Transcriptional activation of Salmonella typhimurium invasion genes by a member of the...

Transcriptional activation of Salmonella typhimuriuminvasion genes by a member of the phosphorylatedresponse-regulator superfamily

Christine Johnston, 1 David A. Pegues, 2 Christoph J.Hueck, 1 Catherine A. Lee 3 and Samuel I. Miller 1*1Departments of Medicine and Microbiology, University ofWashington, Biomedical Health Sciences Building, K-116,Box 357710, Seattle, Washington 98195, USA.2Infectious Disease Unit, Massachusetts GeneralHospital, Harvard Medical School, Fruit Street, Boston,Massachusetts 02114, USA.3Department of Microbiology and Molecular Genetics,Harvard Medical School, 200 Longwood Avenue, Boston,Massachusetts 02115, USA.

Summary

The Salmonella typhimurium PhoP-repressed locusprgHIJK encodes components of a sec-independenttype III secretion apparatus. This apparatus is com-posed of at least 17 proteins encoded on a 40 kbpathogenicity island located at centisome 63 on theS. typhimurium chromosome. The secretion appara-tus and some of its targets, SspB, SspC and SspD,are necessary for epithelial cell invasion. The tran-scription of many invasion genes, including prgHIJK ,is co-ordinately activated by HilA, a transcription fac-tor encoded within the pathogenicity island. In thisreport we identify sirA , a gene located outside thepathogenicity island that is essential for induction ofprgHIJK and hilA transcription. sirA encodes a 234-amino-acid protein that is essential for S. typhimuriumSsp ( Salmonella secreted protein) secretion and inva-sion and is similar to response regulators of two-com-ponent regulatory systems. sirA -mutant phenotypescould be suppressed by two DNA clones fromunlinked loci, designated sirB and sirC . These datasuggest that SirA may be phosphorylated in responseto S. typhimurium sensing a mammalian microenvir-onment. Furthermore, SirA phosphorylation is pre-dicted to initiate a cascade of transcription-factorsynthesis which results in invasion-gene transcription,Ssp secretion, and bacterial invasion of epithelia.

Introduction

During the initial stages of infection, Salmonella typhimur-ium invades the intestinal epithelium by inducing dramaticchanges in the epithelial surface (Takeuchi, 1967). Thesemorphological changes are caused by cytoskeletal rear-rangements that involve filamentous actin and are visual-ized as membrane ruffles adjacent to areas of bacterialadherence (Finlay and Falkow, 1990; Francis et al., 1992;Galan et al., 1992; Garcia-del Portillo and Finlay, 1994;Ginocchio et al., 1994; Takeuchi, 1967). Subsequently,in a process known as bacterial mediated endocytosis(BME) (Portnoy and Smith, 1992), or invasion, mamma-lian cells internalize bacteria in membrane-bound spa-cious phagosomes (SP) which are formed as bacteriaare engulfed within membrane ruffles (Alpuche-Arandaet al., 1994; Garcia-del Portillo and Finlay, 1994; Racoosinand Swanson, 1992). Salmonellae interactions withepithelia can be modelled using cultured epithelial cells(Finlay and Falkow, 1990; Finlay et al., 1988a; McCormicket al., 1993). Such models have been used to identify andunderstand the bacterial, host and environmental factorsimportant to S. typhimurium invasion. Non-invasivemutants of S. typhimurium exhibit virulence defects inmice, consistent with an inability to cross the intestinalmucosa (Behlau and Miller, 1993; Galan and Curtiss,1989). In addition, when tested in an in vitro model ofhuman gastroenteritis, non-invasive mutants of S. typhi-murium are defective in the induction of epithelial cellIL-8 production and do not induce polymorphonuclear leuko-cyte (PMN) translocation across polarized epithelium(McCormick et al., 1995). These results suggest that theability to induce BME is important to human gastroenteritis.

A number of S. typhimurium loci essential for invasionhave been identified (Bajaj et al., 1995; Behlau and Miller,1993; Finlay et al., 1988b; Galan and Curtiss, 1989; Grois-man and Ochman, 1993; Hueck et al., 1995; Jones andFalkow, 1994; Kaniga et al., 1995; Lee et al., 1992;Pegues et al., 1995; Stone et al., 1992). Over 25 suchloci are located at centisome 63 of the S. typhimuriumchromosome which has been referred to as Salmonellapathogenicity island 1 (SPI1) (Collazo et al., 1995; Millset al., 1995; Shea et al., 1996). These genes encodeproteins which are either components or secreted targetsof a sec-independent type III protein-secretion apparatus

Molecular Microbiology (1996) 22(4), 715–727

# 1996 Blackwell Science Ltd

Received 17 June, 1996; revised 9 September, 1996; accepted 13 Sep-tember, 1996. *For correspondence. E-mailmillersi@u. washington.edu;Tel. (206) 616 5107; Fax (206) 616 4295.

