Unexpected results from the application of signature-tagged mutagenesis to identify Yersinia pestis genes required for adherence and invasion.

The signature-tagged mutagenesis (STM) method was applied in a protocol designed to identify genes required for Yersinia pestis invasion into epithelial cells. A library of 3060 mutants of Y. pestis CO99-3015 was made and assayed using an in vitro invasion assay with gentamicin protection. Initial results from the screen identified a set of 23 genes that might be required for invasion; however, screening of individual mutants for decreased invasion, even in a competition assay with the parent strain, failed to reveal obvious invasion defects. Altered colony character or size might have imposed a growth disadvantage for two of the mutants, which could possibly account for apparently decreased invasion. The sensitivity of the mutants to gentamicin was assayed to determine if the presence of the kanamycin-resistance cassette in the STM transposon changed the gentamicin resistance of the individual mutants. It was discovered that the mutants exhibited a variable range of resistance to killing by gentamicin, suggesting that the presence of the kanamycin-resistance cassette or the particular insertion mutation did in many cases affect the bactericidal potency of gentamicin. However, all mutants remained highly sensitive to growth inhibition in a disk assay on plates. These results may warrant precautions for use of kanamycin-resistance markers in studies with fully virulent Y. pestis, since gentamicin has been recommended for treatment of plague. Further, to use STM in the context of invasion assays, a selection other than gentamicin should be applied.

Structure-function analysis of Yersinia pestis YopM's interaction with alpha-thrombin to rule on its significance in systemic plague and to model YopM's mechanism of binding host proteins.

The plague virulence protein YopM of Yersinia pestis KIM5 belongs to the large family of leucine-rich repeat (LRR) proteins. The only activity demonstrated so far for YopM is thrombin-binding, which could be a function of the small amount of YopM that is released into surrounding tissues by the bacteria. This study combined deletional and mutational analysis, chemical crosslinking assays, and in vitro functional tests with molecular modelling to identify key features of YopM necessary for interacting with thrombin. Two Y. pestis strains expressing YopM variants that differed in thrombin binding were used to assess the importance of thrombin-binding for lethality of plague. Both strains suffered a similar decrease in virulence by three orders of magnitude, indicating that thrombin-binding per se was not the major deficiency for lethality in the systemic disease model employed. It remains possible that extracellular YopM could contribute to plague pathology and to early events in peripheral tissues. The structural studies provided a model for how YopM may interact with thrombin and an insight into how YopM's LRR structure may assemble distinct regions for binding different targets.

Invasion of epithelial cells by Yersinia pestis: evidence for a Y. pestis-specific invasin.

The causative agent of plague, Yersinia pestis, is regarded as being noninvasive for epithelial cells and lacks the major adhesins and invasins of its enteropathogenic relatives Yersinia enterocolitica and Yersinia pseudotuberculosis. However, there are studies indicating that Y. pestis invades and causes systemic infection from ingestive and aerogenic routes of infection. Accordingly, we developed a gentamicin protection assay and reexamined invasiveness of Y. pestis for HeLa cells. By optimizing this assay, we discovered that Y. pestis is highly invasive. Several factors, including the presence of fetal bovine serum, the configuration of the tissue culture plate, the temperature at which the bacteria are grown, and the presence of the plasminogen activator protease Pla-encoding plasmid pPCP1, were found to influence invasiveness strongly. Suboptimal combinations of these factors may have contributed to negative findings by previous studies attempting to demonstrate invasion by Y. pestis. Invasion of HeLa cells was strongly inhibited by cytochalasin D and modestly inhibited by colchicine, indicating strong and modest respective requirements for microfilaments and microtubules. We found no significant effect of the iron status of yersiniae or of the pigmentation locus on invasion and likewise no significant effect of the Yops regulon. However, an unidentified thermally induced property (possibly the Y. pestis-specific capsular protein Caf1) did inhibit invasiveness significantly, and the plasmid pPCP1, unique to Y. pestis, was essential for highly efficient invasion. pPCP1 encodes an invasion-promoting factor and not just an adhesin, because Y. pestis lacking this plasmid still adhered to HeLa cells. These studies have enlarged our picture of Y. pestis biology and revealed the importance of properties that are unique to Y. pestis.

