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Intravacuolar bacterial pathogens establish intracellular niches by constructing membrane-encompassed compartments. The vacuoles surrounding the bacteria are remarkably stable, facilitating microbial replication and preventing exposure to host cytoplasmically-localized innate immune sensing mechanisms. To maintain integrity of the membrane compartment, the pathogen is armed with defensive weapons that prevent loss of vacuole integrity and potential exposure to host innate signaling. In some cases, the microbial components that maintain vacuolar integrity have been identified, but the basis for why the compartment degrades in their absence is unclear. In this review, we point out that lessons from the microbial-programmed degradation of the vacuole by the cytoplasmically-localized Shigella flexneri provide critical insights into how degradation of pathogen vacuoles occurs. We propose that in the absence of bacterial-encoded guard proteins, aberrant trafficking of host membrane-associated components results in a dysfunctional pathogen compartment. As a consequence, the vacuole is poisoned and replication is terminated.

Recent excitement regarding immune clearance of intracellular microorganisms has focused on two systems that maintain cellular homeostasis. One system includes cellular autophagy components that mediate degradation of pathogens in membrane-bound compartments, in a process termed xenophagy. The second system is driven by interferon– regulated GTPases that promote rupture of pathogen-containing vacuoles and microbial degradation. In the case of xenophagy, pathogen sequestration and compartmentalization suppress inflammation. In contrast, interferon-driven events can lead to exposure of pathogen-associated molecular patterns to the host cytosol with consequent inflammasome activation. Paradoxically, signals and factors involved in xenophagy also mobilize interferon-regulated GTPases, which drive the inflammatory response, indicating considerable crosstalk between these pathways. How these responses are prioritized remains to be understood. In this review, we describe mechanisms of intracellular pathogen clearance that rely on the autophagy machinery and interferon-regulated GTPases, and speculate how these pathways engage each other to balance pathogen elimination with inflammation.

Intracellular pathogens manipulate host organelles to support replication within cells. For Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. We show here that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as alterations in Rtn4 associated with the replication compartment. These rearrangements are triggered via Sde-promoted ubiquitin transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin transfer requires two sequential enzymatic activities from a single Sde polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4, or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. Therefore, a single bacterial protein drives a multistep biochemical pathway to control ubiquitination and tubular ER function independently of the host ubiquitin machinery.

Many intracellular bacterial pathogens reside within a membrane-bound compartment. The biogenesis of these vacuolar compartments is complex, involving subversion of host cell secretory pathways by bacterial proteins. In recent years it has become clear that disruption of vacuole biogenesis may result in membrane rupture and escape of bacteria into the host cell cytosol. Correct modulation of the host cell cytoskeleton, signalling molecules such as small GTPases and the lipids of the vacuole membrane have all been shown to be critical in the maintenance of vacuole integrity. Increasing evidence suggests that vacuole rupture may result from aberrant mechanical forces exerted on the vacuole, possibly due to a defect in vacuole expansion.

SummarySuccessful pathogens have evolved to evade innate immune recognition of microbial molecules by pattern recognition receptors (PRR), which control microbial growth in host tissues. Upon Legionella pneumophila infection of macrophages, the cytosolic PRR Nod1 recognizes anhydro-disaccharide-tetrapeptide (anhDSTP) generated by soluble lytic transglycosylase (SltL), the predominant bacterial peptidoglycan degrading enzyme, to activate NF-κB-dependent innate immune responses. We show that L. pneumophila periplasmic protein EnhC, which is uniquely required for bacterial replication within macrophages, interferes with SltL to lower anhDSTP production. L. pneumophila mutant strains lacking EnhC (ΔenhC) increase Nod1-dependent NF-κB activation in host cells, while reducing SltL activity in a ΔenhC strain restores intracellular bacterial growth. Further, L. pneumophila ΔenhC is specifically rescued in Nod1- but not Nod2-deficient macrophages, arguing that EnhC facilitates evasion from Nod1 recognition. These results indicate that a bacterial pathogen regulates peptidoglycan degradation to control the production of PRR ligands and evade innate immune recognition.

The hallmark of bubonic plague is the presence of grotesquely swollen lymph nodes, called buboes. This frenzied inflammatory response to Yersinia pestis is poorly understood. In this issue of Immunity, St. John et al. (2014) explore the mechanism by which Y. pestis spreads and thus leads to this striking lymphadenopathy.

The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors.