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(reviewed in Galan, 1996). The secretion apparatus of S.typhimurium, as well as some of the secreted proteinsthemselves, are necessary for BME and are homologousto the type III secretion system found in Shigella spp.(Behlau and Miller, 1993; Collazo et al., 1995; Groismanand Ochman, 1993; Hueck et al., 1995; Jones and Falkow,1994; Kaniga et al., 1995; Pegues et al., 1995). Specifi-cally, three targets of the secretion apparatus in S. typhi-murium, SspB, SspC, and SspD, also known as Sip, arehomologous to the IpaB, IpaC and IpaD proteins ofShigella spp. (Hermant et al., 1995; Hueck et al., 1995;Kaniga et al., 1995). IpaB and IpaC, attached to beadsby specific antibodies, have been shown to initiate rufflingand endocytosis, implying that these proteins, and byhomology possibly SspB and SspC of S. typhimurium,are sufficient to stimulate membrane ruffling and bacterialinvasion (Menard et al., 1996).

The regulation of S. typhimurium invasion appears to bemodulated in vitro by a number of environmental factors.Low oxygen, osmolarity, and bacterial growth state havebeen shown to influence the ability to induce BME (Ernstet al., 1990; Galan and Curtiss, 1990; Garcia-Vescoviet al., 1996; Lee and Falkow, 1990; Lee et al., 1992;MacBeth and Lee, 1993). The mechanism by whichS. typhimurium senses these conditions in order to acti-vate the expression of the invasion phenotype is not wellunderstood. To date, three factors are known that regulatethe expression of invasion genes: PhoP/Q, HilA and InvF(Bajaj et al., 1995; Behlau and Miller, 1993; Eichelberget al., 1996; Pegues et al., 1995). The two-componentregulatory system, PhoP/Q, negatively regulates invasionby repressing the transcription of genes which encodetype III secretion-apparatus proteins and several of theSsp (Salmonella secreted protein(s)) (Bajaj et al., 1996;Behlau and Miller, 1993; Eichelberg et al., 1996; Pegueset al., 1995). Two-component regulatory systems containa sensor kinase (homologous to PhoQ) which responds tochanges in environmental conditions by autophosphoryla-tion and transfer of the phosphate to a response regulator(homologous to PhoP). In this activated state, theresponse regulator is able to activate or repress target-gene transcription (Stock et al., 1989). The PhoP/Q two-component regulatory system activates genes requiredfor intracellular survival within acidified macrophages,named pag (PhoP activated genes) (Alpuche-Arandaet al., 1992; Miller et al., 1989) and represses othergenes, termed prg (PhoP repressed genes) which areessential to BME and macropinocytosis of both profes-sional and non-professional phagocytes (Alpuche-Arandaet al., 1994; Behlau and Miller, 1993; Pegues et al., 1995).

Two transcription factors essential for S. typhimuriuminvasion, InvF and HilA, are encoded in SPI1 at centi-some 63. While InvF specifically promotes transcriptionof sspBCDA (Eichelberg et al., 1996), HilA co-ordinately

activates the expression of essential PhoP-repressedinvasion genes, including prgHIJK, sspA, sspC, invF,and orgA (Bajaj et al., 1995; 1996). HilA is a novel proteinwith an amino-terminal domain that is similar to that ofthe transcription factors ToxR of Vibrio cholerae andCadC of Escherichia coli. ToxR and CadC are inner-mem-brane proteins that have amino-teminal domains thatbind DNA. These DNA-binding domains are similar tothe carboxy-terminal DNA-binding domains of the two-component response-regulator superfamily typified byOmpR of E. coli. Two-component system transcriptionfactors modulate gene expression in response to physio-logical changes in the environment via regulatory phos-phorylation (Albright et al., 1989; Stock et al., 1989).Although HilA is an essential regulator of invasion-genetranscription, it does not appear to contain a membrane-spanning region, like CadC and ToxR, or a phosphoryl-acceptor domain, like OmpR, indicating that it alone maynot sense environmental conditions (Bajaj et al., 1995).Thus, we hypothesized that additional factors thatresponded to environmental cues were involved in thetranscriptional activation of invasion genes. In this reportwe describe the identification of a locus, designated sirA(Salmonella invasion regulator), that is required for theactivation of hilA and prgHIJK transcription, Ssp(Salmonella secreted protein(s)) secretion, and bacterialinvasion of epithelial cells. SirA is similar to the Pseudomo-nas spp. virulence regulator GacA and is a member of thetwo-component response-regulator superfamily.

