Stress responsive bacteria: biosensors as environmental monitors.

The delicate and dynamic balance of the physiological steady state and its maintenance is well characterized by studies of bacterial stress response. Through the use of genetic analysis, numerous stress regulons, their physiological regulators and their biochemical processes have been delineated. In particular, transcriptionally activated stress regulons are subjects of study and application. These regulons include those that respond to macromolecular damage and toxicity as well as to nutrient starvation. The convenience of reporter gene fusions has allowed the creation of biosensor strains, resulting from the fusion of stress-responsive promoters with a variety of reporter genes. Such cellular biosensors are being used for monitoring dynamic systems and can report the presence of environmental stressors in real time. They provide a greater range of sensitivity, e.g. to sub-lethal concentrations of toxicants, than the simple assessment of cell viability. The underlying physiological context of the reporter strains results in the detection of bioavailable concentrations of both toxicants and nutrients. Culture conditions and host strain genotypes can be customized so as to maximize the sensitivity of the strain for a particular application. Collections of specific strains that are grouped in panels are used to diagnose targets or mode of action for unknown toxicants. Further application in massive by parallel DNA and gene fusion arrays greatly extends the information available for diagnosis of modes of action and may lead to development of novel high-throughput screens. Future studies will include more panels, arrays, as well as single reporter cell detection for a better understanding of the population heterogeneity during stress response. New knowledge of physiology gained from further studies of novel systems, or using innovative methods of analysis, will undoubtedly yield still more useful and informative environmental biosensors.

RNase H overproduction allows the expression of stress-induced genes in the absence of topoisomerase I.

Induction of stress proteins in response to heat shock was found to be reduced significantly in Escherichia coli with DeltatopA mutation. RNase H overexpression in the DeltatopA mutant partially restored the sigma(32)-dependent induction of stress genes in response to high temperature and ethanol. The presence of overexpressed RNase H also improved the survival rate of the DeltatopA mutant after high temperature and oxidative challenges. Topoisomerase I is likely required during stress response for preventing accumulation of transcription-driven hypernegative supercoiling and R-loop formation at induced stress genes loci.

Recombinant microorganisms as environmental biosensors: pollutants detection by Escherichia coli bearing fabA'::lux fusions.

A set of genetically engineered Escherichia coli strains was constructed, in which the promoter of the fabA gene is fused to Vibrio fischeri luxCDABE either in a multi-copy plasmid or as a single copy chromosomal integration. The fabA gene codes for beta-hydroxydecanoyl-ACP dehydrase, a key enzyme in the synthesis of unsaturated fatty acids, and is induced when fatty acid biosynthesis pathways are interrupted. A dose-dependent and highly sensitive bioluminescent response to a variety of chemicals was controlled by the fadR gene. A tolC mutant E. coli host displayed generally lower detection threshold for toxicants. A chromosomal integration of a single copy of the fabA'::lux fusion led to a markedly lower background luminescence, but did not yield an improvement in overall performance. It is proposed that these or similarly constructed reporters of fatty acid biosynthesis inhibition may serve as novel microbial toxicity biosensors.

Bioluminescent reporter genes for promoter discovery.

Discovery of promoter elements with previously unknown regulated responses is important for metabolic engineering. For example, promoters responsive to the end product can be useful to regulate expression with increasing levels of product. In addition, such promoters can be used as screens for production strain with increased titers. Use of reporter genes, such as a bioluminescent reporter luxCDABE, can facilitate promoter discovery. Here, protocols for analysis of genome-wide luxCDABE reporter gene collections in Escherichia coli are provided. Further, a protocol for using a selected para-hydroxycinnamic (pHCA)-responsive promoter as detection assay for bioproduced pHCA is provided.

Production of tyrosine from sucrose or glucose achieved by rapid genetic changes to phenylalanine-producing Escherichia coli strains.

Escherichia coli K12 strains producing L-phenylalanine were converted to L-tyrosine-producing strains using a novel genetic method for gene replacement. We deleted a region of the E. coli K12 chromosome including the pheA gene encoding chorismate mutase/prephenate dehydratase, its leader peptide (pheL), and its promoter using a new polymerase chain reaction-based method that does not leave a chromosomal scar. For high level expression of tyrA, encoding chorismate mutase/prephenate dehydrogenase, its native promoter was replaced with the strong trc promoter. The linked DeltapheLA and Ptrc-tyrA::Kan(R) genetic modifications were moved into L-phenylalanine producing strains by generalized transduction to convert L-phenylalanine-producing strains to L-tyrosine-producing strains. Moreover, introduction of a plasmid carrying genes responsible for sucrose degradation into these strains enabled L-tyrosine-production from sucrose.

