Regulation of the Ysa Type III Secretion System of Yersinia enterocolitica by YsaE/SycB and YsrS/YsrR

Yersinia enterocolitica biovar 1B contains two type III secretionsystems (TTSSs), the plasmid-encoded Ysc-Yop system and thechromosomally encoded Ysa-Ysp system. Proteins secreted fromthe Ysa TTSS (Ysps) have only been detected in vitro when cellsare cultured at 26°C in a high-NaCl medium. However, theexact role of the Ysa TTSS is unclear. Thus, investigationsinto the regulation of this system may help elucidate the roleof the Ysps during the life cycle of Y. enterocolitica. Herewe present evidence that the AraC-like regulator YsaE acts togetherwith the chaperone SycB to regulate transcription of the sycByspBCDAoperon, a phenomenon similar to that seen in the closely relatedSalmonella SPI-1 and Shigella flexneri Mxi-Spa-Ipa TTSSs. Deletionof either sycB or ysaE results in a twofold reduction in theactivity of a sycB-lacZ fusion compared to the wild type. Ina reconstituted Escherichia coli system, transcription of sycBwas activated sixfold only when both YsaE and SycB were present,demonstrating that they are necessary for activation. ysrR andysrS are located near the ysa genes and encode a putative two-componentregulatory system. Mutations in either gene indicated that bothYsrR and YsrS were required for secretion of Ysps. In addition,transcription from sycB-lacZ and ysaE-lacZ fusions was decreased6.5- and 25-fold, respectively, in the ysrS mutant comparedto the wild type. Furthermore, in the absence of NaCl, the activityof ysaE-lacZ was reduced 25-fold in the wild-type and ${Delta}$ ysrS strains,indicating that YsrS is probably required for the salt-dependentexpression of the ysa locus. These results suggest that theputative two-component system YsrRS may be a key element inthe regulatory cascade for the Ysa TTSS.

INTRODUCTION

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Introduction

The genus Yersinia has three species that are pathogenic tohumans. Y. pestis is the causative agent of bubonic and pneumonicplagues, and Y. enterocolitica and Y. pseudotuberculosis primarilycause gastroenteritis. Most prevalent of the yersiniae in humans,the Y. enterocolitica infection is usually self-limiting tothe gastrointestinal tract and mesenteric lymph node, causinggastroenteritis and lymphadenitis (12). However, in immunocompromisedindividuals, Y. enterocolitica can become systemic, and it hasa 50% mortality rate in such cases (12).

Consumption of contaminated food or water is the primary sourceof Y. enterocolitica infection. Ingested bacteria are capableof surviving the gastric barrier and then migrate to the terminalileum, where they attach to and subsequently invade the M cellsthat overlie the Peyer's patches (9, 25). Once inside the Peyer'spatches, the bacteria replicate to high titers and can thendisseminate to the mesenteric lymph nodes, spleen, and otherorgans, resulting in systemic disease (6, 8, 42, 51). The abilityof Yersinia spp. to survive and replicate within the host islinked to the presence of a large virulence plasmid (43). Thisplasmid carries genes encoding the Ysc type III secretion apparatusas well as translocators, regulators, and effector proteins(Yops). At 26°C, the optimal growth temperature for Yersiniaspp. outside the host, several copies of the secretion apparatus,called injectisomes, are detectable on the surface of the bacterium(37). At 37°C, in response to contact with target cells(in vivo) or to loss of Ca²⁺ ions (in vitro), the Yops are secreted.The functions of several Yops have been identified; these includetranslocation of effector Yops into host cells, impairment ofphagocytosis, and downregulation of the host's inflammatoryresponse (reviewed in reference 10).

While the virulence plasmid is necessary for full virulence,it is not sufficient, and several chromosomal genes have beenidentified as being important for the progression of disease.The genes encoding invasin, the primary invasion factor forY. enterocolitica and Y. pseudotuberculosis, and its regulator,RovA, are located on the chromosome (26, 39, 40, 44). The highlyvirulent strains of biotype 1B have a high-pathogenicity islandthat contains genes involved in iron uptake (7). In addition,several genes have been identified through various means ashaving a role in virulence, but their functions are not yetunderstood (reviewed in reference 45).

Recently, a chromosomally encoded type III secretion system(TTSS) was discovered in Y. enterocolitica and designated Ysa,for Yersinia secretion apparatus (23). This system is only presentin a subset of Y. enterocolitica strains, the highly virulentbiotype 1B strains (serotypes O:4, O:8, O:13, and O:21) (19).A recent phylogenetic analysis of TTSSs revealed that the Ysasystem is closely related to the Salmonella SPI-1 and Shigellaflexneri Mxi/Spa TTSSs (19). Interestingly, there is a TTSSon the chromosome of Y. pestis, but it is more closely relatedto the Salmonella SPI-2 TTSS, indicating that the chromosomalTTSSs of Y. enterocolitica and Y. pestis were acquired afterdivergence of the species (19).

