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R A LaRossa

Publications and source records attributed to R A LaRossa.

At least 19 recordsLinked to original sources
PubMed2001

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.

Artificial Gene Fusion↗
PubMed2001

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.

Aminohydrolases↗
PubMed2001

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.

Bacterial Proteins↗
PubMed2001

High-density microarray-mediated gene expression profiling of Escherichia coli.

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.

Amino Acids↗
PubMed2001

In vivo titration of mitomycin C action by four Escherichia coli genomic regions on multicopy plasmids.

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.

Antibiotics, Antineoplastic↗
PubMed2001

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.

Bacterial Proteins↗
PubMed2000

Bacterial bioluminescent emission from recombinant Escherichia coli harboring a recA::luxCDABE fusion.

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.

Acyltransferases↗
PubMed2000

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.

Alginates↗
PubMed2000

Detection of radiation effects using recombinant bioluminescent Escherichia coli strains.

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.

Bacterial Proteins↗
PubMed2000

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.

Anti-Bacterial Agents↗
PubMed1999

Distinct responses of a recA::luxCDABE Escherichia coli strain to direct and indirect DNA damaging agents.

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.

DNA Damage↗
PubMed1998

Branched-chain amino acid biosynthesis in Salmonella typhimurium: a quantitative analysis.

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.

Acetolactate Synthase↗
PubMed1997

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.

Adenosine Triphosphatases↗
PubMed1996

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.

Bacterial Proteins↗
PubMed1995

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.

Base Sequence↗
PubMed1994

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.

Bacterial Outer Membrane Proteins↗
PubMed1991

Physiological roles of the DnaK and GroE stress proteins: catalysts of protein folding or macromolecular sponges?

When organisms ranging from microbes to man are subjected to certain environmental stresses a characteristic 'heat shock' response is observed. In Escherichia coli this response is characterized by the induction of several proteins, three of which are the 70 kilodalton product of the dnaK gene, the 60 kilodalton product of the groEL (mopA) gene and the 15 kilodalton product of the groES (mopB) gene. In this review, utilizing enteric bacteria as model organisms, we focus on the role of these proteins within the context provided by well-established functions of other heat shock products. These facts serve as a starting point from which to speculate upon the in vivo role of these proteins during steady-state growth.

Bacterial Proteins↗