Results

Identification of sirA, a locus essential for induction ofexpression of prgHIJK

In order to identify genes that positively regulate theprgHIJK operon, a bank of Tn10d insertions in the wild-type S. typhimurium chromosome was transduced intostrain IB040, which expresses an enzymatically activePrgH–Alkaline Phosphatase (AP) fusion protein. Colonieswere screened for loss of blue colony phenotype on Luria–Bertani (LB) agar plates containing kanamycin, tetra-cycline, and the chromogenic phosphatase substratep-toluidine salt (XP). Three independent strains wereidentified as having a white colony phenotype, indicatingthat the PrgH–AP fusion was no longer optimallyexpressed. To test whether the Tn10d insertions inacti-vated genes such as dsbA encoding the disulphide-bondreductase that would affect the enzymatic activity of AP(Bardwell and Beckwith, 1993), several derivatives ofthese strains were constructed and tested for AP activity.The Tn10d insertion was transduced into strain CS119containing an active PagC–AP fusion protein. In addition,a plasmid expressing the phoA gene was transformed

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into strains with the white colony phenotype. Assay of theAP activity in these strains demonstrated that the loss ofPrgH–AP activity in strains with the white colony pheno-type was not owing to effects on the AP portion of thefusion protein (data not shown). In all three strains, theloss of blue colony phenotype was demonstrated to be100% associated with the acquisition of transposon-encoded tetracycline resistance, indicating that theTn10d and not a spontaneous mutation was responsiblefor the observed phenotype (data not shown).

Southern blot hybridization of chromosomal DNA fromthe three strains, using a fragment of Tn10d as a probe,showed that they contained a mutation in a DNA regionwith the same physical map, indicating that they were likelyto be inserted in the same locus (data not shown). Onestrain, designated DAP3, was chosen for further study.The locus defined by the transposon insertion in DAP3was designated sirA.

To quantify PrgH–AP activity in DAP3, AP assays wereperformed at different time points in the bacterial growthcurve. DAP3 was impaired in activation of PrgH–APexpression, while the expression of PrgH–AP in IB040with wild-type sirA was approx. 20-fold induced in late-logarithmic growth phase when compared with the earlylogarithmic phase (Fig. 1A).

To confirm that sirA::Tn10d influenced expression ofprgHIJK at the transcriptional level, RNA levels of theprgHIJK operon were examined in wild-type and sirA-mutant bacteria. The sirA::Tn10d fusion was transducedinto CS019, a strain with a wild-type protein-secretionprofile and invasion phenotype ((Behlau and Miller,1993) and Fig. 2), to create strain CJ010. A fragment ofTn10d was used to demonstrate by Southern blot hybridi-zation that the transposon insertion in CJ010 was identicalto that in DAP3 (data not shown). The level of prgHIJKtranscript in CS019 (wild type) and CJ010 was determinedby primer-extension analysis. This analysis showed thatthe prgHIJK transcript was reduced to undetectable levelsin CJ010 compared with CS019 (data not shown).

To quantify the reduction of prgHIJK transcription instrains with sirA::Tn10d, a transcriptional gene fusionbetween the prgHIJK operon and the firefly luciferasegene was constructed within vector pKAS32, which isa pir-dependent suicide plasmid (Skorupski and Taylor,1996). This plasmid (pCJ31) was transferred into aCS019 derivative (carrying a recessive streptomycin-resistance allele) by mating. Homologous recombinationevents were identified by selection for plasmid-encodedampicillin resistance. This resulted in isolation of strainCJ034, containing a prgHIJK gene duplication with a sin-gle-copy gene fusion between prgHIJK and the firefly luci-ferase gene. This expected gene arrangement generatedby homologous recombination was confirmed by Southernblot analysis (data not shown). Therefore, in CJ034,

measurement of firefly luciferase activity allows prgHIJKtranscription to be measured from a single chromosomalgene copy in strains with a wild-type invasion phenotype.To create an isogenic sirA mutant, sirA::Tn10d was trans-duced into CJ034 to create CJ035. Measurement ofluciferase activity of these strains in both logarithmic andstationary phases of growth indicated that prgHIJKtranscription was greatly reduced (80-fold) in CJ035 ascompared with CJ034 (Fig. 1B). This analysis confirmedthat sirA::Tn10d significantly reduced prgHIJK transcription.

hilA transcription is regulated by sirA

Because HilA co-ordinately regulates transcription of anumber of invasion genes in the SPI1, including prgHIJK(Bajaj et al., 1995; 1996), we analysed the effect ofsirA::Tn10d on transcription of hilA by use of a lacZYoperon fusion to iagB, a gene located downstream fromand co-transcribed with hilA (C. Lee, unpublished). Thisfusion allows the effects on hilA transcription to be ana-lysed in the presence of the intact hilA gene product.The sirA::Tn10d was crossed into the hilA–iagB ::lacZYstrain by transduction, creating strain CJ020. hilA tran-scription is reduced six- to sevenfold in CJ020 as com-pared with the wild-type strain (Fig. 1C). The pattern ofinduction of hilA transcription in a wild-type backgroundduring late-logarithmic growth phase mirrors that of theactivation of the prgH ::luc transcriptional fusion andPrgH–AP fusion protein.