Virulence role of V antigen of Yersinia pestis at the bacterial surface.

Yersinia pestis, the etiologic agent of plague, secretes a set of environmentally regulated, plasmid pCD1-encoded virulence proteins termed Yops and V antigen (LcrV) by a type III secretion mechanism (Ysc). LcrV is a multifunctional protein that has been shown to act at the level of secretion control by binding the Ysc inner-gate protein LcrG and to modulate the host immune response by altering cytokine production. LcrV also is essential for the unidirectional targeting of Yops to the cytosol of infected eukaryotic cells. In this study, we constructed an in-frame deletion within lcrG (DeltalcrG3) to further analyze the requirement of LcrV in Yop targeting. We confirmed the essentiality of LcrV and found that LcrG may have a facilitative role, perhaps by promoting efficient secretion of LcrV. We also constructed mutants of lcrV expressing LcrV truncated at the N or C terminus. Both the N and C termini of LcrV were required for the secretion of LcrV into the medium and targeting of Yops. LcrV was detected in punctate zones on the surface of fixed Y. pestis by laser-scanning confocal microscopy, and this localization required a functional Ysc. However, the truncated LcrV proteins were not found on the bacterial surface. Finally, we tested the ability of LcrV-specific Fab antibody fragments or full-length antibody to interfere with Yop targeting and found no interference, even though this antibody protects mice against plague. These results indicate that LcrV may function in Yop targeting at the extracellular surface of yersiniae and that the protective efficacy of LcrV-specific antibodies can be manifested without blocking Yop targeting.

LcrV of Yersinia pestis enters infected eukaryotic cells by a virulence plasmid-independent mechanism.

Yersinia pestis is the causative agent of bubonic plague and possesses a set of plasmid-encoded, secretable virulence proteins termed LcrV and Yops which are essential for survival in mammalian hosts. Yops and LcrV are secreted by a type III mechanism (Ysc), and Yops are unidirectionally targeted into the cytosol of associated eukaryotic cells in a tissue culture infection model. LcrV is required for Yops targeting, and recent findings have revealed that it can localize to the bacterial surface; however, its fate in this infection model has not been investigated in detail. In this study, we compared the localization of LcrV to that of the targeted proteins YopE and YopM by immunoblot analysis of fractions of Yersinia-infected HeLa cultures or by laser-scanning confocal microscopy of infected monolayers. Both LcrV and YopE were secreted by contact-activated, extracellularly localized yersiniae and were targeted to the HeLa cell cytosol. Although a significant amount of LcrV partitioned to the culture medium (unlike YopE), this extracellular pool of LcrV was not the source of the LcrV that entered HeLa cells. Unlike targeting of YopE and YopM, targeting of LcrV occurred in the absence of a functional Ysc apparatus and other virulence plasmid (pCD1)-expressed proteins. However, the Ysc is necessary for LcrV to be released into the medium, and our recent work has shown that localization of LcrV on the bacterial surface requires the Ysc. These results indicate that two mechanisms exist for the secretion of LcrV by Y. pestis, both of which are activated by contact with eukaryotic cells. LcrV secreted by the Ysc reaches the bacterial surface and the surrounding medium, whereas the second is a novel, Ysc-independent pathway which results in localization of LcrV in the cytosol of infected cells but not the surrounding medium.

YscP of Yersinia pestis is a secreted component of the Yop secretion system.

