetic material for extended periods of time in natural ecosystems. Pseudolysogeny is a form of phage-host cell interaction in which the nucleic acid of the phage resides within its starved host in an unstable, inactive state. It is hypothesized that pseudolysogeny occurs due to the cell's highly starved condition. In such cells, there is insufficient energy available for the phage to initiate genetic expression leading to either a true temperate response or to the lytic response. However, upon nutrient addition, the pseudolysogenic state is resolved, resulting in either the establishment of true lysogeny or the initiation of the lytic production of progeny virions. The pseudolysogenic state may explain the long-term survival of viruses in unfavourable environments in which the infective half-life of their virions is relatively short.

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.

BackgroundExpression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines.Methodology/Principal FindingsThe lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations.Conclusions/SignificanceOur results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity. This autobioluminescent phenotype allowed for repeated interrogation of populations and self-directed control of bioluminescent activation with detection limits and EC50 values similar to traditional reporter systems, making the autobioluminescent cells amenable to automated monitoring and significantly reducing the time and cost required to perform bioluminescent workflows.

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.