Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 36
Filter
Add more filters










Publication year range
1.
J Bacteriol ; 183(15): 4562-70, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11443091

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , DNA-Binding Proteins , Escherichia coli Proteins , Escherichia coli/genetics , Hydrogen Peroxide/pharmacology , Repressor Proteins/metabolism , Trans-Activators , Transcription Factors/metabolism , Transcription, Genetic , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Base Sequence , DNA, Bacterial , Escherichia coli/drug effects , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Operon , Regulon , Repressor Proteins/genetics , Repressor Proteins/physiology , Transcription Factors/genetics , Transcription Factors/physiology
2.
J Bacteriol ; 183(11): 3353-64, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11344143

ABSTRACT

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.


Subject(s)
Anthranilate Synthase , Enzyme Inhibitors/pharmacology , Escherichia coli/metabolism , Isoxazoles/pharmacology , Nitrogenous Group Transferases/antagonists & inhibitors , Nitrogenous Group Transferases/genetics , Aminohydrolases/genetics , Aminohydrolases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding, Competitive , Culture Media , Enzyme Inhibitors/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/growth & development , Gene Expression Profiling/methods , Glutamine/metabolism , Imidazoles/metabolism , Isoxazoles/metabolism , Nitrogenous Group Transferases/metabolism , Ribonucleotides/metabolism , Transaminases/genetics , Transaminases/metabolism
3.
J Bacteriol ; 183(7): 2259-64, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11244065

ABSTRACT

Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinct E. coli genes (mdfA, gyrl, rob, and sdiA) on high-copy-number vectors. mdfA encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Escherichia coli Proteins , Escherichia coli/drug effects , Genome, Bacterial , Mitomycin/pharmacology , Plasmids , Bacterial Proteins/physiology , DNA Damage , Drug Resistance, Microbial , Escherichia coli/genetics , Gene Dosage , Serine Endopeptidases/physiology , Trans-Activators/genetics
4.
J Bacteriol ; 183(7): 2265-72, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11244066

ABSTRACT

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.


Subject(s)
Carrier Proteins , Cytoskeletal Proteins , Escherichia coli Proteins , Escherichia coli/genetics , Gene Amplification , Gene Expression Profiling , Trans-Activators/genetics , Bacterial Proteins/genetics , DNA Replication , Drug Resistance, Microbial , Escherichia coli/drug effects , Gene Dosage , Gene Expression Regulation, Bacterial , Lipoproteins , Membrane Proteins , Membrane Transport Proteins , Mitomycin/pharmacology , Multidrug Resistance-Associated Proteins , Operon , RNA, Messenger/analysis
5.
Proc Natl Acad Sci U S A ; 98(5): 2555-60, 2001 Feb 27.
Article in English | MEDLINE | ID: mdl-11226277

ABSTRACT

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.


Subject(s)
Artificial Gene Fusion , Escherichia coli/genetics , Genome, Bacterial , Genes, Reporter , Oligonucleotide Array Sequence Analysis , Photorhabdus/genetics , Plasmids , Promoter Regions, Genetic
6.
J Bacteriol ; 183(2): 545-56, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11133948

ABSTRACT

A nearly complete collection of 4,290 Escherichia coli open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from E. coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-beta-D-thiogalactopyranoside (IPTG) treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between E. coli cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species. Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of E. coli physiology.


Subject(s)
Escherichia coli/genetics , Gene Expression Profiling/methods , Oligonucleotide Array Sequence Analysis , Amino Acids/biosynthesis , Culture Media , Gene Expression Regulation, Bacterial , Genes, Bacterial , Isopropyl Thiogalactoside , Operon , RNA, Bacterial/analysis , RNA, Messenger/analysis , Ribosomal Proteins/genetics
7.
Microb Ecol ; 42(4): 635-643, 2001 Dec.
Article in English | MEDLINE | ID: mdl-12024246

ABSTRACT

The release of non-disinfected wastewater into the marine environment is a common practice in many countries; nevertheless, the molecular mechanisms involved in determining the survival of enteric bacteria in seawater are poorly understood, in spite of the obvious public health implications. In a methodological attempt to address this issue, a plasmid-based collection of 687 Escherichia coli distinct promoter::luxCDABE fusions was screened to identify promoters that are induced upon exposure to seawater. The luminescence driven by 22 out of these promoters reproducibly increased at least two-fold in an artificial seawater medium; only 9 of the corresponding genes have previously been assigned a function. The most prominent characteristic of the induced genes was that most (18 out of 22) were under rpoS control. The induction of these seawater-responsive promoters was evaluated in different media to identify the cause of the increased transcription. Salinity or osmolarity was instrumental in only four cases, and in three promoters, increased pH also seemed to play a role; however, the most significant environmental effector in inducing the majority of the seawater-induced promoters appeared to be nutrient limitation.