sirA is essential for secretion of Ssp andS. typhimurium invasion

As sirA was necessary for the transcription of essentialinvasion genes that encode components of the secretionapparatus, we tested whether sirA was necessary forS. typhimurium Ssp secretion. Ssp from CJ010 were iso-lated and compared with those from CS019 by SDS–PAGE analysis and Coomassie-brilliant-blue staining.The sirA mutant showed a secretion defect similar to thedefect observed for the prgHIJK and hilA mutants(Hueck et al., 1995). Specifically, SspA, SspB, SspCand SspD were not detected in the culture supernatantof the sirA mutant (Fig. 2).

To test the role of sirA in S. typhimurium invasion,assays using cultured epithelial cells were performed. Asshown in Table 1, the sirA mutant had an approx. 100-fold reduction in its ability to invade HEp-2 cells when com-pared with wild-type strain 14028s. The defect was thesame magnitude as that found in prgHIJK and hilAmutants (Bajaj et al., 1995; Pegues et al., 1995). Theseresults demonstrate that sirA is essential for both Sspsecretion and S. typhimurium invasion.


Salmonella typhimurium invasion gene regulation 717

Phenotypic complementation of a sirA mutation bycloned sirA and by two unique cloned DNA loci, sirBand sirC

To identify clones which suppressed the sirA::Tn10deffect on PrgH–AP expression, an S. typhimurium genomiclibrary was transformed into DAP3, and resulting colonies

were screened for blue colony phenotype to indicate arecovery of PrgH–AP fusion-protein activity. Out ofapprox. 19 000 colonies, three blue colonies were isolated,containing plasmids designated pCJ13, pCJ20 andpCJ22, respectively. For all three plasmids, the return ofthe blue colony phenotype was 100% associated withthe acquisition of plasmid-encoded ampicillin resistance


Fig. 1. A. SirA is necessary for induction ofPrgH–PhoA. Comparison of the AP activity ofIB040, containing prgH ::TnphoA, and DAP3,containing prgH ::TnphoA and sirA::Tn10d.The x-axis is the culture OD at 600 nm, andthe y-axis is AP units as defined by Miller(1972) for b-galactosidase.B. SirA is necessary for induction of prgHIJKtranscription. Comparison of the fireflyluciferase activity of CJ034, containing aprgH ::luc operon fusion, and CJ035,containing prgH ::luc and sirA::Tn10d. They-axis is luciferase activity expressed asrelative light units, and the x-axis is theculture OD at 600 nm.C. SirA is necessary for induction of hilAtranscription. Comparison of the b-galactosi-dase activity of CL87, containing a lacZYoperon fusion to hilA (hilA–iagB ::lacZY ),and CJ020, containing the hilA–iagB ::lacZYfusion and sirA::Tn10d. The y-axis is units ofb-galactosidase as defined by Miller (1972),and the x-axis is the culture OD at 600 nm.


by DAP3. DAP3 contains a mutation in the phoN gene andthus is defective in the production of acid phosphatase.Therefore, blue colony phenotype could have beenrestored by plasmids containing the phoN gene encodingthe acid phophatase. Upon transformation into CS019, aphoN mutant, none of the three plasmids conferred bluecolony phenotype, indicating that plasmids pCJ13,pCJ20 and pCJ22 did not contain phoN.

Restriction and Southern blot hybridization analysesindicated that the S. typhimurium DNA fragments inpCJ13, pCJ20 and pCJ22 did not contain overlappingpieces of DNA (data not shown). The location of the clonedfragments on the S. typhimurium chromosome wasdetermined by dot blot hybridization to a set of definedchromosomal DNA fragments from S. typhimurium (Ben-son and Goldman, 1992). This analysis indicated that thecloned fragments from pCJ13, pCJ20 and pCJ22 localizedto centisomes 40.2–44.2, 63 and 37.6–40.2, respectively,on the S. typhimurium chromosome. DNA sequenceanalysis of pCJ20 indicated that it includes DNA from apreviously uncharacterized region of SPI1 (J. L. Rakeman,H. R. Bonifield and S. I. Miller, unpublished). Therefore,the gene responsible for complementation in this plasmidis not hilA or invF. Based on these experiments, we con-cluded that we had isolated three unlinked clones, all of

which could independently suppress the effect of sir-A::Tn10d on PrgH–AP expression.