The Yersinia pestis low-Ca2+ response stimulon is responsible for the environmentally regulated expression and secretion of antihost proteins (V antigen and Yops). We have previously shown that yscO encodes a secreted core component of the Yop secretion (Ysc) mechanism. In this study, we constructed and characterized in-frame deletions in the adjacent gene, yscP, in the yscN-yscU operon. The DeltaP1 mutation, which removed amino acids 246 to 333 of YscP, had no effect on Yop expression or secretion, and the mutant protein, YscP1, was secreted, as was YscP in the parent. In contrast, the DeltaP2 strain expressed and secreted less of each Yop than did the parent under the inductive conditions of 37 degrees C and the absence of Ca2+, with an exception being YopE, which was only minimally affected by the mutation. The YscP2 protein, missing amino acids 57 to 324 of YscP, was expressed but not secreted by the DeltaP2 mutant. The effect of the DeltaP2 mutation was at the level of Yop secretion because YopM and V antigen still showed limited secretion when overproduced in trans. Excess YscP also affected secretion: overexpression of YscP in the parent, in either yscP mutant, or in an lcrG mutant effectively shut off secretion. However, co-overexpression of YscO and YscP had no effect on secretion, and YscP overexpression in an lcrE mutant had little effect on Yop secretion, suggesting that YscP acts, in conjunction with YscO, at the level of secretion control of LcrE at the bacterial surface. These findings place YscP among the growing family of mobile Ysc components that both affect secretion and themselves are secreted by the Ysc.

DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5.

The low-Ca2+-response (LCR) plasmid pCD1 of the plague agent Yersinia pestis KIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenic Yersinia spp., that had been identified previously in one or more of Y. pestis or the enteropathogenic yersiniae Yersinia enterocolitica and Yersinia pseudotuberculosis. Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS100 and two new IS elements, IS1616 and IS1617) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case for Y. pestis EV76, and the gene for the lipoprotein YlpA found in Y. enterocolitica likely is a pseudogene in Y. pestis. The yopM gene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream of ypkA-yopJ is discussed as a potential Syc gene, and a previously undescribed ORF downstream of yopE was identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.

YscO of Yersinia pestis is a mobile core component of the Yop secretion system.

The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca2+-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degreesC in the absence of Ca2+. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37 degreesC in the presence of Ca2+. In the absence of Ca2+, the mutant grew independently of Ca2+, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37 degreesC irrespective of the presence of Ca2+ and in the culture medium in the absence of Ca2+. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.

The V antigen of Yersinia pestis regulates Yop vectorial targeting as well as Yop secretion through effects on YopB and LcrG.

Yersinia pestis expresses a set of secreted proteins called Yops and the bifunctional LcrV, which has both regulatory and antihost functions. Yops and LcrV expression and the activity of the type III mechanism for their secretion are coordinately regulated by environmental signals such as Ca2+ concentration and eukaryotic cell contact. In vitro, Yops and LcrV are secreted into the culture medium in the absence of Ca2+ as part of the low-Ca2+ response (LCR). The LCR is induced in a tissue culture model by contact with eukaryotic cells that results in Yop translocation into cells and subsequent cytotoxicity. The secretion mechanism is believed to indirectly regulate expression of lcrV and yop operons by controlling the intracellular concentration of a secreted negative regulator. LcrG, a secretion-regulatory protein, is thought to block secretion of Yops and LcrV, possibly at the inner face of the inner membrane. A recent model proposes that when the LCR is induced, the increased expression of LcrV yields an excess of LcrV relative to LcrG, and this is sufficient for LcrV to bind LcrG and unblock secretion. To test this LcrG titration model, LcrG and LcrV were expressed alone or together in a newly constructed lcrG deletion strain, a delta lcrG2 mutant, of Y. pestis that produces low levels of LcrV and constitutively expresses and secretes Yops. Overexpression of LcrG in this mutant background was able to block secretion and depress expression of Yops in the presence of Ca2+ and to dramatically decrease Yop expression and secretion in growth medium lacking Ca2+. Overexpression of both LcrG and LcrV in the delta lcrG2 strain restored wild-type levels of Yop expression and Ca2+ control of Yop secretion. Surprisingly, when HeLa cells were infected with the delta lcrG2 strain, no cytotoxicity was apparent and translocation of Yops was abolished. This correlated with an altered distribution of YopB as measured by accessibility to trypsin. These effects were not due to the absence of LcrG, because they were alleviated by restoration of LcrV expression and secretion alone. LcrV itself was found to enter HeLa cells in a nonpolarized manner. These studies supported the LcrG titration model of LcrV's regulatory effect at the level of Yop secretion and revealed a further role of LcrV in the deployment of YopB, which in turn is essential for the vectorial translocation of Yops into eukaryotic cells.

