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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.

The expression of invasin, the product of the Yersinia pseudotuberculosis inv gene, allows enteric bacteria to enter cultured mammalian cells. The ability of invasin to bind animal cells and the potential significance of this interaction in the entry process were investigated. It was found that HEp-2 cells could attach to surfaces coated with bacterial membranes containing invasin. By fractionating bacterial membrane proteins on NaDodSO4/polyacrylamide gels and transferring the protein to filters, we demonstrated that the cell-binding component of the membranes comigrated with invasin. Mutations that changed the electrophoretic mobility of the protein also caused a corresponding shift in the migration of the cell-binding activity, showing that the comigrating protein was indeed invasin. Monoclonal antibodies directed against invasin that blocked invasin-HEp-2 cell interaction also inhibited bacteria from penetrating HEp-2 cells, indicating that interaction of this protein with animal cells is critical for cellular penetration.

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.

Bacterial strains harboring the Yersinia pseudotuberculosis inv locus were analyzed in order to investigate the mechanism of host cell penetration by an invasive pathogen. The inv locus was found to be necessary for Y. pseudotuberculosis to enter HEp-2 cells and sufficient to convert E. coli into a microorganism able to penetrate cultured cells. Both E. coli and Y. pseudotuberculosis strains harboring inv mutations were defective for entry into HEp-2 cells. Furthermore, molecular clones containing inv, and little additional DNA, converted E. coli into a microorganism that was indistinguishable from the parental Yersinia strain with regard to the entry of cultured cells. Data from in vitro protein synthesis indicated that a 103 kd protein was synthesized from inv, saturating the coding capacity of the locus. The nucleotide sequence shows an open reading frame corresponding to a protein of similar size. This protein, called invasin, is necessary for the microorganisms to penetrate HEp-2 cells, and is compartmentalized on the outer surface of the bacterium.

A normally stable gene of Escherichia coli was converted into a transposable element. A bacterial strain was constructed in which the malK gene was flanked on each side by the transposable element Tn5. The resulting Tn5-malK+-Tn5 structure (Tn651) became a transposable element with properties very similar to those of Tn5 itself. Tn651 transposes into regions of both the E. coli chromosome and bacteriophage lambda and is able to induce mutations. Transposition of Tn651 does not require the product of recA. Based on a physical analysis of lambda Tn651 DNA it is shown that the two Tn5s flanking the malK gene are in inverted orientation. In these experiments a new derivative of bacteriophage lambda is used that can accept a 14 kilobase insertion in vivo and still yield a plaque-forming transducing particle.

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.

Bacterial populations are often clonal, and even within a bacterial species, the frequency of gene exchange and recombination is quite low. Consequently, mobile genetic elements--plasmids, bacteriophages, and transposons--have been the central factors in the evolution of pathogenic traits. One central feature of pathogenicity, the capacity to enter epithelial cells, is encoded by the bacterial chromosome of Yersinia pseudotuberculosis but by plasmid genes in enteroinvasive Escherichia coli. A single Yersinia gene, inv, which encodes a single 107,000-dalton protein, can be cloned in E. coli K12, and its presence is sufficient to permit the bacteria to enter cultured human cells. In contrast, fully 70 kilobases of a virulence plasmid from enteroinvasive E. coli must be transferred to E. coli K12 to achieve the same result. While researchers are at the early stages of understanding microbial entry into host cells, they can now investigate the molecular basis of this event in greater detail than has previously been possible.

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.

Several transposable elements are able to promote replicon fusions in Escherichia coli . We show here that the transposon, Tn5, also has this property. Tn5 is able to promote fusions between the plasmid pBR322 (on which it resides) and bacteriophage λ at 2% of the frequency of Tn5 transposition onto λ. Plasmid/phage replicon fusions formed in strains that are totally deficient in homologous and integrative recombination always consist of direct duplications of the insertion element at the junctions between λ and pBR322. In order to define those regions within the Tn5 necessary for causing replicon fusions, a series of deletions of Tn5 generated in vitro and in vivo were constructed. Deletion derivatives that remove all but the 1500-base-pair right inverted repetition (stem) are competent to promote fusions. The left inverted repetition is unable to supply this activity, but can be complemented in trans to form replicon fusions. We conclude that both repeats have the sites necessary for replicon fusions, but only the right stem has the required protein(s). This establishes that the right repeat is an insertion sequence.

A series of translational fusions between the Yersinia pseudotuberculosis inv locus and lacZ was constructed. Each Lac+ fusion strain expressed a hybrid protein containing invasin, the product of the inv locus, at its amino-terminal end. Analysis of these gene fusions allowed determination of the direction of translation of the inv gene. Previous studies of Y. pseudotuberculosis invasion have shown that entry into animal cells is temperature regulated. It is shown here that control of expression of the inv gene is also temperature regulated. phoA gene fusions to inv, when present in Y. pseudotuberculosis, were expressed at lower levels when bacteria were grown at 37 degrees C rather than at 28 degrees C. Similar fusions, in contrast, were regulated in a temperature-independent fashion in Escherichia coli, as was the wild-type inv gene. This implies that Y. pseudotuberculosis has chromosomally encoded trans-acting functions that normally thermoregulate expression of inv.