8.
Physiol Genomics ; 3(2): 83-92, 2000 Aug 09.
Article in English | MEDLINE | ID: mdl-11015603

ABSTRACT

The expression pattern of 1,529 yeast genes in response to sulfometuron methyl (SM) was analyzed by DNA microarray technology. SM, a potent herbicide, inhibits acetolactate synthase, a branched-chain amino acid biosynthetic enzyme. Exposure of yeast cells to 0.2 microg/ml SM resulted in 40% growth inhibition, a Gcn4p-mediated induction of genes involved in amino acid and cofactor biosynthesis, and starvation response. The accumulation of intermediates led to the induction of stress response genes and the repression of genes involved in carbohydrate metabolism, nucleotide biosynthesis, and sulfur assimilation. Extended exposure to SM led to a relaxation of the initial response and induction of sugar transporter and ergosterol biosynthetic genes, as well as repression of histone and lipid metabolic genes. Exposure to 5 microg/ml SM resulted in >98% growth inhibition and stimulated a similar initial expression change, but with no relaxation after extended exposure. Instead, more stress response and DNA damage repair genes become induced, suggesting a serious cellular consequence. Other salient features of metabolic regulation, such as the coordinated expression of cofactor biosynthetic genes with amino acid biosynthetic ones, were evident from our data. A potential link between SM sensitivity and ergosterol metabolism was uncovered by expression profiling and confirmed by genetic analysis.


Subject(s)
Amino Acids/biosynthesis , DNA-Binding Proteins , Gene Expression Profiling , Genes, Fungal/genetics , Herbicides/pharmacology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/drug effects , Sulfonylurea Compounds/pharmacology , Acetolactate Synthase/antagonists & inhibitors , Down-Regulation/drug effects , Down-Regulation/genetics , Ergosterol/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungal Proteins/physiology , Gene Deletion , Gene Expression Regulation, Fungal/drug effects , Genes, Fungal/physiology , Microbial Sensitivity Tests , Oligonucleotide Array Sequence Analysis , Open Reading Frames/genetics , Protein Kinases/genetics , Protein Kinases/physiology , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Up-Regulation/drug effects , Up-Regulation/genetics
9.
J Biochem Biophys Methods ; 45(1): 45-56, 2000 Aug 10.
Article in English | MEDLINE | ID: mdl-10899389

ABSTRACT

This paper describes the quantitative evaluation of a bioluminescence assay for DNA damaging agents with respect to the linearity, sensitivity, specificity and dependence on the cell culture status. A recombinant bacterium, DPD2794, harboring a plasmid with a recA promoter fused to the luxCDABE operon, showed a very sensitive response to DNA-damaging stress. DPD2794 was found to show no noticeable response to non-mutagenic agents, i.e. phenol, except for some false responses appearing soon after injection. DPD2794 also showed a highly sensitive response to Mitomycin C, which was found to be a growth-stage-dependent response, not a growth-rate-dependent response. In addition, the relationship between the bioluminescence emitted in vivo, luciferase activity measured in vitro, and the amount of Lux proteins expressed was determined. The intensity of the bioluminescence emitted was found to be proportional to the luciferase activity in vitro, while the bioluminescence also seems to be correlated with the level of Lux proteins expressed in these Escherichia coli cells, up to 230 min post induction.


Subject(s)
Acyltransferases , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Luminescent Measurements , Rec A Recombinases/metabolism , Recombinant Fusion Proteins/metabolism , Cell Culture Techniques , DNA Damage/drug effects , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Kinetics , Luciferases/metabolism , Mitomycin/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Phenol/pharmacology , Plasmids/metabolism , Promoter Regions, Genetic/drug effects , Rec A Recombinases/genetics , Time Factors , Transcription, Genetic/drug effects
10.
Radiat Environ Biophys ; 39(1): 41-5, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10789894

