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R R Isberg

Publications and source records attributed to R R Isberg.

85 records · Page 5Linked to original sources
PubMed1987

Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells.

Salmonella typhimurium, enteroinvasive Escherichia coli, Yersinia pseudotuberculosis, and Yersinia enterocolitica possess the ability to enter intestinal epithelial cells. We used a quantitative tissue culture model employing HEp-2 cells to compare the abilities of these bacteria to enter epithelial cells. S. typhimurium and Yersinia species were highly infective for HEp-2 cells but were unable to replicate extensively intracellularly. Enteroinvasive E. coli exhibited low infectivity but replicated extensively intracellularly. The growth of enteroinvasive E. coli led to destruction of the HEp-2 monolayer, whereas Yersinia spp. and S. typhimurium were maintained intracellularly for prolonged periods without damage to the monolayer. The ability of enteroinvasive E. coli to enter HEp-2 cells required prior growth at 37 degrees C; neither S. typhimurium nor Yersinia spp. exhibited this temperature dependence for cell entry. An E. coli K-12 derivative containing a 230-kilobase plasmid from enteroinvasive E. coli was constructed. This derivative shared all the invasive characteristics of the parental enteroinvasive strain, suggesting that determinants required for cell entry and intracellular multiplication were at least partially plasmid encoded. An HB101 derivative containing a cloned invasion determinant from Y. pseudotuberculosis was constructed in our laboratory. HEp-2 monolayers were coinfected with these two K-12 derivatives to compare invasion determinants from enteroinvasive E. coli with those of Y. pseudotuberculosis in a common genetic background. Results from these experiments suggest that these organisms reside within separate intracellular compartments.

Cells, Cultured↗
PubMed1985

Compartmentalization of the proteins encoded by IS50R.

IS50R is a transposable genetic element that serves as the right inverted repeat of the transposon Tn5. Earlier work has shown that IS50R encodes at least two proteins (called P1 and P2) involved in transposition. In this paper, we describe the localization properties of the proteins encoded on this repeat. Strains were constructed that overproduced either these two proteins or hybrids between beta-galactosidase and the IS50R proteins. An antiserum was raised against the hybrid proteins, and this was used to study the localization of P1 and P2. Based on studies in maxicells as well as in growing cells, we show that P1 and P2 are localized differently in the cell. P2 is a cytoplasmic protein, while P1 largely fractionates with the membrane.

Bacterial Proteins↗
PubMed1985

Tn5 transposes independently of cointegrate resolution. Evidence for an alternative model for transposition.

The replicon fusion model for transposition predicts that each transposable element encodes a site-specific recombination activity (resolvase). A series of experiments were performed to test for the presence of such a resolvase in Tn5. None could be found when assays were devised to detect inter- or intramolecular recombination. An experiment was performed to determine if expression of resolvase occurs simultaneously with the transposition process, thus explaining the undetectable levels of site-specific recombination. In this case, no resolvase activity could be detected in cells that had undergone transposition. Instead, rearrangements were found that could be explained best by an alternative model of transposition. The rearrangements were consistent with a pathway that invokes excising the transposon from its initial site of insertion, followed by circularization of the element before its eventual transposition. The excision event may occur either with or without DNA synthesis.

Ampicillin↗
PubMed1985

New gene in Escherichia coli K-12 (drpA): does its product play a role in RNA synthesis?

The mutation drpA1 defines a new gene in Escherichia coli K-12 that maps at about 5.2 min. This mutation was obtained after enriching a population of cells for temperature sensitive dna mutations with the [3H]thymidine "suicide" technique followed by screening for mutants defective in transposon Tn5 precise excision. When growing cells carrying the drpA1 allele were shifted to the nonpermissive temperature, we showed that DNA, RNA, and protein syntheses shut off quickly, with the cessation of RNA synthesis occurring first. A recombinant plasmid between pBR322 and an HindIII fragment from wild-type E. coli restores the growth defect in drpA1 mutants. Using transposon Tn5 mutagenesis of this plasmid, we have been able to correlate the presence of a 68-kilodalton protein, as observed with the maxicell technique, with the ability of this plasmid to restore growth to drpA1 mutants.

