Dana R. Smulski

DNA Microarray-Mediated Transcriptional Profiling of the Escherichia coli Response to Hydrogen Peroxide

The genome-wide transcription profile of Escherichia coli cells treated with hydrogen peroxide was examined with a DNA microarray composed of 4,169 E. coli open reading frames. By measuring gene expression in isogenic wild-type and oxyR deletion strains, we confirmed that the peroxide response regulator OxyR activates most of the highly hydrogen peroxide-inducible genes. The DNA microarray measurements allowed the identification of several new OxyR-activated genes, including the hemH heme biosynthetic gene; the six-gene suf operon, which may participate in Fe-S cluster assembly or repair; and four genes of unknown function. We also identified several genes, including uxuA, encoding mannonate hydrolase, whose expression might be repressed by OxyR, since their expression was elevated in the DeltaoxyR mutant strain. In addition, the induction of some genes was found to be OxyR independent, indicating the existence of other peroxide sensors and regulators in E. coli. For example, the isc operon, which specifies Fe-S cluster formation and repair activities, was induced by hydrogen peroxide in strains lacking either OxyR or the superoxide response regulators SoxRS. These results expand our understanding of the oxidative stress response and raise interesting questions regarding the nature of other regulators that modulate gene expression in response to hydrogen peroxide.

A Panel Of Stress-Responsive Luminous Bacteria For The Detection Of Selected Classes Of Toxicants

Abstract A panel of bacteria, each genetically engineered to respond by increased luminescence to a different type of environmental stress, is presented. Members of the panel were shown to be sensitive to several groups of chemicals including phenols, halomethanes and several oxidants. The increase in light emission depended upon toxicant concentration and could, thus, be used to calculate a characteristic value, EC 200 , designating the sample concentration causing a two-fold luminescence induction. In almost all cases, EC 200 values were lower than the corresponding Microtox™ EC 50 values, indicating a generally higher sensitivity. One of the panel members, DPD2794, a designated DNA-damage sensor, responded within 2 h to the presence of genotoxicants, including metabolically activated nitropropane. It is suggested that these or similarly constructed strains can be used for the rapid and sensitive detection of potentially toxic and genotoxic pollutants and that the concept of genetically engineering a panel of microbial toxicity sensors can readily be implemented for environmental monitoring.

Global Impact of sdiA Amplification Revealed by Comprehensive Gene Expression Profiling of Escherichia coli

In Escherichia coli the amplification of sdiA, a positive activator of ftsQAZ, genes that are essential for septation, results in mitomycin C resistance. To help us understand this resistance phenotype, genes whose expression was altered by increased sdiA dosage were identified using a DNA microarray-based, comprehensive transcript profiling method. The expression of 62 genes was reduced by more than threefold; of these, 41 are involved in motility and chemotaxis. Moreover, the expression of 75 genes, 36 of which had been previously characterized, was elevated at least threefold. As expected, increased sdiA dosage led to significantly elevated sdiA and ddlB-ftsQAZ-lpxC operon expression. Transcription of two genes, uvrY and uvrC, located downstream of sdiA and oriented in the same direction, was elevated about 10-fold, although the intervening gene, yecF, of opposite polarity was unaffected by increased sdiA dosage. Three genes (mioC and gidAB) flanking the replication origin, oriC, were transcribed more often when sdiA dosage was high, as were 12 genes within 1 min of a terminus of replication, terB. Transcription of the acrABDEF genes, mapping in three widely spaced loci, was elevated significantly, while several genes involved in DNA repair and replication (e.g., nei, recN, mioC, and mcrC) were moderately elevated in expression. Such global analysis provides a link between septation and the response to DNA-damaging agents.

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 strains response to the other inhibitors was quite similar to that of the wild-type strain.

Combined, Functional Genomic-Biochemical Approach To Intermediary Metabolism: Interaction Of Acivicin, A Glutamine Amidotransferase Inhibitor, With Escherichia Coli K-12

Acivicin, a modified amino acid natural product, is a glutamine analog. Thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. This molecule prevented the growth of Escherichia coli in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the HisHF enzyme, a glutamine amidotransferase, was the target of acivicin action. This enzyme, purified from E. coli, was inhibited by low concentrations of acivicin. Acivicin inhibition was overcome by the presence of three distinct genetic regions when harbored on multicopy plasmids. Comprehensive transcript profiling using DNA microarrays indicated that histidine biosynthesis was the predominant process blocked by acivicin. The response to acivicin, however, was quite complex, suggesting that acivicin inhibition resonated through more than a single cellular process.

Interaction of lead nitrate and cadmium chloride with Escherichia coli K-12 and Salmonella typhimurium global regulatory mutants.

SummaryTo investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb2+ with inorganic phosphate. Using this medium, spontaneous mutants ofEscherichia coli resistant to addition of Pb(NO3)2 were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response ofE. coli andSalmonella typhimurium mutants altered in global regulatory networks to Pb(NO3)2) and CdCl2. Strains bearing mutations inoxyR andrpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperonedefective mutants suggests that these metals damage proteins in vivo.

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.

Transcript profiling of Escherichia coli using high-density DNA microarrays

Publisher Summary The chapter describes the techniques that use for comprehensive transcript profiling of Escherichia coli ( E. coli ) using high-density DNA microarrays. High-density DNA microarrays represent a powerful tool with which to simultaneously measure the expression of every gene in a cell. By identifying the genes that are expressed differentially in response to either specific mutations or modified environmental conditions, we are provided with a view of global responsiveness that improves our understanding of microbial physiology and homeostasis on a genomic scale. This technology complements other methods that measure the cellular content of polypeptides and small molecules. The Escherichia coli genome is composed of more than 4.5 megabase pairs of DNA and is predicted to encode 4290 proteins. Even though this organism has been studied extensively for over 50 years and its genome is completely sequenced, the function of about 40% of these genes remains unknown. High-density gene arrays are now providing important insights into gene function and microbial physiology and are fast becoming an essential tool to link information from genomics, bioinformatics, proteomics, metabolite analyses, and flux analyses into a cohesive “snapshot” of cellular metabolism.