The plague is caused by the bacterium Yersinia pestis. Plague bacteria are thought to inject effector Yop proteins into host cells via the type III pathway. The identity of the host cells targeted for injection during plague infection is unknown. We found, using Yop β-lactamase hybrids and fluorescent staining of live cells from plague-infected animals, that Y. pestis selected immune cells for injection. In vivo, dendritic cells, macrophages, and neutrophils were injected most frequently, whereas B and T lymphocytes were rarely selected. Thus, it appears that Y. pestis disables these cell populations to annihilate host immune responses during plague.Yersinia pestis, the causative agent of plague or black death, harbors a virulence plasmid that encodes a type III secretion machine and its Yop protein substrates (1, 2). The essential contribution of type III secretion to the pathogenesis of plague was revealed by comparing lethal infectious doses of wild-type and mutant strains (3). Yersinia type III injection of Yop proteins into tissue culture cells has been detected with fluorescent microscopy, adenylate cyclase or Elk tag fusions, and fractionation techniques (4-7). These technologies, however, have not been useful for measuring the selection of host cells as targets of type III injection during infection.CCF2-AM, a β-lactamase substrate, has been used to detect bacterial type HI reporter injection into tissue culture cells(8). CCF2-AM is a membrane-permeant ester with two fluorophores attached to cephalosporin that exhibit fluorescence resonance energy transfer (FRET). Excitation of coumarin (409 nm) results in green fluorescence emission from fluorescein (520 nm) in intact CCF2-AM (9). β-Lactamase cleaves CCF2-AM, thereby disrupting FRET and establishing blue fluorescence emission. To investigate the usefulness of β-lactamase as a reporter for in vivo target cell selection by Y. pestis, we transformed plasmids carrying translational fusions between YopM or glutathione S-transferase (GST) and the mature domain of TEM-1 β-lactamase (YopM-Bla and GST-Bla, respectively) into Y. pestis strain KIM D27(10) Bacterial cultures were induced for type III secretion via the depletion of calcium at 37°C and then centrifuged to separate the extracellular medium from bacterial cells (11). Immunoblotting of cell-associated and secreted proteins identified YopM-Bla in the extracellular medium of KIM D27 cultures (Fig. 1A). In contrast, GST-Bla or RpoA, the α subunit of RNA polymerase, were not secreted. Disruption of yscU, which encodes a secretion machine component, abrogates all type III secretion (12).

Members of the rhizobia are distinguished for their ability to establish a nitrogen-fixing symbiosis with leguminous plants. While many details of this relationship remain a mystery, much effort has gone into elucidating the mechanisms governing bacterium-host recognition and the events leading to symbiosis. Several signal molecules, including plant-produced flavonoids and bacterially produced nodulation factors and exopolysaccharides, are known to function in the molecular conversation between the host and the symbiont. Work by several laboratories has shown that an additional mode of regulation, quorum sensing, intercedes in the signal exchange process and perhaps plays a major role in preparing and coordinating the nitrogen-fixing rhizobia during the establishment of the symbiosis. Rhizobium leguminosarum, for example, carries a multitiered quorum-sensing system that represents one of the most complex regulatory networks identified for this form of gene regulation. This review focuses on the recent stream of information regarding quorum sensing in the nitrogen-fixing rhizobia. Seminal work on the quorum-sensing systems of R. leguminosarum bv. viciae, R. etli, Rhizobium sp. strain NGR234, Sinorhizobium meliloti, and Bradyrhizobium japonicum is presented and discussed. The latest work shows that quorum sensing can be linked to various symbiotic phenomena including nodulation efficiency, symbiosome development, exopolysaccharide production, and nitrogen fixation, all of which are important for the establishment of a successful symbiosis. Many questions remain to be answered, but the knowledge obtained so far provides a firm foundation for future studies on the role of quorum-sensing mediated gene regulation in host-bacterium interactions

Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing symbiosis with the legume Medicago sativa. Recent work has identified a pair of genes, sinR and sinI, which represent a potential quorum-sensing system and are responsible for the production of N-acyl homoserine lactones (AHLs) in two S. meliloti strains, Rm1021 and Rm41. In this work, we characterize the sinRI locus and show that these genes are responsible for the synthesis of several long-chain AHLs ranging from 12 to 18 carbons in length. Four of these, 3-oxotetradecanoyl HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel structures. This is the first report of AHLs having acyl chains longer than 14 carbons. We show that a disruption in sinI eliminates these AHLs and that a sinR disruption results in only basal levels of the AHLs. Moreover, the same sinI and sinR mutations also lead to a decrease in the number of pink nodules during nodulation assays, as well as a slight delay in the appearance of pink nodules, indicating a role for quorum sensing in symbiosis. We also show that sinI and sinR mutants are still capable of producing several shortchain AHLs, one of which was identified as octanoyl HL. We believe that these short-chain AHLs are evidence of a second quorum-sensing system in Rm1021, which we refer to here as the mel system, for "S. meliloti."Quorum sensing is a widespread phenomenon among gramnegative bacteria (for recent reviews see references 9, 16, and 33). This form of cell density-dependent gene regulation is mediated by sensing the concentrations of low-molecularweight compounds called autoinducers, which are produced by the bacteria. A few organisms such as Photobacterium fischeri and Pseudomonas aeruginosa serve as model systems for quorum sensing. P. fischeri, in which autoinduction was first identified, has a relatively simple quorum-sensing system. An autoinducer synthase, LuxI, produces the N-(3-oxohexanoyl)-Lhomoserine lactone (oxo-C 6 -HL), which is recognized by the LuxR regulator (11,22,25). LuxR then induces expression of the lux operon, which causes bioluminescence (47,48). This model has recently become slightly more complicated with the identification of a second autoinducer synthase, AinS, and the regulatory protein LuxO, both of which serve to modulate quorum-sensing-induced luminescence in P. fischeri (25,26,34). On the other hand, P. aeruginosa has two quorum-sensing systems (lasR/lasI and rhlR/rhlI) organized into a complex hierarchy, which together regulate numerous genes required for virulence (2, 36).The most common and well-characterized gram-negative bacterial autoinducers are N-acyl homoserine lactones (AHLs) (see above reviews). AHLs consist of a variable acyl chain attached to a conserved homoserine lactone head group. The acyl chains can vary in length from 4 to 14 carbons. They can also vary in the nature of the substituent on the third carbon, from hydrogen to a hydroxyl or oxo group. One last characteristic that can confer variability is the presence of a do...

Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C 16:1 -HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.

Sinorhizobium meliloti is a free-living soil bacterium which is capable of establishing a symbiotic relationship with the alfalfa plant (Medicago sativa). This symbiosis involves a network of bacterium-host signaling, as well as the potential for bacterium-bacterium communication, such as quorum sensing. In this study, we characterized the production of N-acyl homoserine lactones (AHLs) by two commonly used S. meliloti strains, AK631 and Rm1021. We found that AK631 produces at least nine different AHLs, while Rm1021 produces only a subset of these molecules. To address the difference in AHL patterns between the strains, we developed a novel screening method to identify the genes affecting AHL synthesis. With this screening method, chromosomal groEL (groELc) was shown to be required for synthesis of the AHLs that are unique to AK631 but not for synthesis of the AHLs that are made by both AK631 and Rm1021. We then used the screening procedure to identify a mutation in a gene homologous to traM of Agrobacterium tumefaciens, which was able to suppress the phenotype of the groELc mutation. A traR homolog was identified immediately upstream of traM, and we propose that its gene product requires a functional groELc for activity and is also responsible for inducing the synthesis of the AHLs that are unique to AK631. We show that the traR/traM locus is part of a quorum-sensing system unique to AK631 and propose that this locus is involved in regulating conjugal plasmid transfer. We also present evidence for the existence of a second quorum-sensing system, sinR/sinI, which is present in both AK631 and Rm1021.Cell density-dependent gene expression, termed quorum sensing, is recognized as a widespread phenomenon in both gram-negative and gram-positive bacteria. The model organism for this form of gene regulation in gram-negative bacteria is Photobacterium fischeri, a luminescent marine bacterium. The process begins with the low-level production of an autoinducer signal at low cell densities, like those found in seawater. The autoinducers are thought to pass through the cell membrane by diffusion, so as the cell density increases during symbiotic association with the squid host, autoinducers accumulate in and around the cells (28). When a threshold level of autoinducers within the cell is reached, the LuxR regulator becomes activated by binding the autoinducer (23,28). LuxR then induces expression of the autoinducer synthase gene, luxI, along with the genes necessary for luminescence (8,9).In addition to P. fischeri, quorum sensing has been identified in numerous other organisms, including Agrobacterium tumefaciens, Pseudomonas aeruginosa, Erwinia carotovora, and several others (for recent reviews see references 5, 15, and 36). Most of the quorum-sensing systems characterized so far occur in bacteria that are able to establish relationships, either pathogenic or symbiotic, with plant or animal hosts. The quorum-sensing mechanism in these cases usually regulates one or more genes that play a role in pathogenesis or symbiosis....