Southern blot analysis indicated that pCJ13 containedsirA, because DAP3 chromosomal DNA demonstratedan altered physical map when probed with a pCJ13-specific probe (data not shown). In addition, this Southernblot hybridization analysis confirmed that sirA and thegenes responsible for the complementing phenotype onpCJ20 (sirC ) and pCJ22 (sirB ) contained single-copycontinuous pieces of S. typhimurium chromosomal DNA.

To quantify the complementation of sirA::Tn10d by thethree plasmids, alkaline phosphatase assays were perfor-med. sirA, sirB and sirC consistently suppressed theeffects of sirA::Tn10d by restoration of wild-type levelsof PrgH–AP activity to DAP3 (Table 2). The three plasmidswere tested for the ability to complement the defect on hilAtranscription that was the result of sirA::Tn10d. Therefore,these plasmids were transformed into CJ020, whichcontains a hilA–iagB ::lacZY fusion and the sirA::Tn10d,and b-galactosidase activity was measured. All plasmidssuppressed the effect of sirA::Tn10d on hilA tran-scription (Table 2), shown through the restoration ofb-galactosidase activity to wild-type levels.

Definition of sirA DNA necessary forcomplementation of the Ssp secretion and invasiondefects of sirA mutants

To identify the DNA on pCJ13 which contained the sirAgene, fragments from pCJ13 were subcloned into thelow-copy-number vector pWKS30 and transformed intostrain DAP3. These strains were assayed for comple-mentation as measured by AP activity. The results of thisanalysis are shown in Fig. 3. pCJ13b, containing a 1.5 kbPst I–SmaI fragment, was the smallest fragment obtainedin this experiment which suppressed the effect of sir-A::Tn10d on expression of PrgH–AP.

To confirm that pCJ13b could complement the effect ofsirA::Tn10d on protein secretion and invasion, the Ssp pro-files and invasiveness of CJ012, containing the sirA::Tn10dfusion and pCJ13b, were examined. pCJ13b suppressedthe effect of the sirA mutation, restoring Ssp secretion


Fig. 2. SirA is necessary for Ssp secretion. SDS–PAGE of TCA-precipitated material from culture supernatants of various strains(indicated above each lane). Each lane contains Ssp concentratedfrom 2 ml of stationary-phase culture supernatant. Sizes ofmolecular mass markers in kDa are shown to the left . DefinedSsp are indicated by arrows.

Table 1. sirA is required for S. typhimurium invasion.

Strain Mutant allele Plasmid Invasion (%)

14028 Wild type None 18.7IB040 prgH None 0.49CJ010 sirA None 0.19CJ012 sirA pCJ13b 3.8CJ025 sirA pCJ13d 7

Invasion experiments were performed three times in triplicate withessentially the same results. The per cent invasion is expressed asa percentage of total bacteria which survived gentamicin treatment.


(Fig. 2, lane 4). pCJ13b also restored the ability of the sirAmutant to invade HEp-2 epithelial cells (Table 1) to almostwild-type levels. The ability of the cloned sirA to complementthe phenotypes of CJ010 provides further evidence of theimportance of sirA to bacterial invasion.

sirA encodes a transcriptional response regulator ofthe two-component regulatory system superfamily

The DNA sequence of the 1.5 kb insert of pCJ13b wasobtained and is available from GenBank under accessionnumber U67869. Analysis of this sequence revealed anopen reading frame (ORF) designated sirA, which is pre-dicted to encode a protein of 234 amino acids expressedfrom its own promoter, and not from the plasmid-encodedlac promoter. The site of the Tn10d insertion of CJ010 wasdetermined by DNA cloning and sequencing and wasfound to be located at bp 51 of the predicted sirA DNA-coding sequence. The predicted protein sequence ofSirA exhibited 58% identity to the gacA gene productfrom Pseudomonas fluorescens and 96% identity to theuvrY gene product from E. coli (Fig. 4), both of which aremembers of the FixJ family of transcriptional activators.A second ORF was identified 3 ' to sirA that had high simi-larity to a DNA-repair protein encoded by uvrC, which isfound in the same relative locations in E. coli and Pseudo-monas spp. (Moolenaar et al., 1987; Rich et al., 1994;Sharma et al., 1986). Fine-structure mapping of thisDNA region by P22 transduction linkage analysis indicatedthat sirA was located between the fliA and flhDC operons(Fig. 3), which are involved in regulation of flagellar syn-thesis. Strain TH2969, containing a fliC ::MudA insertion,showed 66% linkage to sirA::Tn10d. pCJ13b was


Table 2. Effect of plasmids that suppress the sirA::Tn10d mutationon prgH ::TnphoA fusion protein activity, and on an iagB ::lacZYtranscriptional fusion which is controlled by hilA.