Several proteins, referred to as Ysps (Yersinia secreted proteins),can be detected in the supernatants of cultures grown at 26°Cin the presence of high NaCl concentrations (20, 23). No proteinswere detected when genes encoding putative Ysa apparatus componentswere disrupted or when cultures were grown at 37°C (20,23, 55). Analysis by 50% lethal dose of a strain carrying amutation in a putative apparatus gene, ysaV, showed no attenuationby intraperitoneal injection. However, by an oral route it wasattenuated 10-fold, suggesting that the Ysa TTSS played a rolein the early stages of infection (23). Seven Ysps have beenidentified to date. Three were identified as YopE, YopN, andYopP, which are encoded by the virulence plasmid (20, 54). Theamounts of these Yops secreted under Ysa secretion conditionsare significantly less than under Yop secretion conditions,and the relevance of their secretion is not understood. However,YopP, secreted only through the Ysa TTSS, was able to suppressthe production of tumor necrosis factor alpha by infected macrophages(54). The other identified Ysps are encoded by the yspBCDA genesjust downstream of the ysa apparatus genes (20). YspB, YspC,and YspD are homologous to proteins involved in translocation,but YspA is a unique protein (20). In addition, SycB has beendemonstrated to function as a chaperone for YspB (20).

While a number of the proteins secreted by the Ysa TTSS havebeen identified and the functions for many genes in the locushave been inferred based on homology, nothing is known aboutthe regulation of this system other than a requirement for growthat 26°C in high NaCl concentrations (23). The closely relatedSalmonella SPI-1 and S. flexneri Mxi/Spa TTSSs have an interestingregulation system that has not been observed in other TTSSs:each utilizes an AraC-like regulator and a chaperone to regulatethe transcription of genes encoding secreted effectors (14,15, 34). In Salmonella spp., it has been shown that InvF (AraC-likeregulator) and SicA (chaperone) interact, and this interactionis likely to be required for the transcriptional activationbecause InvF alone can bind DNA but not activate transcription(15). The ysa locus has homologs of InvF and SicA, designatedYsaE and SycB, respectively, and the genetic organization ofthese and surrounding genes is very similar (Fig. 1). Transcriptionof the TTS apparatus genes is regulated by HilA in Salmonellaspp. (3, 31) and by VirB in S. flexneri (4), both of which arethemselves regulated by various environmental conditions (reviewedin references 17 and 32). It appears that HilA and VirB serveas the focal point for transmitting the environmental signalsthat lead to expression of these type III secretion systemsand their effectors. These regulators do not show any homologyto each other, and no homologue of either protein exists inY. enterocolitica. Therefore, identifying regulators upstreamof the Ysa system is of interest and may facilitate an understandingof the role of the Ysa TTSS.

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FIG. 1. Organization of the ysa operon. Black arrows indicate putative apparatus genes, gray arrows indicate genes encoding regulators, and white arrows indicate genes encoding secreted proteins. Speckled genes and those labeled with numbers are unique. Open reading frames 8 and 9/10 are believed to encode proteins that are part of the TTSS apparatus (19) and are therefore colored black. sycB and sicA have dual functions as chaperone and regulator. Dotted lines indicate homologous genes between the two systems (only a portion of SPI-1 is shown). The intergenic region between ysaU and sycB is 96 bp; the analogous region in SPI-1 (spaS to sicA) is 137 bp. No terminator structure was predicted to exist in a 300-bp region that includes the ysaU-sycB intergenic region with Mfold (http://www.bioinfo.rpi.edu/applications/mfold/).

In this work, we investigated the transcriptional regulationof the ysa and ysp genes. We show that the AraC-like protein,YsaE, and chaperone, SycB, are both required to activate transcriptionof the sycByspBCDA operon, a phenomenon similar to that seenin Salmonella spp. and S. flexneri (13-15, 27, 34). In addition,we show that YsrS, the putative sensor protein of a two-componentsystem, is required for expression of the ysaE promoter andthat this activation is NaCl dependent. These results indicatethat the putative two-component system YsrRS may be a key componentin the regulatory cascade for the Ysa secretion apparatus.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and growth conditions. The bacterial strains and plasmids used in this work are listedin Table 1 and described in detail below. Overnight cultureswere typically grown in Luria broth (LB) (170 mM NaCl; Difco)at 26°C for Y. enterocolitica or 37°C for Escherichiacoli, unless otherwise stated. For examination of secreted proteins,cultures were grown overnight in L-broth (1% tryptone, 0.5%yeast extract; referred to hereafter as LB-0) and subculturedinto L-broth containing 290 mM NaCl (referred to hereafter asLB-290). Antibiotics were added as needed at the following concentrations:ampicillin, 100 µg/ml; kanamycin, 100 µg/ml; nalidixicacid, 20 µg/ml; chloramphenicol, 12.5 µg/ml; spectinomycin,50 µg/ml; streptomycin, 50 µg/ml; and tetracycline,7.5 µg/ml. 5-Bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-Gal) was added to a final concentration of 40 µg/ml.

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TABLE 1. Strains and plasmids used in this work

Construction of plasmids. The plasmids used to generate in-frame deletion mutants of sycB,ysrR, ysaE, and ysrS were constructed as follows. Primers sycB-delAand -delB were used to amplify a ${approx}$ 500-bp region containing afew N-terminal codons and upstream sequence. This fragment wasdigested with SalI and BamHI and cloned into the same sitesof pSR47S. Primers sycB-delC and -delD were used to generatea similarly sized fragment containing a few C-terminal codonsand additional downstream sequence. This product was digestedwith BamHI and NotI and cloned into those sites of pSR47S containingthe upstream fragment, resulting in plasmid pKW10. Plasmid pKW16was constructed in an identical fashion with primer pairs ysrR-delA/delBand ysrR-delC/delD.