YopD of Yersinia pestis plays a role in negative regulation of the low-calcium response in addition to its role in translocation of Yops.

Yersinia pestis produces a set of virulence proteins (Yops and LcrV) that are expressed at high levels and secreted by a type III secretion system (Ysc) upon bacterium-host cell contact, and four of the Yops are vectorially translocated into eukaryotic cells. YopD, YopB, and YopK are required for the translocation process. In vitro, induction and secretion occur at 37 degrees C in the absence of calcium. LcrH (also called SycD), a protein required for the stability and secretion of YopD, had initially been identified as a negative regulator of Yop expression. In this study, we constructed a yopD mutation in both wild-type and secretion-defective (ysc) Y. pestis to determine if the lcrH phenotype could be attributed to the decreased stability of YopD. These mutants were constitutively induced for expression of Yops and LcrV, despite the presence of the secreted negative regulator LcrQ, demonstrating that YopD is involved in negative regulation, regardless of a functioning Ysc system. Normally, secretion of Yops and LcrV is blocked in the presence of calcium. The single yopD mutant was not completely effective in blocking secretion: LcrV was secreted equally well in the presence and absence of calcium, while there was partial secretion of Yops in the presence of calcium. YopD is probably not rate limiting for negative regulation, as increasing levels of YopD did not result in decreased Yop expression. Overexpression of LcrQ in the yopD mutant had no significant effect on Yop expression, whereas increased levels of LcrQ in the parent resulted in decreased levels of Yops. These results indicate that LcrQ requires YopD to function as a negative regulator.

Targeting of the Yersinia pestis YopM protein into HeLa cells and intracellular trafficking to the nucleus.

The YopM virulence protein of Yersinia pestis has been described as binding human alpha-thrombin and inhibiting thrombin-induced platelet aggregation in vitro. However, recent studies have shown that a YopM-CyaA fusion protein could be targeted vectorially into eukaryotic cells through the Yersinia type III secretion system. In this study, our objective was to characterize YopM's fate in more detail. We followed YopM in the culture medium and inside infected HeLa cells. We confirmed that the native YopM is targeted into HeLa cells, where it is insensitive to exogenous trypsin. The bacteria must be surface located to target YopM, and YopB and YopD are necessary, whereas the LcrE protein (called also YopN) makes this process more efficient. Immunofluorescence localization revealed that YopM, in contrast to YopE, is not only targeted to the cytoplasm but also trafficks to the cell's nucleus by means of a vesicle-associated pathway that is strongly inhibited by brefeldin A, perturbed by monensin or bafilomycin A1 and dependent upon microtubules (decreased by colchicine and nocodazole). These findings revealed a novel interaction of Yersinia pestis with its eukaryotic host.

Failure to detect binding of LcrH to the V antigen of Yersinia pestis.

V antigen of Yersinia pestis has been reported to bind the chaperone LcrH. We were unable to demonstrate this interaction. Despite methodological differences between our study and an earlier study, we believe that the previous findings were artifactual. One likely confounding element was the tendency of LcrH to adhere on its own to metal chelation chromatographic resin.

Effect of Yersinia pestis YopM on experimental plague.

YopM of Yersinia pestis has previously been shown to be necessary for full virulence in mice and to be able to bind human alpha-thrombin. This activity prompted the hypothesis that YopM, functioning extracellularly during plague, might be accessible to neutralization by antibody and hence might be a protective antigen. This study tested this hypothesis and found that YopM was not protective, either by passive or active immunization, in inbred or outbred mice. These findings showed that either YopM-specific antibody does not have access to YopM during experimental plague or the function of extracellular YopM is not neutralizable by antibody. Exogenously supplied YopM partially restored virulence to a YopM- strain of Y. pestis while having no effect on lethality of Listeria monocytogenes. These findings indicate that YopM does not significantly alter host defenses important for resistance against heterologous infection (Listeria monocytogenes) but raise the possibility that YopM has a minor extracellular function specific to homologous infection (Y. pestis).

Yersinia pestis LcrV forms a stable complex with LcrG and may have a secretion-related regulatory role in the low-Ca2+ response.

