LuxArray, a high-density, genomewide transcription analysis of Escherichia coli using bioluminescent reporter strains.

A sequenced collection of plasmid-borne random fusions of Escherichia coli DNA to a Photorhabdus luminescens luxCDABE reporter was used as a starting point to select a set of 689 nonredundant functional gene fusions. This group, called LuxArray 1.0, represented 27% of the predicted transcriptional units in E. coli. High-density printing of the LuxArray 1.0 reporter strains to membranes on agar plates was used for simultaneous reporter gene assays of gene expression. The cellular response to nalidixic acid perturbation was analyzed using this format. As expected, fusions to promoters of LexA-controlled SOS-responsive genes dinG, dinB, uvrA, and ydjM were found to be upregulated in the presence of nalidixic acid. In addition, six fusions to genes not previously known to be induced by nalidixic acid were also reproducibly upregulated. The responses of two of these, fusions to oraA and yigN, were induced in a LexA-dependent manner by both nalidixic acid and mitomycin C, identifying these as members of the LexA regulon. The responses of the other four were neither induced by mitomycin C nor dependent on lexA function. Thus, the promoters of ycgH, intG, rihC, and a putative operon consisting of lpxA, lpxB, rnhB, and dnaE were not generally DNA damage responsive and represent a more specific response to nalidixic acid. These results demonstrate that cellular arrays of reporter gene fusions are an important alternative to DNA arrays for genomewide transcriptional analyses.

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.

A genomic approach to gene fusion technology.

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

Cationic peptide antimicrobials induce selective transcription of micF and osmY in Escherichia coli.

Cationic antimicrobial peptides, such as polymyxin and cecropin, activated transcription of osmY and micF in growing Escherichia coli independently of each other. The micF response required the presence of a functional rob gene. It is intriguing that in this and other assays an identical response profile was also seen with hyperosmotic salt or sucrose gradient, two of the most commonly used traditional food preservatives. The osmY and micF transcription was not induced by hypoosmotic gradient, ionophoric peptides, uncouplers, or with other classes of membrane perturbing agents. The antibacterial peptides did not promote transcription of genes that respond to macromolecular or oxidative damage, fatty acid biosynthesis, heat shock, or depletion of proton or ion gradients. These and other results show that the antibacterial cationic peptides induce stasis in the early growth phase, and the transcriptional efficacy of antibacterial peptides correlates with their minimum inhibitory concentration, and also with their ability to mediate direct exchange of phospholipids between vesicles. The significance of these results is developed as the hypothesis that the cationic peptide antimicrobials stress growth of Gram-negative organisms by making contacts between the two phospholipid interfaces in the periplasmic space and prevent the hyperosmotic wrinkling of the cytoplasmic membrane. Broader significance of these results, and of the hypothesis that the peptide mediated contacts between the periplasmic phospholipid interfaces are the primary triggers, is discussed in relation to antibacterial resistance.

Quaternary ammonium functionalized poly(propylene imine) dendrimers as effective antimicrobials: structure-activity studies.

Quaternary ammonium functionalized poly(propyleneimine) dendrimers were synthesized and their antibacterial properties were evaluated using a bioluminescence method. These quaternary ammonium dendrimers are very potent biocides. The antibacterial properties depend on the size of the dendrimer, the length of hydrophobic chains in the quaternary ammonium groups, and the counteranion. Since these dendrimers are well characterized and monodisperse, they also serve as an effective system to study the structure-activity relationship. The antimicrobial properties of these dendrimer biocides have a parabolic dependence on molecular weight, which is different from the bell-shaped molecular weight dependence of conventional polymer biocides. The dependence on the hydrophobic chain of the quaternary ammonium structure is similar to conventional polymer biocides, and shows a parabolic relationship with dendrimer biocides carrying C10 hydrophobes the most potent. The antimicrobial properties of these novel biocides with bromide anions are more potent than those with chloride anions. Biocides derived from hyperbranched polymers were also synthesized and found to possess somewhat lower effectiveness.

Improved bacterial SOS promoter∷lux fusions for genotoxicity detection.

Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.

Cecropins induce the hyperosmotic stress response in Escherichia coli.