The PCR products generated with ysaE-delA/delB and ysaE-delC/delDwere digested with ClaI and BglII and with BglII and XbaI, respectively.They were sequentially cloned into pEP185.2, generating pKW4.The SalI-NotI fragment from pKW4, which contained the ysaE inserts,was subcloned into pSR47S to make pKW6. PCR products generatedwith ysrS-delA/delB and ysrS-delC/delD were digested with KpnIand XbaI and with XbaI and SacI, respectively, and sequentiallycloned into pEP185.2 to make pKW3. Primers ysrS-delA and ysrS-delDwere used to amplify the ysrS inserts from pKW3, and the productwas cloned into pCR2.1 TOPO. The SalI-NotI fragment from thisplasmid was then subcloned into pSR47S, generating pKW7. Followingligation, each plasmid was transformed into E. coli strain S17-1 ${lambda}$ pirby electroporation. All constructs were confirmed by restrictiondigestion and sequenced to ensure that no errors were generatedduring amplification.

For generating a disruption in the ysaC gene, an internal regioncontaining approximately 500 bp of the ysaC gene was amplifiedwith primers ysaC-F1 and ysaC-R1 (Table 2) and cloned into thepCR2.1-TOPO vector (Invitrogen). The KpnI-XhoI fragment wascleaved out of that plasmid and cloned into the same sites ofpEP185.2, resulting in pSAH1. This plasmid was transformed intoE. coli strain S17-1 ${lambda}$ pir by electroporation and confirmed byrestriction digestion.

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TABLE 2. Primers used in this work

Transcriptional lacZ fusions were constructed by cloning putativepromoter regions into pKN8 (18). For the ysaE and sycB promoters,approximately 300 bp of promoter sequence and 250 bp of codingsequence were amplified, digested with XbaI and BglII, and clonedinto those sites of pKN8, resulting in plasmids pKW5 and pKW11,respectively. The plasmids were transformed into S17-1 ${lambda}$ pir byelectroporation, confirmed by restriction digestion, and sequencedto ensure that no errors were generated during amplification.Plasmid pKW21 was made by digesting a PCR-generated fragmentof the sycB promoter region with EcoRI and BamHI and ligatingit into the same sites of pRW50. The ligated plasmid was transformedinto E. coli strain DH5 ${alpha}$ , confirmed by restriction digestion,and sequenced. The primer sequences and pairs used for theseconstructs are listed in Tables 2 and 3, respectively.

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TABLE 3. Primer pairs used for transcriptional fusions and complementing clones

Two plasmids carrying the sycB coding region were constructed.In the first, primers sycB-FP3 and -RP2 were used to amplifythe sycB gene and promoter region. The product was digestedwith EcoRI and HindIII, cloned into those sites of pHG329, andtransformed into DH5 ${alpha}$ , giving pKW23. The second PCR product,generated with primers sycB-FP1 and -RP2, was digested withBglII and HindIII and cloned into those sites in pKW27, givingpKW28. Plasmid pKW27 is pACYC184 with a streptomycin-spectinomycinresistance cassette from p34-Sm (16) cloned into the BamHI site;the tetracycline resistance cassette was disrupted by this insertion.The ysaE-complementing clone was made by digesting a PCR productfrom primers ysaE-delA and -delD with SalI and NotI and cloningit into those sites of pWKS130 to give pKW22. The ysrR-complementingclone was made by digesting a PCR product from primers ysrR-delAand -RP1 with SalI and KpnI and cloning it into those sitesof pWKS130 to give pKW24. For the ysrS-complementing clone,a PCR product generated by primers ysrS-OEF1 and -RP3 was digestedwith SalI and NotI and cloned into those sites of pWKS130 togive pKW31. All plasmids were transformed into DH5 ${alpha}$ by electroporation,confirmed by restriction digestion, and sequenced to ensurethat no errors occurred during amplification. Primer sequencesand pairs used for these constructs are given in Tables 2 and3, respectively.

Primers yspC 1.1 and yspC 2.2 (Table 2) were used to amplifythe entire coding sequence of YspC. The PCR product was digestedwith SalI and NotI and cloned into the same sites of pGEX-6P-1(Amersham). The resulting plasmid, pYW2, encodes a glutathioneS-transferase (GST)-YspC fusion protein.

Strain construction. (i) In-frame deletions. Strains YVM969, YVM932, YVM981, and YVM1006 containing chromosomalin-frame deletions in ysrS, ysaE, sycB, and ysrR, respectively,were made by conjugation as follows. Equal volumes of saturatedcultures of E. coli carrying the desired plasmid (pKW6, -7,-10, or -16) and Y. enterocolitica JB580v were mixed, platedon LB agar, and allowed to incubate at 26°C overnight. Theresulting lawn of cells was scraped into 1 ml of 1x PBS, diluted1:100, and plated on LB agar plates containing nalidixic acidto select against E. coli and kanamycin to select against Y.enterocolitica lacking the plasmid. Replication of pSR47S requiresthe pir protein; all Y. enterocolitica strains used lack pir,and thus survivors would have undergone site-specific recombination.Several transconjugants were streaked onto LB agar plates containingnalidixic acid and 5% sucrose for selection of colonies thathad undergone a second recombination step and lost the vector.Cells retaining a functional sacB gene should not grow in thepresence of sucrose. Several of these colonies were then screenedfor kanamycin sensitivity, of which 10 to 20 were picked forconfirmation of the in-frame deletion by colony PCR (see below).