Strain Allele Plasmid Activitya

Effect on prgH ::TnphoA Units of AP

IB040 prgH ::TnphoA None 133DAP3 prgH ::TnphoA, sirA::Tn10d None 21CJ001 prgH ::TnphoA, sirA::Tn10d pWKS30 24CJ002 prgH ::TnphoA, sirA::Tn10d PCJ13 188CJ003 prgH ::TnphoA, sirA::Tn10d pCJ20 229CJ004 prgH ::TnphoA, sirA::Tn10d pCJ22 194CJ018 prgH ::TnphoA, sirA::Tn10d pCJ13d 201

Effect on iagB ::lacZY Units of b-gal

CL87 iagB ::lacZY None 646CJ020 iagB ::lacZY, sirA::Tn10d None 80CJ021 iagB ::lacZY, sirA::Tn10d pCJ13b 558CJ022 iagB ::lacZY, sirA::Tn10d pCJ20 696CJ025 iagB ::lacZY, sirA::Tn10d pCJ13d 705

a. Activities were measured from stationary-phase cultures. The num-bers denote representative values of experiments performed on fourseparate occasions and represent activity as defined by Miller for b-galactosidase (b-gal).

Fig. 3. Physical map of the chromosomalregion containing sirA and the flagellarregulatory genes fliA and flhD. The dottedline represents chromosomal DNA ofundetermined size. Horizontal arrows indicatethe direction of transcription. DNA of variousplasmids derived from pCJ13 and resultingphenotypic complementaion of sirA::Tn10dare indicated by ‘+’ (complementation) or ‘7’(no complementation). Abbreviations ofrestriction sites are as follows: V, EcoRV;S, SmaI; P, Pst I. Horizontal arrows indicatethe direction of transcription of the sirA anduvrC genes. The box on either side of theclone indicates the location of the plasmid-encoded lac promoter.


predicted to contain 275 amino-terminal amino acids outof the predicted 588 amino acids of the uvrC gene pro-duct. Sequencing upstream of the first encoded aminoacid of sirA revealed a putative Shine–Dalgarno site 9 bpupstream of the first codon and potential 710 and 735sequences. The putative promoter sequences upstreamof sirA are 100% identical to the defined promoters ofuvrY in E. coli (Moolenaar et al., 1987), suggesting thatthese sequences might represent the promoter for sirA.

To ensure that the partial uvrC gene product found onpCJ13b was not responsible for the observed supressionof the sirA::Tn10d, polymerase chain reaction (PCR) wasutilized to obtain a 1068 bp fragment containing only thesirA gene and its putative promoter. This fragment wascloned into pWKS30, creating pCJ13d. pCJ13d wasshown to complement the sirA::Tn10d defect in PrgH–AP production (Table 2), hilA–iagB ::lacZY activity (Table2), Ssp production (data not shown), and invasion, to thesame extent as pCJ13b. Taken together, these datademonstrate the role of SirA as an essential transcriptionalactivator of S. typhimurium invasion genes.

Discussion

The ability of S. typhimurium to invade epithelial cellsdepends on the function of a complex sec-independenttype III secretion system, encoded by at least 17 genes,which are clustered at centisome 63 of the S. typhimuriumchromosome. The invasive phenotype and secretion ofSsp is regulated by bacterial growth conditions (Behlauand Miller, 1993; Galan and Curtiss, 1990; Lee andFalkow, 1990; MacBeth and Lee, 1993). In this report weshow that the acquisition of invasion competence corre-lates with the transcriptional activation of invasion genesand depends on sirA, which encodes a transcriptional acti-vator of the two-component response-regulator family. Wedemonstrate that a sirA ::Tn10d mutant is impaired intranscriptional activation of the prgHIJK operon encodingpart of the secretion apparatus (Pegues et al., 1995),and also of a global activator of invasion genes encodedby hilA (Bajaj et al., 1995). Consequently, the sirA::Tnl0dmutant was shown to be impaired in Ssp secretion andepithelial cell invasion. The sirA::Tn10d phenotypescould be suppressed by cloned DNA from three loci, onewhich encodes sirA and two which are from unlinkedloci, designated sirB and sirC.

The deduced amino acid sequence of SirA shows 96%sequence identity to UvrY from E. coli and 58% identityto GacA of P. fluorescens. SirA, UvrY and GacA belongto the FixJ subfamily of response regulators of two-compo-nent regulatory systems. Proteins of the FixJ family arecharacterized by a carboxy-terminal helix-turn-helix DNA-binding domain and an amino-terminal regulatory domain(Kahn and Ditta, 1991). It has been shown that FixJ of Rhi-zobium meliloti activates target gene transcription afterregulatory phosphorylation by the sensor kinase FixL inresponse to changes in environmental oxygen concentra-tion (reviewed in Agron and Helinski, 1995). Although FixJhas not formally been proven to bind DNA (Agron, 1993),overexpression of FixJ induces the expression of the tar-get genes nifK and fixK under non-permissive conditionsand in the absence of its environmental sensor, which isconsistent with the prediction that FixJ is a transcriptionalactivator (Hertig et al., 1989).