Cecropin A and B, below or near their minimum inhibitory concentrations in viable Escherichia coli, interfered with the rapid NaCl-induced hyperosmotic shrinkage of the cytoplasmic volume (plasmolysis), and also activated the promoter of the hyperosmotic stress gene osmY. The same promoter was also expressed by hyperosmolar NaCl or sucrose, two of the most commonly used antimicrobial food preservatives. Stress responses were monitored during the logarithmic growth phase of E. coli strains that contain specific promoters fused to a luxCDABE operon on a plasmid. The luminescence assay, developed to monitor the transcriptional response to stresses, is based on the premise that organisms often respond and adapt to sublethal environmental adversities by increased expression of stress proteins to restore homeostasis. The luminescence response from these fusion strains to a specific stress occurs as the transcription at the promoter site is activated. Cecropins induced luminescence response only from the osmY-luxCDABE fusion, but not the corresponding stress promoter activation associated with macromolecular or oxidative damage, or leakage of the cytoplasmic content including the proton gradient. The inhibitory effect of cecropins on plasmolysis is interpreted to suggest that the primary locus of action of these antimicrobial peptides in the periplasmic space is on the coupling between the inner and outer membrane.

Osmotic stress in viable Escherichia coli as the basis for the antibiotic response by polymyxin B.

Cationic antimicrobial peptides, such as polymyxin B (PxB), below growth inhibitory concentration induce expression of osmY gene in viable E. coli without leakage of solutes and protons. osmY expression is also a locus of hyperosmotic stress response induced by common food preservatives, such as hypertonic NaCl or sucrose. High selectivity of PxB against Gram-negative organisms and the basis for the hyperosmotic stress response at sublethal PxB concentrations is attributed to PxB-induced mixing of anionic phospholipid between the outer layer of the cytoplasmic membrane with phospholipids in the inner layer of the outer membrane. This explanation is supported by PxB-mediated rapid and direct exchange of anionic phospholipid between vesicles. This mechanism is consistent with the observation that genetically stable resistance against PxB could not be induced by mutagenesis.

Constricted flux through the branched-chain amino acid biosynthetic enzyme acetolactate synthase triggers elevated expression of genes regulated by rpoS and internal acidification.

The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM). To further understand the physiological consequences of flux alterations at this point in metabolism, Escherichia coli genes whose expression was induced by partial inhibition of ALS were sought. Plasmid-based fusions of random E. coli DNA fragments to Photorhabdus luminescens luxCDABE were screened for bioluminescent increases in actively growing liquid cultures slowed 25% by the addition of SM. From more than 8,000 transformants, 12 unique SM-inducible promoter-lux fusions were identified. The lux reporter genes were joined to seven uncharacterized open reading frames, f253a, f415, frvX, o513, o521, yciG, and yohF, and five known genes, inaA, IdcC, osmY, poxB, and sohA. Inactivation of the rpoS-encoded sigma factor, sigmaS, reduced basal expression levels of six of these fusions 10- to 200-fold. These six genes defined four new members of the sigmaS regulon, f253a, IdcC, yciG, and yohF, and included two known members, osmY and poxB. Furthermore, the weak acid salicylate, which causes cytoplasmic acidification, also induced increased bioluminescence from seven SM-inducible promoter-lux fusion-containing strains, namely, those with fusions of the sigmaS-controlled genes and inaA. The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the sigmaS-dependent stress response.

Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids.

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair.

Oxidative stress detection with Escherichia coli harboring a katG'::lux fusion.

A plasmid containing a transcriptional fusion of the Escherichia coli katG promoter to a truncated Vibrio fischeri lux operon (luxCDABE) was constructed. An E. coli strain bearing this plasmid (strain DPD2511) exhibited low basal levels of luminescence, which increased up to 1,000-fold in the presence of hydrogen peroxide, organic peroxides, redox-cycling agents (methyl viologen and menadione), a hydrogen peroxide-producing enzyme system (xanthine and xanthine oxidase), and cigarette smoke. An oxyR deletion abolished hydrogen peroxide-dependent induction, confirming that oxyR controlled katG'::lux luminescence. Light emission was also induced by ethanol by an unexplained mechanism. A marked synergistic response was observed when cells were exposed to both ethanol and hydrogen peroxide; the level of luminescence measured in the presence of both inducers was much higher than the sum of the level of luminescence observed with ethanol and the level of luminescence observed with hydrogen peroxide. It is suggested that this construction or similar constructions may be used as a tool for assaying oxidant and antioxidant properties of chemicals, as a biosensor for environmental monitoring and as a tool for studying cellular responses to oxidative hazards.

