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 Escherichia coli strains for the quantitative detection of phosphate and ammonia in coastal and suburban watersheds.

Accumulation of phosphate and ammonia in estuarine systems and subsequent dinoflagellate and algal blooms has been implicated in fish kills and in health risks for fishermen. Analytic chemistry kits are used to measure phosphate and ammonia levels in water samples, but their sensitivity is limited due to specificity for inorganic forms of these moieties. An Escherichia coli bioluminescent reporter system measured the bioavailability of inorganic nutrients through fusion of E. coli promoters (phoA or glnAp2) to the luxCDABE operon of Vibrio fischeri carried either on the chromosome or on a multicopy plasmid vector, resulting in emission of light in response to phosphate or ammonia starvation. Responses were shown to be under the control of expected physiological regulators, phoB and glnFG, respectively. Standard curves were used to determine the phosphate and ammonia levels in water samples from diverse watersheds located in the northeastern United States. Bioluminescence produced in response to nutrient starvation correlated with concentrations of phosphate (1-24 ppm) and ammonia (0.1-1.6 ppm). While the ammonia biosensor measured nutrient concentrations in tested water samples that were comparable to the amounts reported by a commercial kit, the phosphate biosensor reported higher levels of phosphate in Chesapeake water samples than did the kit.

Gene expression analysis of the response by Escherichia coli to seawater.

Gene expression of Escherichia coli cells exposed to seawater for 20 h was compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA microarray technology. The expression of most (ca. 3,000) of the 4,228 open reading frames on the microarray remained unchanged; the relative expression of about 320 genes decreased in seawater, whereas that of ca. one fourth (937) increased. Clearly coherent expression patterns were observed for several functional gene groups. Induced genes were numerous in groups specifying the degradation of small molecules (carbon compounds, amino acids and fatty acids), energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures and phage related functions. Repressed genes were clustered in two groups, cell division and nucleotides biosynthesis, indicating a cessation of growth.

Interfering with different steps of protein synthesis explored by transcriptional profiling of Escherichia coli K-12.

Escherichia coli responses to four inhibitors that interfere with translation were monitored at the transcriptional level. A DNA microarray method provided a comprehensive view of changes in mRNA levels after exposure to these agents. Real-time reverse transcriptase PCRanalysis served to verify observations made with microarrays, and a chromosomal grpE::lux operon fusion was employed to specifically monitor the heat shock response. 4-Azaleucine, a competitive inhibitor of leucyl-tRNA synthetase, surprisingly triggered the heat shock response. Administration of mupirocin, an inhibitor of isoleucyl-tRNA synthetase activity, resulted in changes reminiscent of the stringent response. Treatment with kasugamycin and puromycin (targeting ribosomal subunit association as well as its peptidyl-transferase activity) caused accumulation of mRNAs from ribosomal protein operons. Abundant biosynthetic transcripts were often significantly diminished after treatment with any of these agents. Exposure of a relA strain to mupirocin resulted in accumulation of ribosomal protein operon transcripts. However, the relA strain's response to the other inhibitors was quite similar to that of the wild-type strain.