To ensure that recombination occurred in the proper location,strains selected for experiments were analyzed by Southern blotting.Strains used for analysis of Ysa-dependent secretion were subsequentlycured of the virulence plasmid by inoculation on LB agar containing20 mM MgCl₂ and 20 mM NaC₂O₄ and grown at 37°C. Loss ofthe virulence plasmid was verified by visualization of plasmidDNA preparations on 0.8% agarose gels. The test strains werealways compared to plasmid preparations from JB580v and JB580c,which served as positive and negative controls, respectively.The cured strains of YVM969, YVM932, YVM981, and YVM1006 aredesignated YVM972, YVM971, YVM996, and YVM1089, respectively.

(ii) Plasmid integrations. Strain YVM886 was generated by conjugating pSAH1 into JB580vas described above but with selection on LB agar containingnalidixic acid and chloramphenicol. Proper insertion of theplasmid was confirmed by Southern blotting. YVM886 was thencured of pYVe8081 as described above to yield YVM917. Strainscarrying chromosomal promoter-lacZ fusions were constructedby conjugating either pKW5 or pKW11 into the desired Y. enterocoliticastrain, followed by selection on LB agar containing nalidixicacid and chloramphenicol, giving YVM925 and YVM987, respectively.Because of the region that was cloned, merodiploid strains weregenerated by the recombination event. Proper integration ofthe plasmid was confirmed by Southern blotting prior to analysis.These strains were not subsequently cured of pYVe8081.

(iii) Plasmid transformation. For Y. enterocolitica, the strain to be transformed was inoculatedinto LB broth containing 1% glucose and grown overnight. Approximately500 µl of the saturated culture was washed twice withan equal volume of ice-cold distilled H₂O and resuspended in40 µl of 10% glycerol; 1 to 2 µl of plasmid DNAwas added to the cells and electroporated by standard procedures.Ten percent of the recovered culture was plated on LB agar withappropriate antibiotics. When two plasmids were needed, bothwere transformed simultaneously. For E. coli strains VM1265,VM1266, VM1267, and VM1272, electrocompetent CC118 ${lambda}$ pir cellswere simultaneously transformed with all three desired plasmids.Ten percent of the recovered culture was plated on LB agar withappropriate antibiotics.

PCR and DNA sequencing. Standard methods for PCR were conducted under the conditionsspecified by the supplier with either Pfu polymerase (Stratagene,La Jolla, Calif.) or Taq polymerase (Qiagen, Valencia, Calif.).Colony PCR was performed in a standard reaction in 50 µl.A single colony was resuspended in 50 µl of distilledH₂O and vortexed, and 5 µl was used as the template. A5-min incubation at 95°C preceded the cycling reactionsto ensure cell lysis. DNA sequencing was performed with theBig Dye termination cycle sequencing ready reaction system underthe conditions specified by the supplier (PE Applied Biosystems,Foster City, Calif.). Reactions were analyzed at the Proteinand Nucleic Acid Chemistry Laboratory at the Washington UniversitySchool of Medicine.

ß-Galactosidase assays. Saturated cultures grown overnight in LB-0 were diluted intofresh LB-290 to an initial optical density at 600 nm (OD₆₀₀)of 0.2 and grown for 4 h at 26°C on a roller drum. Antibioticswere added as necessary to retain plasmids. Assays were performedas described before (38).

Total RNA extraction and RT-PCR. A saturated culture of JB580v grown overnight in LB-0 was dilutedinto fresh LB-290 to an initial OD₆₀₀ of 0.2 and grown for 4h at 26°C on a roller drum. RNAprotect bacterial reagent(Qiagen) was added to the cell sample as described by the manufacturer.The cells were collected by centrifugation, and total RNA wasextracted with the MasterPure RNA extraction kit from Epicenter.DNA was removed from 20 µg of sample with DNA-free (Ambion)following the manufacturer's protocol. For cDNA synthesis, 2µg of RNA was used as a template with 200 U of SuperscriptIII as described by the supplier (Invitrogen). PCR was performedwith the cDNA synthesis products as the template with primersKW114 and KW115 (Table 2) and Taq polymerase (Qiagen) in a 50-µlreaction volume. For controls, PCR was performed without cDNAtemplate as well as with genomic DNA to show the expected sizeof the product generated with these primers. One fifth of thereaction was separated on a 1.2% agarose gel and stained withethidium bromide.

Preparation of secreted proteins, SDS-PAGE, and Western blot analysis. Extracellular proteins were collected as described before (55).Briefly, saturated cultures grown in LB-0 were diluted intofresh LB-290 to an initial OD₆₀₀ of 0.2 and grown for 6 h at26°C on a roller drum. The longer culture time for proteinpreparations than for expression studies was chosen becausethe preparations were cleaner, making detection of individualbands easier. Antibiotics were added as necessary to retainplasmids. The cells were removed from 4.5 ml of culture by centrifugationin microcentrifuge tubes for 1 min at 13,000 rpm. The supernatantwas centrifuged a second time, followed by passage through a0.22-µm syringe filter. Ice-cold trichloroacetic acidwas added to a final concentration of 10% (vol/vol) and incubatedon ice for 10 to 20 min. The samples were centrifuged at 4°Cfor 10 min at 13,000 rpm, washed once with ice-cold acetone,and resuspended in 1 M Tris-HCl, pH 9.0. The proteins were boiledfor 5 min in 1x sample buffer (46), and OD₆₀₀ equivalents wereloaded onto sodium dodecyl sulfate (SDS)-polyacrylamide gels.Proteins were visualized by staining with silver nitrate (Bio-Rad)or transferred to nitrocellulose for Western analysis with aBio-Rad Trans-Blot SD semidry transfer apparatus as specifiedby the supplier. Blots were blocked in 1x phosphate-bufferedsaline (PBS) with 0.1% Tween 20 and 5% skim milk (PBST-milk)for 1 h at room temperature. Primary antibody directed againstYspC was diluted 1:1,000 in PBST-milk and allowed to react overnightat 4°C. The membranes were washed several times with PBST-milkand then incubated with goat anti-rabbit immunoglobulin G-horseradishperoxidase at 1:25,000 in PBST-milk for 1 h at room temperature.The membranes were washed again in PBST-milk, and proteins weredetected by chemiluminescence (ECL; Amersham).