ABSTRACT

Effects of ionizing radiation (0.1-500 Gy) on recombinant Escherichia coli cells containing the stress promoters recA, grpE, or katG, fused to luxCDABE, were characterized by monitoring transcriptional responses reflected by the bioluminescent output. The minimum dose of gamma-irradiation detected by E. coli DPD2794 (recA::luxCDABE) was about 1.5 Gy, while the maximum response was obtained at 200 Gy. The amount of emitted bioluminescence increased proportionally with the gamma-ray doses which were found to elicit a DNA damage response in a range of 1-50 Gy. In addition, the cell growth rate was severely, but transiently, retarded by about 50 Gy. Quantification of the gamma-ray dose may be possible using the recA promoter fusion, since linear enhancement of the bioluminescence emission with increasing gamma-ray dose was observed. Other irradiated strains (50 Gy) responsive to either oxidative stress (DPD2511, katG::luxCDABE) or protein-damaging stress (TV1061, grpE::luxCDABE) did not display an increased bioluminescent output, while DPD2794 irradiated by the same dose of gamma-rays gave a significant bioluminescent output. This indicates that the recA promoter is the one most suitable for developing a biosensor for ionizing radiation.


Subject(s)
Escherichia coli Proteins , Escherichia coli/radiation effects , Bacterial Proteins/genetics , Dose-Response Relationship, Radiation , Escherichia coli/genetics , Escherichia coli/growth & development , Gamma Rays , Heat-Shock Proteins/genetics , Luminescent Measurements , Operon , Peroxidases/genetics , Rec A Recombinases/genetics , Recombination, Genetic
11.
Appl Biochem Biotechnol ; 89(2-3): 151-60, 2000.
Article in English | MEDLINE | ID: mdl-11209459

ABSTRACT

Escherichia coli strains containing plasmid-borne fusions of the recA promoter-operator region to the Vibrio fischeri lux genes were previously shown to increase their luminescence in the presence of DNA damage hazards, and thus to be useful for genotoxicant detection. The present study expands previous work by demonstrating and investigating the luminescent response of these strains to ultraviolet radiation. Several genetic variants of the basic recA'::lux design were examined, including a tolC modification of membrane efflux capacity, a chromosomal integration of the recA'::lux fusion, a different lux reporter (Photorhabdus luminescens instead of V. fischeri, allowing the assay to be run at 37 degrees C), and a different host bacterium (Salmonella typhimurium instead of E. coli). Generally, two modifications provided the fastest responses: the use of the S. typhimurium host or the P. luminescens lux reporter. Highest sensitivity, however, was demonstrated in an E. coli strain in which a single copy of the V. fischeri lux fusion was integrated into the bacterial chromosome.


Subject(s)
Bacterial Proteins/genetics , Biosensing Techniques/instrumentation , Rec A Recombinases/genetics , Transcription Factors/genetics , Ultraviolet Rays , Chromosomes/genetics , Chromosomes/radiation effects , DNA/radiation effects , DNA Damage/drug effects , DNA Damage/radiation effects , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Escherichia coli/genetics , Escherichia coli/radiation effects , Genes, Reporter , Kinetics , Mutation , Nalidixic Acid/pharmacology , Photorhabdus/genetics , Plasmids/metabolism , Salmonella typhimurium/metabolism , Temperature , Time Factors , Topoisomerase II Inhibitors , Vibrio/genetics
12.
Biomacromolecules ; 1(3): 473-80, 2000.
Article in English | MEDLINE | ID: mdl-11710139

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Polypropylenes/chemistry , Quaternary Ammonium Compounds/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Escherichia coli/drug effects , Microbial Sensitivity Tests , Molecular Weight , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/pharmacology , Structure-Activity Relationship
13.
Mutat Res ; 466(1): 97-107, 2000 Mar 03.
Article in English | MEDLINE | ID: mdl-10751731

ABSTRACT

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.


Subject(s)
Bacteria/genetics , Mutagenicity Tests/methods , Promoter Regions, Genetic/genetics , Repressor Proteins/genetics , SOS Response, Genetics/genetics , Trans-Activators/genetics , Alginates , Bacteria/drug effects , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Escherichia coli Proteins , Genes, Reporter/genetics , Glucuronic Acid , Hexuronic Acids , Hydrogen Peroxide/toxicity , Industrial Waste , Kinetics , Luminescent Measurements , Membrane Transport Proteins , Mutation , Photorhabdus/genetics , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Replicon , SOS Response, Genetics/drug effects , Salmonella typhimurium/genetics , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Species Specificity , Temperature , Time Factors , Vibrio/genetics , Water Pollutants/toxicity
14.
Mutat Res ; 442(2): 61-8, 1999 Jun 25.
Article in English | MEDLINE | ID: mdl-10393274

ABSTRACT

The recombinant Escherichia coli strain DPD2794 containing a recA::luxCDABE fusion is used to detect genotoxicity of various chemicals. Genotoxic agents were previously categorized into two groups, Direct DNA Damaging (DDD) agents and Indirect DNA Damaging (IDD) agents; these two groups have been distinguished with this strain. Minimum detectable concentrations of the DDD agents were about one to five orders of magnitude lower than those of the IDD agents. The response patterns of this strain to DDD agents differed from those to IDD agents in terms of kinetics and the forms of the dose-dependent response.