Gas- and solid-phase infrared spectra and liquid- and solid-phase Raman spectra of cis-dimethyldiazene-d/sub 0/, -1,1,1-d/sub 3/, and -d/sub 6/(cis-CH/sub 3/N=NCH/sub 3/,cis-CD/sub 3/N=NCH/sub 3/, and cis-CD/sub 3/N=NCD/sub 3/) have been recorded. A virtually complete assignment of the vibrational fundamentals has been made for the set of molecules with frequencies being given for all but four of the 72 fundamentals. The vibrational spectra support C/sub 2//sub v/ symmetry for the d/sub 0/ and d/sub 6/ molecules but not distinguish between the two possible conformers of this symmetry. The frequencies have been computed at the zero-order level with a potential function transferred from trans-dimethyldiazene. These predicted frequencies aided the assignments for the cis molecules and provided a basis for analyzing some of the observed frequencies. The observed spectra contain a number of features which have been interpreted as evidence of crowding between the two methyl groups in cis-dimethyldiazene. The fundamentals for the undeuterated molecule are as follows, in cm/sup -1/: (a/sub 1/) 3006,2904,1561,1438,1370,1087,861,398; (a/sub 2/) 2960,1477,1170,463,242; (b/sub 1/) 3019,2920,1430,1359,1159,935,624; (b/sub 2/) 2960,1460,935,....

SummaryBacterial pathogens express virulence-specific transcriptional programs that allow tissue colonization. Although phenotypic variation has been noted in the context of antibiotic exposure, no direct evidence exists for heterogeneity in virulence-specific transcriptional programs within tissues. In a mouse model of Yersinia pseudotuberculosis infection, we show that at least three subpopulations of bacteria develop within a single tissue site in response to distinct host signals. Bacteria growing on the exterior of spleen microcolonies responded to soluble signals and induced the nitric oxide (NO)-detoxifying gene, hmp. Hmp effectively eliminated NO diffusion and protected the interior bacterial population from exposure to NO-derived inducing signals. A third subpopulation, constituting the most peripherally localized bacteria, directly contacted neutrophils and transcriptionally upregulated a virulence factor. These studies demonstrate that growth within tissues results in transcriptional specialization within a single focus of microbial replication, facilitating directed pathogen counterattack against the host response.

The dominant conceptual framework for understanding innate immunity has been that host cells respond to evolutionarily conserved molecular features of pathogens called pathogen-associated molecular patterns (PAMPs). Here, we propose that PAMPs should be understood in the context of how they are naturally presented by pathogens. This can be experimentally challenging, since pathogens, almost by definition, bypass host defense. Nevertheless, in this review, we explore the idea that the immune system responds to PAMPs in the context of additional signals that derive from common "patterns of pathogenesis" employed by pathogens to infect, multiply within, and spread among their hosts.

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.

The recent identification of E-cadherin as the cell-surface receptor for the Listeria monocytogenes adhesin, internalin, indicates that pathogenic bacteria exploit a variety of host surface molecules for entry and that they may share some common strategies for uptake into host cells.

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.

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 fractioned 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 time to a plasma dependent growth arrest point. Moreover, plasma controls the concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c-3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.

The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a “simple” transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.

Summary The entry of enteropathogenic Yersinia into cultured mammalian ceils has been studied in order to gain insight into the mechanism of bacterial penetration into host cells during infection. There exist at least three pathways for entry by Yersinia into mammalian cells, the most efficient of which is promoted by invasin, the product of the inv gene. Invasin is an outer membrane protein that attaches to a mammalian cell receptor, initiating the entry process. Several receptors that bind invasin have been identified, and each is a member of the VLA family of integrin cell adhesion molecules. The role of integrins in the entry process is discussed, as is the ability of invasin to stimulate uptake by binding to its integrin receptor.

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.

Yersinia pseudotuberculosis inv mutants were analyzed for their ability to bind and penetrate mammalian cell lines. Strains defective for the production of invasin and cured of the Yersinia virulence plasmid pIB1 were extremely defective for entry into the HEp-2 cell line. inv strains harboring the virulence plasmid partially overcame this defect, indicating that the virulence plasmid mediates an invasin-independent pathway for low-level entry into cultured cells. Plasmid-cured inv mutants were able to attach efficiently to mammalian cells after bacterial culture at 37 degrees C but not after culture at a lower temperature. The enhanced cellular binding of inv mutants grown at 37 degrees C did not result in efficient cellular penetration, indicating that invasin-mediated entry is the primary chromosomally encoded pathway responsible for Y. pseudotuberculosis penetration into both HEp-2 and Chinese hamster ovary cells under the assay conditions described here.

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.

The Legionella pneumophila Sde family of translocated proteins promote host tubular endoplasmic reticulum (ER) rearrangements that are tightly linked to phosphoribosyl-ubiquitin (pR-Ub) modification of Reticulon 4 (Rtn4). Sde proteins have two additional activities of unclear relevance to the infection process: K63 linkage-specific deubiquitination and phosphoribosyl modification of polyubiquitin (pR-Ub). We show here that the deubiquitination activity (DUB) stimulates ER rearrangements while pR-Ub protects the replication vacuole from cytosolic surveillance by autophagy. Loss of DUB activity was tightly linked to lowered pR-Ub modification of Rtn4, consistent with the DUB activity fueling the production of pR-Ub-Rtn4. In parallel, phosphoribosyl modification of polyUb, in a region of the protein known as the isoleucine patch, caused an absolute block in binding by the autophagy adapter p62. An inability of Sde mutants to modify polyUb resulted in immediate p62 association, a critical precursor to autophagic attack. The ability of Sde WT to block p62 association decayed quickly after bacterial infection, as predicted by the presence of previously characterized L. pneumophila effectors that inactivate Sde and remove polyUb. In sum, these results show that the accessory Sde activities act to stimulate ER rearrangements and protect from host innate immune sensing in a temporal fashion.