Bacterial Proteins↗
PubMed1982

DNA gyrase is a host factor required for transposition of Tn5.

We show that DNA gyrase is required for transposition of Tn5. Coumermycin, a potent inhibitor of DNA gyrase subunit B, inhibits transposition in a wild-type strain, but has no effect on strains carrying a coumermycin-resistant allele in gyrB. In addition, strains containing a thermolabile subunit A of gyrase (gyrA43) are defective for transposition at a nonpermissive temperature. The requirement for gyrase is due to a requirement for supercoiled DNA. We showed this by introducing into the gyrA43 strain a deletion of the gene encoding topoisomerase I. The introduction of the second mutation caused an increase in the superhelical density of DNA as well as an increase in the transposition frequency. This also implies that if the DNA is supercoiled there is no further requirement for gyrase. Experiments with coumermycin support this, because the drug does not inhibit transposition if the recipient DNA remains supercoiled. This indicates that if the DNA acting as recipient of the transposon is deficient in supercoils, it will be a poor substrate for transposition. We also describe a system in which a gene on a multicopy plasmid can be efficiently introduced into the Escherichia coli chromosome.

Aminocoumarins↗
PubMed1982

Regulation of Tn5 by the right-repeat proteins: control at the level of the transposition reaction?

The transposon Tn5 consists of inverted repeats, called IS50R and IS50L, each of which encode two proteins. We show here that the larger protein encoded on IS50R, protein 1, is absolutely required for transposition. Deletion or insertion mutants that fail to make this protein fail to promote gene movement. In addition, his protein acts in cis preferentially. We also show that the smaller protein encoded on IS50R, protein 2, is competent to inhibit transposition of a Tn5 freshly introduced into the cell on a lambda phage. In contrast, the proteins from IS50L possess neither of these two activities. By assaying expression of proteins that are hybrids between beta-galactosidase and IS50R proteins, we find that the regulation of transposition cannot be due to the inhibitor repressing synthesis of Tn5 proteins. Control experiments, in which we assay synthesis of IS50 proteins synthesized from a lambda::IS50R that has been infected into cells carrying the transposition inhibitor, confirm this conclusion.

Bacterial Proteins↗
PubMed1980

Recombination genes on the Escherichia coli sex factor specific for transposable elements.

The Escherichia coli sex factor stimulates precise excision of transposons Tn5 and Tn10 from sites either within the bacterial chromosome or within the factor itself. We have isolated two kinds of mutations that affect this activity. The ferA mutations eliminate the stimulation; the ferB mutations enhance it in the presence of FerA+. We conclude that ferA defines a sex factor gene that stimulates precise excision. The ferB mutations also specifically increase the rate of recombination between two IS3 elements on F' lac-pro (F'128) in a reaction that requires the product of recA. The stimulation of this recombination by ferB also requires an active ferA gene, which implies that the ferA gene stimulates this reaction as well as precise excision. A ferA mutation was mapped at 84.2 kilobases on the F factor, and a ferB mutation was mapped at 82.5 kilobases. The fer mutants were obtained by an approach that permits the isolation of mutants affecting precise excision.

Chromosome Mapping↗
PubMed1979

Control of the Balb/c-3T3 cell cycle by nutrients and serum factors: analysis using platelet-derived growth factor and platelet-poor plasma.

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractionated into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.

Animals↗
PubMedDate not supplied

A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12.

For many species of pathogenic bacteria, invasion and survival within animal cells is central to establishing a successful host-parasite relationship. Localization within host cells protects the microorganism from host defences, or permits it to cross epithelial barriers and subsequently become systemically distributed. The precise mechanisms that permit entry of bacteria into host tissues are unclear, therefore we have been studying the invasion of epithelial cells by Yersinia pseudotuberculosis. As a first step towards identifying the factors required for this process, we report here the identification of a single genetic locus from this organism that is sufficient to convert the innocuous Escherichia coli K-12 strain into an organism capable of invading cultured animal cells.

Carcinoma, Squamous Cell↗