Rho family GTPases are important regulators of the actin cytoskeleton. Activation of these proteins can be promoted by guanine nucleotide exchange factors containing Dbl and Pleckstrin homology domains resulting in membrane insertion of a Rho family member, whereas the inactive GDP-bound form is sequestered primarily in the cytoplasm, bound to the guanosine dissociation inhibitor RhoGDI. Dominant interfering variants of Rac1, but not Cdc42, inhibit beta1 integrin-promoted uptake of Yersinia pseudotuberculosis. Unexpectedly, we found that the Rac1(W56F) guanine nucleotide exchange factors specificity switch mutant blocked invasin-promoted uptake as well as Cdc42-dependent uptake of enteropathogenic Escherichia coli. Fluorescence resonance energy transfer experiments demonstrated that Rac1(W56F) retained the ability to be loaded with GTP, bind a downstream effector, and interact with RhoGDI. Mutational analyses of intragenic suppressors and coexpression studies demonstrated that binding of the Rac1(W56F) mutant to RhoGDI appeared to play a role in the inhibition of uptake. As RhoGDI inhibits RhoA, overactivation of RhoA may account for the uptake interference caused by Rac1(W56F). Consistent with this model, a dominant interfering form of RhoA restored significant uptake in the presence of the Rac1(W56F) mutant but had no effect on another interfering Rac1 form. Furthermore, the cellular GTP-RhoA level was elevated by the presence of Rac1(W56F) mutant protein. These data are consistent with the proposition that Rac1(W56F) blocks invasin-promoted uptake by preventing RhoGDI from inactivating RhoA. We conclude that RhoGDI allows cross-talk between Rho family members that promote potentially antagonistic processes, and disruption of this cross-talk can interfere with invasin-promoted uptake.

The social amoeba Dictyostelium discoideum is increasingly being used as a simple model for the investigation of problems that are relevant to human health. This article focuses on several recent examples of Dictyostelium-based biomedical research, including the analysis of immune-cell disease and chemotaxis, centrosomal abnormalities and lissencephaly, bacterial intracellular pathogenesis, and mechanisms of neuroprotective and anti-cancer drug action. The combination of cellular, genetic and molecular biology techniques that are available in Dictyostelium often makes the analysis of these problems more amenable to study in this system than in mammalian cell culture. Findings that have been made in these areas using Dictyostelium have driven research in mammalian systems and have established Dictyostelium as a powerful model for human-disease analysis.

SummaryThe intracellular pathogen Legionella pneumophila replicates in a vacuole that recruits material from the host cell endoplasmic reticulum (ER). Biogenesis of this unique vacuole depends on the bacterial Dot/Icm type IV secretion system that translocates proteins across host cell membranes. Here, we show that two translocated substrates, SidM and LidA, target host cell Rab1, a small GTPase regulating ER-to-Golgi traffic. SidM is a guanosine nucleotide exchange factor for Rab1 that recruits Rab1 to Legionella-containing vacuoles, a process that is enhanced by LidA. Expression of sidM in mammalian cells interferes with the secretory pathway and causes Golgi fragmentation. Consistent with a collaborative relationship between the two proteins, immobilized SidM and LidA synergize to promote Rab1-dependent binding of early secretory vesicles. These results indicate that proteins translocated into the host cell by the intravacuolar pathogen L. pneumophila are able to recapitulate events involved in host secretory trafficking.

Mutations in bacterial pathogens have been isolated using many strategies. In contrast, the hosts they attack are significantly less tractable. To overcome this problem, a number of model host systems have been developed for isolation and investigation of mutations that modulate pathogen growth. These novel host models are either unicellular organisms, intact invertebrates or cells derived from invertebrates.

Enteropathogenic Yersinia species encode invasin, which promotes uptake into host cells by binding beta1 integrins. Invasin may cluster integrin heterodimers extracellularly and cause the integrin alpha and beta chains to splay apart in the cytoplasm. Cdc42 signaling is not essential for Yersinia uptake, whereas invasin crucially triggers Rac1-mediated signals that enable internalization. The signals linking invasin-mediated adhesion to Rac1 activation are not clear, but a novel kinase may release it from RhoGDI so that Rac1 can be activated, for example by Dock180. Rac1 may act via Arp2/3, phosphatidylinositol 4,5-bisphosphate and capping-proteins in the formation of nascent phagosomes during Yersinia uptake.