Subject(s)
DNA Damage , DNA, Bacterial/drug effects , Escherichia coli/drug effects , Mutagens/toxicity , Rec A Recombinases/genetics , Recombinant Fusion Proteins/genetics , Transcription Factors/genetics , Dose-Response Relationship, Drug , Escherichia coli/genetics , Luminescent Measurements , Mutagenicity Tests , SOS Response, Genetics/drug effects , SOS Response, Genetics/genetics , Vibrio/genetics
15.
J Bacteriol ; 180(16): 4056-67, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9696751

ABSTRACT

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.


Subject(s)
Amino Acids, Branched-Chain/biosynthesis , Salmonella typhimurium/metabolism , Acetolactate Synthase/antagonists & inhibitors , Acetolactate Synthase/metabolism , Amino Acids/metabolism , Amino Acids, Branched-Chain/analysis , Bacterial Proteins/metabolism , Cell Division , Enzyme Inhibitors/pharmacology , Enzymes/metabolism , Glyoxylates/pharmacology , Isoenzymes/metabolism , Keto Acids/metabolism , Salmonella typhimurium/chemistry , Salmonella typhimurium/enzymology
16.
J Bacteriol ; 180(4): 785-92, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9473030

ABSTRACT

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.


Subject(s)
Acetolactate Synthase/metabolism , Amino Acids, Branched-Chain/metabolism , Bacterial Proteins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Sigma Factor/metabolism , Acetolactate Synthase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Genes, Reporter , Isoleucine/metabolism , Luminescent Measurements , Promoter Regions, Genetic , Salicylates/pharmacology , Sulfonylurea Compounds/pharmacology , Valine/metabolism
17.
Appl Environ Microbiol ; 63(7): 2566-71, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9212407

ABSTRACT

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.


Subject(s)
DNA Damage/drug effects , DNA Damage/radiation effects , DNA Glycosylases , DNA, Bacterial/drug effects , DNA, Bacterial/radiation effects , Escherichia coli Proteins , Escherichia coli/genetics , Repressor Proteins , Trans-Activators , Adenosine Triphosphatases/genetics , Bacterial Proteins/genetics , Biosensing Techniques , DNA-Binding Proteins/genetics , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Lac Operon , Luminescence , Methylnitronitrosoguanidine/pharmacology , Mitomycins/pharmacology , Molecular Sequence Data , Mutagens/pharmacology , N-Glycosyl Hydrolases/genetics , Plasmids/genetics , Promoter Regions, Genetic , Rec A Recombinases/genetics , Recombination, Genetic , Ultraviolet Rays
18.
Appl Environ Microbiol ; 62(7): 2252-6, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8779563

ABSTRACT

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.


Subject(s)
Bacterial Proteins , Biosensing Techniques , Escherichia coli/genetics , Escherichia coli/metabolism , Oxidative Stress , Base Sequence , Cloning, Molecular , DNA Primers/genetics , Escherichia coli/drug effects , Genes, Bacterial , Hydrogen Peroxide/pharmacology , Luminescence , Molecular Sequence Data , Operon , Peroxidases/genetics , Plasmids/genetics , Promoter Regions, Genetic , Smoke , Vibrio/genetics
19.
Biotechnol Prog ; 12(3): 393-7, 1996.
Article in English | MEDLINE | ID: mdl-8652123

ABSTRACT

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.


Subject(s)
Biosensing Techniques , Biotechnology/methods , Escherichia coli/drug effects , Ethanol/toxicity , Luminescent Measurements , Escherichia coli/genetics , Recombination, Genetic , Reproducibility of Results
20.
Biotechnol Prog ; 12(3): 387-92, 1996.
Article in English | MEDLINE | ID: mdl-8652122

ABSTRACT

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.


Subject(s)
Biosensing Techniques , Biotechnology/methods , Heat-Shock Response , Luminescent Measurements , Escherichia coli/cytology , Escherichia coli/metabolism , Ethanol/pharmacology , Temperature
SELECTION OF CITATIONS
SEARCH DETAIL
...