While the function of uvrY has not been elucidated inE. coli, gacA has been shown to be required for patho-genicity in Pseudomonas aeruginosa (Rahme et al.,1995) and Pseudomonas syringae (Rich et al., 1994),and for the synthesis of antifungal factors in P. fluorescens(Laville et al., 1992). The P. syringae GacA is thought toactivate the transcription of target genes after phosphory-lation by the genetically unlinked environmental sensorLemA, because gacA and lemA mutants exhibit thesame defects in protease- and syringomycin productionand in plant-lesion formation (Rich et al., 1994). Similarto the genetic organization of gacA of Pseudomonas


Fig. 4. Comparison and alignment of SirA (S. typhimurium ), UvrY(E. coli ), and GacA (P. fluorescens ) predicted gene products.Conserved residues are indicated by vertical bars. The site of theTn10d insertion in sirA at amino acid 17 is underlined. Amino acidsthat are strictly conserved in response regulators are indicated bybold dots above the residues. The helix-turn-helix domain isoverlined.


spp., we did not identify sequences encoding a putativesensor protein immediately adjacent to sirA. Based onthe homology between SirA and GacA, we hypothesizethat S. typhimurium contains an unlinked sensor proteinwhich senses an environmental signal(s) and activatesSirA by regulatory phosphorylation.

In both E. coli and P. fluorescens, the uvrC gene, whichis required for the repair of UV-induced DNA damage, isdirectly downstream of the uvrY/gacA gene (Laville et al.,1992; Rich et al., 1994; Sharma et al., 1986). We foundthis gene organization to be conserved in S. typhimuriumand this raised the question of whether DNA damage couldbe an in vivo signal recognized by virulent bacteria thatresults in phosphorylation of SirA/UvrY/GacA. However,neither uvrC nor uvrY transcription was induced by UVlight or other DNA-damaging agents when single-copyrecorders were tested (Walker, 1996). Studies in E. colihave shown that there are a number of putative promotersupstream of the uvrC gene that may be utilized for thetranscription of uvrC in vivo. One promoter is located infront of the uvrY gene. A second promoter is found inthe C-terminal coding sequence of uvrY (Moolenaaret al., 1987; Sharma et al., 1986). Although Northernblot hybridization and S1 nuclease mapping experimentshave suggested that both promoters are capable of pro-moting uvrC transcription, an E. coli uvrY-insertion mutanthas wild-type UV sensitivity, indicating that transcriptionfrom the promoter in the C-terminus of uvrY produces suf-ficient uvrC to protect against UV in vivo (Moolenaar et al.,1987). These C-terminal promoter sequences are con-served in sirA (nucleotides 447–453, 471–477), indicatingthat the sirA::Tn10d insertion is probably not polar onuvrC transcription. These data, coupled with the fact thatsuppression of the sirA::Tn10d phenotypes could beaccomplished with plasmids that do not contain uvrC, donot point to DNA damage as a signal for S. typhimurium toinduce invasion-gene transcription via SirA phosphorylation.

Because hilA directly regulates prgHIJK transcription,as well as that of many other invasion genes (Bajaj etal., 1996), the demonstration that sirA regulates hilA indi-cates that a cascade of transcription-factor synthesis isimportant for invasion (see Fig. 5). The residual transcrip-tion of hilA and prgH (80 and 25 units of b-galactosidaseand AP activity, respectively) in the sirA mutant indicatesthat there may be control of hilA by factors other thansirA. The fact that two DNA fragments (sirB and sirC ), inaddition to sirA, were isolated that suppressed the effectof sirA::Tn10d on hilA–iagB ::lacZY activity suggests thatthis regulatory cascade could include other factorsbesides sirA and hilA. Therefore, SirA may not directly reg-ulate hilA transcription, but may, in fact, do so by activationof sirB and sirC transcription. The presence of intact sirBand sirC could, in part, explain the residual transcription ofhilA. Interesting in this regard is the fact that a plasmid

containing sirC increased hilA transcription to greaterthan wild-type levels in the absense of sirA (Table 2).Because the suppression of the sirA::Tn10d phenotypeby sirB and sirC was observed with multicopy plasmids(6–8 copies per cell), and with the possibility of geneexpression from the constitutive lac promoter (Fu andKushner, 1991), further work is necessary to determinethe role of sirB and sirC in the regulation of invasion-gene expression. Therefore, although we hypothesizethat SirA phosphorylation is an early step in a regulatorycascade of multiple transcription factors that results inbacterial invasion, there may be SirA-independent path-ways to activate hilA transcription.