Legionella pneumophila proliferates within alveolar macrophages as a central property of Legionnaires' disease. Intracellular growth involves formation of a replicative phagosome, which requires the bacterial Dot/Icm system, a multiprotein secretion apparatus that translocates proteins from the bacterium across the macrophage plasma membrane. Two components of this system, IcmR and IcmQ, are proposed to exhibit a chaperone/substrate relationship similar to that observed in other protein translocation systems. We report here that IcmQ inserts into lipid membranes and forms pores that allow the efflux of the dye calcein but not Dextran 3000. Both membrane insertion and pore formation were inhibited by IcmR. Trypsin digestion mapping demonstrated that IcmQ is subdivided into two functional domains. The N-terminal region of IcmQ was necessary and sufficient for insertion into lipid membranes and calcein efflux. The C-terminal domain was necessary for efficient association of the protein with lipid bilayers. IcmR was found to bind to the N-terminal portion of the protein thus providing a mechanism for its ability to inhibit IcmQ pore-forming activity. Localization of IcmQ on the surface of the L. pneumophila shortly after infection as well as its pore-forming capacities suggest a role for IcmQ in forming a channel that leads translocated effectors out of the bacterium.

Most bacteria that colonize eukaryotes must bind directly to host cells to establish a replicative niche. In enteric bacteria, adhesion to host cells is often promoted by a lectin found on surface-localized pili. Some pili promote efficient adhesion only when they are subjected to shear stress, as found during the flow of blood over endothelium or mucous over the surface of the epithelium.

The invasin protein encoded by enteropathogenic Yersinia allows entry of bacteria into intestinal M cells by binding to integrin receptors. In cultured cells, invasin-mediated uptake requires proteins involved in endocytosis and signaling to the cell cytoskeleton. At least four different factors have been demonstrated to play a role in regulating the efficiency of invasin-promoted uptake. These include receptor-ligand affinity, receptor clustering, signaling through focal adhesion kinase, and stimulation of cytoskeletal rearrangements by small GTP binding proteins.

Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires' disease. L. pneumophila survives and replicates inside macrophages by preventing phagosome-lysosome fusion. A large number of L. pneumophila genes, called dot or icm, have been identified that are required for intracellular growth. It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway.

To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human alpha3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the alpha3 chain, alpha3-W220A, failed to promote adhesion to the natural alpha3 beta1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, alpha3-Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, alpha3-D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-alpha3 antibody, indicating that the second and third repeats of the alpha3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin alpha chain mutations, as alpha4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.

Des bacteries pathogenes ont adopte des strategies de survie chez leur hote vivant, en produisant des facteurs d'adhesion qui medient une association etroite avec les cellules hotes et leur internalisation. L'invasine, proteine multimerique de Y pseudotuberculosis , permet l'invasion de ce microorganisme, par l'intermediaire de sa liaison avec un sous-ensemble de recepteurs de l'integrine β1 de la cellule eucaryote. Nous presentons ici des modeles complementaires illustrant le mecanisme moleculaire et cellulaire de la penetration de Yersinia , mediee par l'invasine, dans les cellules de mammiferes.

The entry of Yersinia pseudotuberculosis into cultured mammalian cells is mediated by the bacterial protein invasin. The mammalian receptors for invasin are five β1 chain integrins. Site-directed mutagenesis of the aspartate and lysine residues in the 192-amino acid integrin binding domain of invasin was performed to identify regions, in addition to the previously characterized 903-913 region, that are important for integrin binding. One mutation, D811A, resulted in depressed ability of invasin to bind purified α5β1 and to promote bacterial entry. Further mutational analysis of Asp-811 indicated that an oxygen-containing side chain is required at this position. A second nearby residue, Phe-808, was also shown to be important for integrin binding, as an alanine substitution at this site had properties similar to the Asp-811 mutation. This mutational analysis has therefore identified a second region that, in conjunction with residues 903-913, is required for wild type levels of integrin binding. The contribution to binding by two noncontiguous sites in the primary sequence parallels results that indicate two domains of fibronectin are involved in integrin binding.

The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene.

Many pathogenic bacterial species produce factors that promote their internalization by host cells. The crucial components for uptake of one such pathogen, Yersinia pseudotuberculosis, have been identified. Efficient uptake of this microorganism requires tight binding of the bacterial invasin protein to integrins on the cell surface. Internalization also involves coordination of signals responsible for cytoskeletal rearrangements and those involved in receptor-mediated endocytosis. A start is being made to define the proteins that are required for efficient completion of the internalization process.

The Yersinia pseudotuberculosis invasin protein is a 986-amino acid protein that promotes bacterial penetration into mammalian cells by avidly binding multiple beta 1-chain integrins. A 192-amino acid carboxyl-terminal domain of invasin was previously shown to be sufficient for binding. Evidence is presented here that a 76-amino acid disulfide loop in the integrin binding domain of invasin is required for invasin-mediated cell binding and entry. Bacterial mutants that were altered at either of 2 cysteine residues in the binding domain of invasin were completely defective for entry. Purified invasin protein derivatives altered at either of these cysteines, in contrast to the wild-type invasin, did not promote either cell binding or penetration. Analysis of proteolytic products of invasin in the presence or absence of reducing agent provided evidence of an intra-chain disulfide bond near the carboxyl terminus of the protein. Alkylation of invasin derivatives with [3H]iodoacetate indicated that these 2 cysteines were normally disulfide-bonded. A treatment that resulted in the maximal reduction of the disulfide bond also resulted in maximal loss of cell attachment activity. These results indicate that the 76-amino acid disulfide loop at the carboxyl terminus of invasin is required for recognition by integrins.