Yersinia pestis contains a virulence plasmid, pCD1, that encodes many virulence-associated traits, such as the Yops (Yersinia outer proteins) and the bifunctional LcrV, which has both regulatory and antihost functions. In addition to LcrV and the Yops, pCD1 encodes a type III secretion system that is responsible for Yop and LcrV secretion. The Yop-LcrV secretion mechanism is believed to regulate transcription of lcrV and yop operons indirectly by controlling the intracellular concentration of a secreted repressor. The activity of the secretion mechanism and consequently the expression of LcrV and Yops are negatively regulated in response to environmental conditions such as Ca2+ concentration by LcrE and, additionally, by LcrG, both of which have been proposed to block the secretion mechanism. This block is removed by the absence of Ca2+ or by contact with eukaryotic cells, and some Yops are then translocated into the cells. Regulation of LcrV and Yop expression also is positively affected by LcrV. Previously, LcrG was shown to be secreted from bacterial cells when the growth medium lacks added Ca2+, although most of the LcrG remains cell associated. In the present study, we showed that the cell-associated LcrG is cytoplasmically localized. We demonstrated that LcrG interacts with LcrV to form a heterodimeric complex by using chemical cross-linking and copurification of LcrG and LcrV. Additionally, we found that small amounts of LcrV and YopE can be detected in periplasmic fractions isolated by cold osmotic shock and spheroplast formation, indicating that their secretion pathway is accessible to the periplasm or to these procedures for obtaining periplasmic fractions. We propose that the cytoplasmically localized LcrG blocks the Yop secretion apparatus from the cytoplasmic side and that LcrV is required to remove the LcrG secretion block to yield full induction of Yop and LcrV secretion and expression.

Interaction between Yersinia pestis YopM protein and human alpha-thrombin.

YopM, a 41.5 kDa virulence protein of Yersinia pestis is believed to have an anti-inflammatory role in bubonic plague. It has been shown previously that YopM binds human alpha-thrombin but not prothrombin and inhibits thrombin-induced platelet aggregation in vitro. In the present studies we carried out crosslinking reactions between purified YopM and alpha-thrombin or its blocked form FPR-alpha-thrombin in the presence of various competitors to identify where on thrombin YopM binds. We found that thrombin cleaves YopM at the C-terminus, indicating that this part of YopM must interact with thrombin's catalytic site. Hirudin, a 65 amino acid natural thrombin inhibitor, prevents both the YopM degradation and the formation of a ca. 75 kDa crosslinking complex between YopM and alpha-thrombin. A similar effect is observed when hirugen, a short peptide corresponding to hirudin's C-terminus (amino acids 58-64), is used as a synthetic thrombin inhibitor. A 15 bp long specific oligonucleotide known to block alpha-thrombin successfully competes with YopM for the thrombin-binding site, whereas a control, scrambled sequence aptamer does not. As these competitors block a fibrinogen binding site (also called anion binding exosite I), our crosslinking data indicate that YopM binds not only to the active site of alpha-thrombin but also to the abeI.

Environmental modulation of gene expression and pathogenesis in Yersinia.

The yersiniae are a useful model for understanding how environmental modulation of gene expression allows pathogens to inhabit a wide range of niches. This review follows the enteropathogenic yersiniae, Yersinia enterocolitica and Yersinia pseudotuberculosis, and the agent of plague, Yersinia pestis, through their life cycles, describing how adaptive gene expression may promote successful pathogenesis.

Mutations in yscC, yscD, and yscG prevent high-level expression and secretion of V antigen and Yops in Yersinia pestis.