To generate the anti-YspC antibody, a GST-YspC fusion proteinwas purified from E. coli carrying pYW2 with the bulk GST purificationmodule as specified by the supplier (Amersham). Approximately1 mg of GST-YspC was sent to Covance Research Products for immunizationof a New Zealand White rabbit.

RESULTS

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Results

Regulation of the sycB-yspBCDA operon. The organization of the ysa-syc-ysp genes in Yersinia spp. isquite similar to that in Salmonella. SPI-1 and to a lesser degreeto the S. flexneri mxi-spa-ipa genes. Given that both the Salmonellaand Shigella systems employ an AraC-like regulator (InvF andMxiE, respectively) and a TTSS chaperone (SicA and IpgC, respectively)to regulate the expression of genes encoding secreted proteins,it was of interest to investigate if the analogous proteins,YsaE and SycB, functioned similarly in the Ysa system. To testthis hypothesis, in-frame deletions of ysaE and sycB were constructed,and the strains were examined for their ability to secrete proteinsafter growth in L-broth with 290 mM NaCl (LB-290). Both strainsshowed a loss of most protein bands (Fig. 2A). This indicatesthat YsaE and SycB are required for wild-type levels of secretion.

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FIG. 2. Strains lacking sycB or ysaE do not secrete Ysps. Proteins were precipitated from culture supernatants and separated by SDS-10% polyacrylamide gel electrophoresis as described in the text. (A) Silver-stained gel showing loss of Ysps from the culture supernatants of ${Delta}$ sycB and ${Delta}$ ysaE strains. ${Delta}$ ysaC carries a disruption in the ysaC gene and is thought to have a defective apparatus. The culture equivalent of 2 OD units was loaded in each lane. (B) Complementation of the mutant strains as determined by Western blotting with anti-YspC antibody. YspC is indicated by the arrow. The culture equivalent of 1 OD unit was loaded in each lane.

Western blots probed with anti-YspC antibody, which recognizesthe secreted protein YspC, showed that secretion was restoredwhen the wild-type gene was provided in trans, demonstratingthat the reduced secretion was due to the loss of the deletedgene (Fig. 2B). Curiously, the complemented ${Delta}$ ysaE strain consistentlyappeared to secrete less YspC than the wild-type strain. Thepresence of kanamycin in the medium did not significantly impairthe growth of the cultures (not shown). However, the antibiotic(or plasmid carriage) may somehow interfere with secretion orprecipitation of Ysps, since the wild-type strain carrying pWKS130or pKW22 secreted less YspC than the strain without either plasmid(Fig. 2B). A similar phenomenon was observed with the sycB-complementingclone (not shown).

In related TTSSs, InvF/SicA and MxiE/IpgC act by stimulatingthe transcription of genes encoding secreted proteins (13-15,27, 34). To test the hypothesis that YsaE and SycB were actingas transcriptional regulators, a sycB-lacZ fusion was introducedinto the ${Delta}$ sycB and ${Delta}$ ysaE strains, and ß-galactosidaseactivities were determined. The activity of the sycB promoterdecreased about twofold in the ${Delta}$ ysaE and ${Delta}$ sycB strains, suggestingthat both proteins play a role in the transcription of the sycByspBCDAoperon (Table 4). The ysaE and sycB mutants could be fully complementedfor sycB expression when the respective wild-type gene was providedon a plasmid; strains carrying the vector alone showed no changein activity from the mutants. A similar twofold reduction intranscription was observed in the ysaE sycB double mutant, andactivity of sycB-lacZ was only restored when both ysaE and sycBwere provided in trans. Promoter activity in each of the complementedstrains was much higher than in the wild type, indicating thatYsaE or SycB may be limiting in the wild type under the conditionsexamined. Expression in the wild-type strain carrying plasmidpSycB, pYsaE, or both was also increased, but no differencewas observed with the vectors (not shown). The observed effectsof SycB and YsaE were independent of genes encoded by the virulenceplasmid. Loss of the virulence plasmid had no effect on sycB-lacZtranscription in a wild-type strain (not shown).