Escherichia coli strains encoding the Yersinia pseudotuberculosis invasin protein are efficiently internalized by mammalian cells. Bacterial uptake into cultured cell lines became defective, however, if invasin was altered by fusion of its carboxyl terminus to E. coli alkaline phosphatase or by the addition of two hydrophobic amino acids to its carboxyl-terminal end. Probing with anti-invasin monoclonal antibodies revealed that the amino-terminal end of invasin was properly localized on the bacterial cell surface in strains encoding invasin with 2 additional amino acids, whereas the carboxyl terminus was not accessible to the monoclonal antibody. Therefore, the 2 additional amino acids interfered with the folding or orientation of the carboxyl terminus in the outer membrane. Alkylation experiments in the absence of reduction indicated that this defect was not caused by a gross inability to form a critical disulfide bond. Revertants were selected from a strain encoding this mutant protein by enriching for organisms able to enter cultured mammalian cells. The vast majority of revertants that were isolated following this enrichment contained a stop codon at the usual position found in the wild type inv gene. The most efficient of the remaining revertants resulted in the introduction of a glycine residue at the site of the wild type stop codon, presumably restoring proper conformation of the carboxyl-terminal region.

Binding to β1 chain integrin receptors can result in bacterial extracellular location or bacterial internalization In cultured cells. Using monoclonal antibodies (mAbs) to coat bacteria in vitro, we have shown that receptor-ligand affinity as well as receptor density determines the fate of the bacterium. In this report, we describe techniques used to coat the Gram-positive bacteria Staphylococcus aureus with anti-integrin mAbs and their application to the study of integrin-mediated bacterial internalization. The affinity of the purified anti-integrin mAb is estimated by determination of the ED50 of the mAb by using cell monolayers, and the efficiency of mAb-promoted bacterial internalization is determined by a gentamicin survival assay. The efficiency of bacterial internalization promoted by various anti-β1 chain integrin mAbs reflects differential expression of β1 chain integrin receptors in various cell lines studied. Analysis of β1 chain integrin receptor expression in HEp-2 cells transfectants shows that the level of bacterial internalization is directly related to the integrin receptor density.

The Yersinia pseudotuberculosis invasin protein promotes bacterial penetration into mammalian cells by binding to several beta 1 chain integrins. We show here that proteins containing the cell-binding domain of invasin bind to the fibronectin receptor alpha 5 beta 1 isolated from human placenta and immobilized on a filter membrane. Two forms of the receptor, each having a molecular weight of about 290,000, were immunodepleted by monoclonal antibodies specific for the beta 1 subunit or the alpha 5 beta 1 heterodimer. The binding of invasin to the receptor immobolized on the filter, or to whole JAR cells, reaches saturation after 90 min and has an apparent dissociation constant (Kd) of 5.0 x 10(-9) M. Invasin binding to alpha 5 beta 1 is inhibited by the 120-kDa chymotryptic fragment of fibronectin in a competitive manner with an inhibition constant (Ki) of 7.5 x 10(-7) M. Furthermore, invasin-receptor binding is also inhibited by the hexapeptide GRGDSP, and monoclonal antibodies that block cell attachment to invasin-coated surfaces also block cell attachment to fibronectin-coated surfaces. These results indicate that invasin and fibronectin bind to the same, or closely located sites on alpha 5 beta 1, although invasin binds with a much higher affinity than does fibronectin.

Mammalian cell receptors that promote entry of intracellular bacteria into nonphagocytic cells have not been identified. We show here that multiple members of the integrin superfamily of cell adhesion receptors bind the Y. pseudotuberculosis invasin protein prior to bacterial penetration into mammalian cells. Affinity chromatography of crude detergent extracts demonstrated that integrins containing the subunit structures alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 bound to immobilized invasin. Furthermore, phospholipid vesicles containing isolated integrin proteins were able to attach to invasin. Specificity for invasin binding to the identified integrin receptors was also demonstrated, as immunoprobing and phospholipid reconstitution studies showed that the alpha 2 beta 1 integrin, beta 2 chain integrins, and vitronectin receptor (alpha v beta 3) were not involved in cellular attachment to invasin.

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 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.

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.