The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca(2+)-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degrees C in the absence of Ca2+. In this study, we constructed and characterized mutants with in-frame deletions in yscC, yscD, and yscG of the ysc operon that contains yscA through yscM. All three mutants lost the Ca2+ requirement for growth at 37 degrees c, expressed only basal levels of V antigen and YopM in the presence or absence of Ca2+, and failed to secrete these proteins to the culture supernatant. Overproduction of YopM in these mutants failed to restore YopM export, showing that the mutations had a direct effect on secretion. The protein products of yscC, yscD, and yscG were identified and localized by immunoblot analysis. YscC was localized to the outer membrane of Y. pestis, while YscD was found in the inner membrane. YscG was distributed equally between the soluble and total membrane fractions. Double mutants were characterized to assess where YscC and YscD act in low-Ca2+ response (LCR) regulation. lcrH::cat-yscC and lcrH::cat-yscD double mutants were constitutively induced for expression of V antigen and YopM; however, these proteins were not exported. This finding showed that the ysc mutations did not directly decrease induction of LCR stimulon genes. In contrast, lcrE-yscC, lcrG-yscC, lcrE-yscD, and lcrG-yscD double mutants as well as an lcrE-lcrD double mutant expressed only basal levels of V antigen and YopM and also failed to secrete these proteins to the culture supernatant. These results indicated that a functional LCR secretion system was necessary for high-level expression of LCR stimulon proteins in the lcrE and lcrG mutants but not in an lcrH::cat mutant. Possible models of regulation which incorporate these results are discussed.

Differential effects of deletions in lcrV on secretion of V antigen, regulation of the low-Ca2+ response, and virulence of Yersinia pestis.

The Yersinia pestis V antigen is necessary for full induction of low-calcium response (LCR) stimulon virulence gene transcription, and it also is a secreted protein believed to have a direct antihost function. We made four nonpolar deletions in lcrV of Y. pestis to determine if secretion, regulation, and virulence functions could be localized within the V antigen (LcrV). Deletion of amino acids 25 to 40 caused secretion of LcrV to be decreased in efficiency; however, removal of residues 108 to 125 essentially abolished LcrV secretion. Neither mutation had a significant effect on LCR regulation. This showed that LcrV does not have to be secreted to have its regulatory effect and that the internal structure of V antigen is necessary for its secretion. Both mutants were avirulent in mice, showing that the regulatory effect of LcrV could be separated genetically from its virulence role and raising the possibility that residues 25 to 40 are essential for the virulence function. This study provides the best genetic evidence available that LcrV per se is necessary for the virulence of Y. pestis. The repressed LCR phenotype of a mutant lacking amino acids 188 to 207 of LcrV raised the possibility that the deleted region is necessary for regulation of LCR induction; however, this mutant LcrV was weakly expressed and may not have been present in sufficient amounts to have its regulatory effect. In double mutants containing this mutant lcrV and also lacking expression of known LCR negative regulators (LcrG, LcrE, and LcrH), full induction of the LCR occurred in the absence of functional LcrV, indicating that LcrV promotes induction not as an activator per se but rather by inhibiting negative regulators.

A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis.

The causative agent of plague, Yersinia pestis, contains a 75-kb plasmid, pCD1, which carries a virulence-related stimulon called the low-Ca2+ response stimulon (LCRS). LCRS operons are regulated by the environmental signals of temperature and Ca2+. This study characterized a portion of the lcrB region of pCD1, known to contain at least one gene necessary for the regulation of LCRS operons by Ca2+. The sequence of a 2-kb region revealed three open reading frames, designated yscQ, yscR, and yscS, predicted to encode acidic proteins of 34.4, 24.4, and 8.5 kDa. All three proteins were homologous to proteins involved in flagellar function or virulence. An antipeptide antibody specific for YscR was used to localize YscR to the inner membrane of Y. pestis. Analysis of yscR-phoA fusions supported a model for yscR which predicts four transmembrane regions and a large, central hydrophilic domain. In-frame deletion mutations of yscQ and yscR were constructed and moved into Y. pestis. Both mutants failed to show the restriction of growth that normally accompanies maximal LCRS induction. Unlike the parent Y. pestis, the yscR mutant did not respond to the absence of Ca2+ by increasing the net transcription or translation of the LCRS-encoded V antigen, YopM, or LcrG. The yscR mutant also was defective for secretion of V antigen, YopM, and LcrG. These findings implicate a dual role for YscR in regulation of LCRS operons and secretion of LCRS proteins and add to the developing picture of how secretion of virulence proteins may be coupled to transcriptional regulation in yersiniae.