Summary The pathogenic Yersinia species share a conserved type III secretion system, which delivers cytotoxic effectors known as Yops into target mammalian cells. In all three species, YopK (also called YopQ) plays an important role in regulating this process. In cell culture infections, yopK mutants inject higher levels of Yops, leading to increase cytotoxicity; however, in vivo the same mutants are highly attenuated. In this work, we investigate the mechanism behind this paradox. Using a β-lactamase reporter assay to directly measure the effect of YopK on translocation, we demonstrated that YopK controls the rate of Yop injection. Furthermore, we find that YopK cannot regulate effector Yop translocation from within the bacterial cytosol. YopE is also injected into host cells and was previously shown to contribute to regulation of the injectisome. In this work we show that YopK and YopE work at different steps to regulate Yop injection, with YopK functioning independently of YopE. Finally, by expressing YopK within tissue culture cells, we confirm that YopK regulates translocation from inside the host cell, and we show that cells pre-loaded with YopK are resistant to Yop injection. These results suggest a novel role for YopK in controlling the Yersinia type III secretion system.

In contrast to Yersinia pestis LcrV, the recombinant V10 (rV10) variant (lacking residues 271 to 300) does not suppress the release of proinflammatory cytokines by immune cells. Immunization with rV10 generates robust antibody responses that protect mice against bubonic plague and pneumonic plague, suggesting that rV10 may serve as an improved plague vaccine.

Yersinia species, as well as many other Gram-negative pathogens, use a type III secretion system (T3SS) to translocate effector proteins from the bacterial cytoplasm to the host cytosol. This T3SS resembles a molecular syringe, with a needle-like shaft connected to a basal body structure, which spans the inner and outer bacterial membranes. The basal body of the injectisome shares a high degree of homology with the bacterial flagellum. Extending from the T3SS basal body is the needle, which is a polymer of a single protein, YscF. The distal end of the needle serves as a platform for the assembly of a tip complex composed of LcrV. Though never directly observed, prevailing models assume that LcrV assists in the insertion of the pore-forming proteins YopB and YopD into the host cell membrane. This completes a bridge between the bacterium and host cell to provide a continuous channel through which effectors are delivered. Significant effort has gone into understanding how the T3SS is assembled, how its substrates are recognized and how substrate delivery is controlled. Arguably the latter topic is the least understood; however, recent advances have provided new insight, and therefore, this review will focus primarily on summarizing the current state of knowledge regarding the control of substrate delivery by the T3SS. Specifically, we will discuss the roles of YopK, as well as YopN and YopE, which have long been linked to regulation of translocation. We also propose models whereby the YopK regulator communicates with the basal body of the T3SS to control translocation.