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TABLE 4. Regulation of sycB promoter by SycB and YsaE

The possibility existed that transcription initiating at theysaE promoter may also transcribe the sycByspBCDA genes, asis thought to be the case in SPI-1 (13). If this is so, thenthe decrease in transcription of the sycB promoter observedin the ${Delta}$ ysaE and ${Delta}$ sycB strains may not be an accurate measureof the contribution of these two proteins on transcription initiatingat the sycB promoter. To determine if a transcript existed thatinitiated upstream of ysaU, RT-PCR was used to amplify a regionthat encompassed the ysaU-sycB intergenic region. A productwas detected only when genomic DNA or cDNA was added to thereaction, but not from reactions containing no template or templatefrom a cDNA synthesis reaction lacking reverse transcriptase(Fig. 3). These data demonstrate that transcription of sycByspBCDAgenes can indeed initiate at a promoter upstream of ysaU andcontributes to the transcription of this operon.

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FIG. 3. Transcription of sycB originates at a promoter upstream of ysaU. (A) Schematic of the ysa locus encompassing the sycB promoter region. The approximate locations of primers KW114 and KW115 are indicated. (B) RT-PCR was performed with primers KW114 and KW115 and cDNA generated from 2 µg of total RNA that was isolated from JB580v as described in the text; 20% of the reaction was loaded on a 1.2% agarose gel and stained with ethidium bromide. Templates for the PCR are listed above each lane and were as follows: gDNA, genomic DNA; +RT, products from cDNA synthesis reaction with Superscript III added; no RT, products from cDNA synthesis reaction with no Superscript III; nt, no DNA or cDNA added.

In order to analyze the effects of SycB and YsaE on sycB-lacZexpression without the contributions from the upstream promoter,a plasmid-based system was reconstituted in E. coli. Transcriptionfrom sycB-lacZ was at background levels if only SycB or YsaEwas present (Table 5). However, when both YsaE and SycB werepresent, the sycB promoter was activated about sixfold. Thissuggests that both proteins are necessary and probably sufficientto stimulate transcription from the sycB promoter, althoughwe cannot exclude the existence of additional regulators.

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TABLE 5. YsaE and SycB are sufficient to activate the sycB promoter in E. coli CC118 ${lambda}$ pir

Furthermore, these data, combined with the RT-PCR data, indicatethat at least two promoters are transcribing the sycByspBCDAgenes.

Regulation of the ysa operon. Since Y. enterocolitica does not appear to have a homologueto known key regulators of the SPI-1 and Mxi-Spa TTSSs, thereis no obvious regulator for the expression of the ysa genes.However, proximal to the ysa locus are two genes that encodea putative two-component regulatory system, ysrR and ysrS (23)(Fig. 1). These genes are closely related to the rcsB and rcsCgenes, respectively, which encode a two-component regulatorysystem that regulates gene transcription in a number of entericorganisms, often in response to osmotic shock (2, 47). YsrSis the putative hybrid sensor component and has regions similarto the conserved His- and Asp-containing domains of sensor proteinsbut, like RcsC, lacks the conserved His residue in the phosphotransfer(HPt) domain. YsrR has regions similar to the Asp-containingreceiver domain and a putative helix-turn-helix DNA bindingdomain. To see if YsrRS is involved in regulating the ysa genes,in-frame deletions were constructed in the ysrS and ysrR genes.Examination of proteins in culture supernatants after growthin LB-290 indicated that both the ${Delta}$ ysrS and ${Delta}$ ysrR strains weredefective in the secretion of all of the Ysps (Fig. 4A). Thesemutant phenotypes could be complemented by providing the wild-typegene in trans, indicating that the loss of secretion was dueto the absence of functional YsrR or YsrS (Fig. 4B).

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FIG. 4. Strains lacking ysrS or ysrR do not secrete Ysps. Proteins were precipitated from culture supernatants and separated by SDS-10% polyacrylamide gel electrophoresis as described in the text. (A) Silver-stained gel showing the loss of all Ysps from the culture supernatants of the ${Delta}$ ysrS and ${Delta}$ ysrR strains. The culture equivalent of 2 OD units was loaded in each lane. (B) Complementation of the mutant strains as determined by Western blotting with anti-YspC antibody. YspC is indicated by the arrow. The culture equivalent of 1 OD unit was loaded in each lane.

To determine if the defect in Ysp secretion observed in the ${Delta}$ ysrS and ${Delta}$ ysrR strains was due to decreased transcription, theactivity of the ysaE-lacZ and sycB-lacZ fusions was examinedin the ${Delta}$ ysrS strain. The ß-galactosidase activitiesof the sycB and ysaE promoters were down 6.5- and 25-fold, respectively,compared to the wild-type strain, indicating that YsrS is requiredfor full activity of both promoters (Table 6). Addition of ysrSin trans restored promoter activity, demonstrating that theobserved decrease was indeed due to loss of ysrS; no changein activity was observed in strains carrying the vector. However,the YsrS regulatory pathway could be acting indirectly at thesycB promoter by activating expression of the upstream ysaEpromoter and thus production of YsaE and SycB. To determineif the reduced sycB-lacZ activity resulted from a direct lossof activation at the sycB promoter, the ${Delta}$ ysrS strain was transformedwith pSycB and pYsaE. If the ysrS defect can be overcome byexpressing ysaE and sycB in trans, this would suggest that theYsrRS system acts indirectly at the sycB promoter. Indeed, theactivity of the sycB-lacZ fusion was restored in the ${Delta}$ ysrS straincarrying pYsaE and pSycB, indicating that the observed decreasein activity at this promoter in the ${Delta}$ ysrS strain is probablya downstream effect from the loss of activation at the ysaEpromoter.