A miniature bioreactor for sensing toxicity using recombinant bioluminescent Escherichia coli cells.

A miniature bioreactor was fabricated as a contactor between biosensing cells and toxic materials. This miniature bioreactor (58 mL working volume) showed performance similar to that of a conventional bioreactor, as well as the advantages of easy installation, facile operation, and small medium requirements during long-term continuous operation. A performance evaluation measured the response to ethanol in continuous operation by using a recombinant bioluminescent Escherichia coli strain. Continuous cultures were repeatedly induced by the ethanol challenge. Steady-state cell concentrations (OD) were found to be decreased, the induced specific bioluminescence (SBL) peak value was found to be increased, and the peak response time, which is the time constant of this continuous monitoring system, was found to be decreased with increasing dilution rate. Finally on- and off-line bioluminescence monitoring was shown to be reliable, suggesting that this system is suitable for applications such as monitoring the influent and effluent streams of waste water biotreatment plants.

Characterization of the stress response of a bioluminescent biological sensor in batch and continuous cultures.

The effects of temperature, growth stage, and inducer (ethanol) concentration on the kinetics and magnitude of the stress response were investigated by using an Escherichia coli strain with the grpE heat shock promoter fused to the Vibrio fischeri lux genes. When stressed, the cells responded by changing the level of specific light emission, which was measured both on- and off-line. These measurements were used to characterize and optimize the sensitivity of the construct by determining the conditions at which the culture exhibited maximum specific bioluminescence and minimum response time to ethanol induction in batch cultivation. The results of the batch study were then applied to continuous cultivation, and the effect of dilution rate was determined. These results are of considerable interest in the development of an on-line biological sensor system for the detection and toxicity assessment of chemical pollutants.

Responses to toxicants of an Escherichia coli strain carrying a uspA'::lux genetic fusion and an E. coli strain carrying a grpE'::lux fusion are similar.

A transcriptional fusion of the Escherichia coli uspA promoter to luxCDABE was characterized and compared with a heat shock-responsive grpE'::lux fusion. Similarities in range and rank order of inducing conditions were observed; however, the magnitude of induction was typically greater for the grpE'::lux fusion strain.

Synergistic induction of the heat shock response in Escherichia coli by simultaneous treatment with chemical inducers.

Escherichia coli strains carrying transcriptional fusions of four sigma 32-controlled E. coli heat shock promoters to luxCDABE or lacZ reporter genes were stressed by chemicals added singly or in pairs. Much more than additive induction resulted from combinations of cadmium chloride, copper sulfate, ethanol, formamide, 4-nitrophenol, and pentachlorophenol.

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

To 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 of Escherichia 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 of E. coli and Salmonella typhimurium mutants altered in global regulatory networks to Pb(NO3)2 and CdCl2. Strains bearing mutations in oxyR and rpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitivity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperone-defective mutants suggests that these metals damage proteins in vivo.

Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions.

Heat shock gene expression is induced by a variety of environmental stresses, including the presence of many chemicals. To address the utility of this response for pollutant detection, two Escherichia coli heat shock promoters, dnaK and grpE, were fused to the lux genes of Vibrio fischeri. Metals, solvents, crop protection chemicals, and other organic molecules rapidly induced light production from E. coli strains containing these plasmid-borne fusions. Introduction of an outer membrane mutation, tolC, enhanced detection of a hydrophobic molecule, pentachlorophenol. The maximal response to pentachlorophenol in the tolC+ strain was at 38 ppm, while the maximal response in an otherwise isogenic tolC mutant was at 1.2 ppm. Stress responses were observed in both batch and chemostat cultures. It is suggested that biosensors constructed in this manner may have potential for environmental monitoring.