In situ measurement of bioluminescence and fluorescence in an integrated microbioreactor.

Reporter strains of bacteria that emit light or a fluorescent marker in response to specific conditions in their environment are having a significant impact in many areas of biology, including toxicity assays for environmental pollutants, chemical detection, and gene expression profiling. We have demonstrated methods for in situ measurements of bioluminescence and fluorescence from bacterial cultures grown in 50 microL instrumented microbioreactors. Results from microbioreactors were compared to results obtained from conventional 500 mL batch bioreactors and shake flasks. Experiments were conducted with reporter strains of Escherichia coli in which luxCDABE or gfp was fused to a promoter that was either expressed constitutively, or that responded to oxygen limitation. With these reporter strains, we have demonstrated the ability to obtain information on growth conditions within the microbioreactor. We have also shown that the large aspect ratio of the microbioreactor provides a unique advantage over measurements in larger bioreactors by reducing the inner filter effect in on-line measurements and eliminating the need for error-prone off-line dilutions. In addition, continuous on-line monitoring of genes in real-time, when expanded to include entire reporter libraries, could potentially provide a true dynamic picture of cellular gene expression from which the kinetics of gene expression can be untangled and elucidated.

Impact of genomic technologies on studies of bacterial gene expression.

The ability to simultaneously monitor expression of all genes in any bacterium whose genome has been sequenced has only recently become available. This requires not only careful experimentation but also that voluminous data be organized and interpreted. Here we review the emerging technologies that are impacting the study of bacterial global regulatory mechanisms with a view toward discussing both perceived best practices and the current state of the art. To do this, we concentrate upon examples using Escherichia coli and Bacillus subtilis because prior work in these organisms provides a sound basis for comparison.

Characterization of the Escherichia coli AaeAB efflux pump: a metabolic relief valve?

Treatment of Escherichia coli with p-hydroxybenzoic acid (pHBA) resulted in upregulation of yhcP, encoding a protein of the putative efflux protein family. Also upregulated were the adjacent genes yhcQ, encoding a protein of the membrane fusion protein family, and yhcR, encoding a small protein without a known or suggested function. The function of the upstream, divergently transcribed gene yhcS, encoding a regulatory protein of the LysR family, in regulating expression of yhcRQP was shown. Furthermore, it was demonstrated that several aromatic carboxylic acid compounds serve as inducers of yhcRQP expression. The efflux function encoded by yhcP was proven by the hypersensitivity to pHBA of a yhcP mutant strain. A yhcS mutant strain was also hypersensitive to pHBA. Expression of yhcQ and yhcP was necessary and sufficient for suppression of the pHBA hypersensitivity of the yhcS mutant. Only a few aromatic carboxylic acids of hundreds of diverse compounds tested were defined as substrates of the YhcQP efflux pump. Thus, we propose renaming yhcS, yhcR, yhcQ, and yhcP, to reflect their role in aromatic carboxylic acid efflux, to aaeR, aaeX, aaeA, and aaeB, respectively. The role of pHBA in normal E. coli metabolism and the highly regulated expression of the AaeAB efflux system suggests that the physiological role may be as a "metabolic relief valve" to alleviate toxic effects of imbalanced metabolism.

FlhD/FlhC is a regulator of anaerobic respiration and the Entner-Doudoroff pathway through induction of the methyl-accepting chemotaxis protein Aer.

The regulation by two transcriptional activators of flagellar expression (FlhD and FlhC) and the chemotaxis methyl-accepting protein Aer was studied with glass slide DNA microarrays. An flhD::Kan insertion and an aer deletion were independently introduced into two Escherichia coli K-12 strains, and the effects upon gene regulation were investigated. Altogether, the flhD::Kan insertion altered the expression of 29 operons of known function. Among them was Aer, which in turn regulated a subset of these operons, namely, the ones involved in anaerobic respiration and the Entner-Doudoroff pathway. In addition, FlhD/FlhC repressed enzymes involved in aerobic respiration and regulated many other metabolic enzymes and transporters in an Aer-independent manner. Expression of 12 genes of uncharacterized function was also affected. FlhD increased gltBD, gcvTHP, and ompT expression. The regulation of half of these genes was subsequently confirmed with reporter gene fusions, enzyme assays, and real-time PCR. Growth phenotypes of flhD and flhC mutants were determined with Phenotype MicroArrays and correlated with gene expression.