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TABLE 6. Regulation by YsrS in Y. enterocolitica

Regulation by YsrS requires NaCl. Because the function of sensor proteins is generally to detectenvironmental cues and secretion of Ysps is only observed whencells are cultured in high NaCl, we hypothesized that YsrS respondsto the NaCl in the growth medium. To test the idea that YsrSsenses NaCl, wild-type and ${Delta}$ ysrS strains containing the ysaE-lacZfusion were grown in LB-0 and LB-290 and assayed for ß-galactosidaseactivity (Table 7). In LB-0, ß-galactosidase activityin the wild-type strain was the same as in the ${Delta}$ ysrS strain.In addition, no induction by NaCl was observed in the ${Delta}$ ysrS strain,yet the wild-type strain showed 25-fold induction. Furthermore,induction of ysaE by NaCl could be restored in the ${Delta}$ ysrS strainby providing ysrS in trans. This revealed that, in the absenceof YsrS or in the absence of NaCl, there is no activation ofthe ysaE promoter.

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TABLE 7. Regulation of the ysaE promoter by YsrS requires NaCl

To confirm that YsrS was required for the NaCl-dependent activation,the ysaE-lacZ fusion was similarly tested in the ${Delta}$ ysaE strain.Consistent with previous studies on InvF (22), loss of YsaEdid not lead to a significant reduction in activity of its ownpromoter when cultured in LB-290. Loss of SycB also did notaffect the expression of ysaE-lacZ (not shown). However, justas in the wild-type strain, activity from the ysaE promoterwas reduced when it was cultured in LB-0. This indicates thatthe NaCl-dependent activation of the ysa operon requires YsrSbut not YsaE. This effect could be a response to changes inosmolarity rather than a specific NaCl-dependent effect, butthis has not been fully explored.

Intriguingly, the growth rate of the ${Delta}$ ysrS and ${Delta}$ ysrR strains wasslightly but reproducibly faster than that of the wild-typestrain when grown in LB-290. Doubling times for the ${Delta}$ ysrS and ${Delta}$ ysrR strains was typically about 10 min faster than for thewild type ( ${approx}$ 83 min for the ${Delta}$ ysrS and ${Delta}$ ysrR strains and ${approx}$ 93 min forthe wild type). This increased doubling time was not observedfor these strains grown in LB-0, nor was it observed for the ${Delta}$ ysaE or ${Delta}$ ysaC strains grown in LB-290, indicating that it isspecific to the ${Delta}$ ysrS and ${Delta}$ ysrR strains when cultured in the presenceof NaCl. This also suggests that it is not related to the secretionof Ysps.

DISCUSSION

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Discussion

In this work, we showed that the AraC-like regulator YsaE andthe chaperone SycB are involved in the regulation of a subsetof the Ysps. Further examination of this phenomenon showed thatthe sycByspBCDA operon is transcriptionally regulated by YsaEand SycB. Loss of either activator resulted in a reduction insycB-lacZ activity in Y. enterocolitica. Similarly, a reconstitutedsystem in E. coli showed a sixfold activation of sycB-lacZ onlyin the presence of both regulators. These data indicate thatYsaE and SycB are necessary and likely sufficient to activatetranscription from this promoter, although the existence ofadditional regulators cannot be excluded. The increased activationobserved in the isolated E. coli system compared to that observedin Y. enterocolitica is probably a consequence of transcriptionof sycByspBCDA initiating at the upstream promoter (ysaE) thatis not subject to regulation by YsaE and SycB. This is alsoprobably the case in Salmonella SPI-1, where transcription ofthe sicAsipBCDA genes can occur through initiation at an upstreampromoter (possibly the invF promoter), as well as at the sicApromoter (13).

A similar mechanism of type III effector gene regulation byMxiE and IpgC exists in S. flexneri. Here, it is not the translocatoroperon that is affected but a set of proteins whose secretionis only observed under conditions of active secretion (27, 34).By incrementally overexpressing IpgC, Mavris et al. showed thatthe expression of these proteins increased as the concentrationof IpgC increased (34). The authors concluded that the levelof free IpgC, which would be found when its cognate proteinshad been secreted, is the signal that leads to increased transcriptionof the secreted proteins. This report marks the third exampleof an AraC-like regulator acting with a type III chaperone tostimulate transcription of secreted proteins. Thus, it is likelythat this is a conserved mechanism by which the cell monitorsits secretion state and links it to transcriptional regulation.

The mechanism(s) behind this activation is not well understood.It has been shown that InvF can bind DNA in the absence of SicA,but SicA by itself does not bind DNA (15). The DNA binding sitesfor InvF and MxiE have been identified and are strikingly similar;in the center of each site, there is a T-rich region (15, 35),which may facilitate bending of the DNA by the protein. Thesepromoters also lack an obvious –35 consensus sequence.In the region thought to contain the sycB promoter, a T-richregion is in roughly the same location as in the InvF- and MxiE-regulatedgenes, and there is no definable –35 region. However,much work remains to determine if this is indeed a YsaE bindingsite.

Many bacteria, both gram-negative and gram-positive, use two-componentregulatory systems to regulate virulence genes. For example,RcsBC is required for the expression of capsule genes in E.coli (49), Erwinia amylovora (5), and Klebsiella pneumoniae(1) as well as Vi antigen expression in Salmonella entericaserovar Typhi (2). In S. enterica serovar Typhimurium, the typeIII secretion systems encoded on both SPI-1 and SPI-2 are regulatedby multiple two-component systems (29, 32, 33, 41). In thiswork, we present data suggesting that expression of the ysaEpromoter requires YsrS and most likely YsrR. YsrS and YsrR areencoded by genes adjacent to the ysa locus and comprise a putativetwo-component regulatory system (19, 23). Analysis of secretedproteins as well as the lacZ fusion to ysaE in the ysrS mutantrevealed that YsrS is a key component in the expression of theysa locus. Loss of YsrS also resulted in lower sycB promoteractivity. However, providing ysaE and sycB in trans in the ${Delta}$ ysrSstrain complemented sycB activity. Together, these results indicatethat the ysaE promoter is regulated by YsrS and also suggeststhat initiation at ysaE can lead to transcription of sycByspBCDA.YsrR is also a critical component, as an in-frame deletion ofysrR similarly resulted in complete loss of all secreted proteinsin culture supernatants. Secretion of Ysps has not been observedin the absence of NaCl in the culture medium, and stimulationof ysaE transcription by YsrS requires NaCl. Thus, YsrS maybe functioning as an environmental sensor of NaCl or osmolarity.Furthermore, since YsrS is probably a membrane-bound protein,it is more likely that the actual transcriptional regulatoris YsrR; however, this has yet to be experimentally tested.

BLAST searches with YsrS revealed homology to RcsC in E. coliand other enteric bacteria. While much of the similarity waslimited to the conserved domains, YsrS is also similar to RcsCin that they both lack a histidine phosphotransferase (HPt)domain. This domain contains the second His site that transfersthe phosphate to the Asp residue on the response regulator (53).In E. coli, the HPt-containing protein YojN has been identifiedas the intermediate between RcsC and RcsB (50). Interestingly,YojN lacks the other necessary His and Asp residues typicallyfound in sensors, suggesting that the only functional domainis the HPt (50). Another example of a third partner in phosphorelayis with the LuxN-LuxU-LuxO system in Vibrio harveyi. LuxN lacksan HPt domain, while LuxU contains the appropriate His residue(21). There are several open reading frames that contain HPtdomains within the Y. enterocolitica genome, suggesting thatsuch an intermediate protein providing this domain for the YsrS-YsrRphosphorelay does indeed exist. YsrR has homology to a numberof response regulators, including RcsB. However, most of theconserved residues are in the LuxR-type helix-turn-helix DNA-bindingmotif.

Although the activity of YsrR and YsrS appears to require NaCl,they probably do not have a role in osmoprotection, as is suggestedfor the RcsC-YojN-RcsB system (56). This is evidenced by theincreased growth rate observed in the ${Delta}$ ysrS and ${Delta}$ ysrR strainsin the presence of high NaCl concentrations. In fact, the Y.enterocolitica genome contains genes that are probably the trueRcsBC orthologues, based on amino acid similarity and geneticorganization (http://www.sanger.ac.uk/Projects/Y_enterocolitica/).Thus, YsrRS represent a new and uncharacterized two-componentsystem. In virulence plasmid-containing Yersinia strains, growthof the bacterium slows when Yop secretion is induced (11 andreferences therein). The ${Delta}$ ysaC strain, which is defective insecretion but not transcription, does not display altered growth.Therefore, the faster growth of the ${Delta}$ ysrS and ${Delta}$ ysrR strains mightbe a consequence of not using metabolic resources for expressingthe ysa and ysp genes.

Based on the results presented here, a schematic model for YsaTTSS regulation is proposed in Fig. 5. We hypothesize that YsrSsenses an environmental cue (NaCl), transfers phosphate to anHPt-containing intermediate (HPt), which then transfers thephosphate to YsrR. The activated phospho-YsrR then stimulatestranscription of the ysaE promoter, either directly or indirectly.Once levels of YsaE and free SycB are sufficient, they stimulatetranscription of the sycB promoter and possibly others. Fromthe experiments conducted with InvF and MxiE (15, 35), we caninfer that YsaE probably binds DNA in the absence of SycB. Itwill be of interest to investigate if SycB enhances the DNAbinding affinity of YsaE, if SycB makes any contact with theDNA, and if either protein contacts RNA polymerase. Similarquestions arise surrounding the regulation by YsrRS. It remainsto be demonstrated that YsrR directly regulates promoter activityand if it is indeed involved in a phosphorylation cascade withYsrS. It also remains to be demonstrated whether or not YsrSdirectly senses NaCl and other environmental cues for activatingthis system.

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FIG. 5. Model for activation of the ysaE and sycB promoters. YsrS senses NaCl in the culture medium by an unknown mechanism and initiates a phosphorelay that leads to phosphorylation of YsrR. The activated YsrR then stimulates transcription of the ysaE promoter, either directly or indirectly. Once sufficient levels of YsaE and SycB have accumulated, they stimulate transcription of the sycB promoter.

ACKNOWLEDGMENTS

We are indebted to Yanli Wu for the cloning and purificationof GST-YspC and for arranging the preparation of antibody withCovance Research Products. We thank Scott Handley for the constructionand initial characterization of YVM886 ( ${Delta}$ ysaC). We thank DamonEllison for critical review of the manuscript.

This research was supported by National Institutes of Healthgrant AI42736 awarded to V. L. Miller.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Microbiology, Campus Box 8230, 660 S. Euclid Ave., Washington University School of Medicine, St. Louis, MO 63110. Phone: (314) 286-2891. Fax: (314) 286-2896. E-mail: virginia{at}borcim.wustl.edu.

REFERENCES

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