An intriguing feature of the sirA locus is that, like PhoP/PhoQ (located at centisome 27.4), its chromosomallocation (centisome 42.4) is outside the centisome 63


Fig. 5. Schematic diagram of hyphothesized regulatory cascadenecessary for S. typhimurium invasion of epithelial cells. OM andIM represent the outer and inner membrane, respectively. ‘+’indicates promotion of transcription and ‘7’ indicates negativeregulation of transcription. The negative regulation of invasiongenes by PhoP/PhoQ is shown at multiple levels, but where PhoP/PhoQ acts in the regulatory cascade is unknown. HilA is shown toregulate invF which regulates sspBCDA, although HilA regulation ofsspBCDA remains a possibility. SirA–PO4 is shown to directlyregulate hilA, although this regulation may occur only via sirBand/or sirC.


pathogenicity island. The pathogenicity island is present inall invasive Salmonella strains (reviewed in Galan, 1996)and was probably acquired by horizontal gene transferfrom another microorganism after the evolutionary splitof S. typhimurium from E. coli (Galan et al., 1992; Ginno-chio et al., 1992; Groisman and Ochman, 1993; Li et al.,1995). Therefore, the ability of SirA and PhoP/Q to regu-late invasion genes probably adapted independentlyfrom the acquisition of the pathogenicity island.

The morphology of epithelial cell invasion is similar in S.typhimurium and Shigella flexneri. Both organisms utilizetype III secretion apparati and homologous secreted pro-teins to enter epithelial cells by induction of membraneruffling. Temperature, DNA supercoiling, and osmolarityare known environmental signals that are sensed by S.flexneri to promote invasion (reviewed in Galan and San-sonetti, 1996). The genes involved in this regulation arefound on both the virulence plasmid and on the chromo-some of S. flexneri. However, it is possible that other regu-latory factors of S. flexneri invasion genes remain to bediscovered. As the SirA/GacA family appears to be con-served in a wide number of Gram-negative bacteria, andregulates invasion of epithelial cells by S. typhimuriumand effects in both plant and animal Pseudomonas sp.virulence (Rahme et al., 1995; Rich et al., 1994), it willbe exciting to determine whether a sirA/gacA homologueis essential to virulence of S. flexneri and other intestinalpathogens. It is interesting to speculate that commonenvironmental stimuli from the mammalian intestine medi-ate the activation of invasion genes of a wide variety ofintestinal pathogens.

Experimental procedures

Bacterial strains, plasmids and libraries

The bacterial strains and plasmids used are listed in Table 3.All aerobically cultured bacterial strains were grown in LBbroth at 378C in a roller drum (New Brunswick Scientific).For oxygen-limiting growth conditions, bacteria were grownat 378C without rolling or shaking. The antibiotics used wereampicillin (50mg ml71), kanamycin (45mg ml71) and tetracy-cline (20mg ml71). For screening purposes, the chromogenicsubstrates Xgal, IPTG and XP were used in LB agar platesat 40mg ml71.

The JS82 library of 8–10 kb Sau 31A fragments from S.typhimurium cloned into the low-copy-number pBluescriptderivative, pWKS30 (Fu and Kushner, 1991), was generouslyprovided by Dr Jim Slauch, University of Illinois, Champagne-Urbana, USA. Plasmid DNA from this library was isolatedusing the Qiagen procedure.

Plasmid pCJ31 containing a transcriptional fusion ofprgHIJK to the firefly luciferase gene was constructed asfollows. A 5 kb Bgl II–HindIII fragment containing the prgH pro-moter was end filled using the Klenow fragment of DNA poly-merase I and cloned into the end-filled Bgl II and EcoRI sitesof pKAS32 (Skorupski and Taylor, 1996). A 485 bp EcoRV

fragment internal to prgK was removed from this constructand replaced with a 1.5 kb SmaI–HindIII fragment frompGPL01, containing a promoterless firefly luciferase gene.

DNA techniques

Restriction endonucleases were purchased from New Eng-land Biolabs. T4 DNA Ligase was purchased from GibcoBRL. Shrimp alkaline phosphatase was purchased fromUSB. All plasmid DNA isolation was performed using the alka-line lysis method (Maniatis et al., 1982) or Qiagen columns(Qiagen). DNA sequencing of both strands was performedusing the procedure for the Sequenase Version 2.0 kit (USB)and sequences were analysed using the package from theUniversity of Wisconsin Genetics Computer Group (GCG),version 7. Oligonucleotide primers that hybridized to thepWKS30 vector and S. typhimurium DNA were synthesizedby Universal DNA and used for DNA sequencing and PCR.Primers used to amplify the sirA gene by PCR were: SirA–1His, 5 '-GAAAGCTTGCGTCAAATATTTCACTCACTGG-3 'and M-13 plus, 5 '-AAACGACGGCCAGTAGCGCGCGTA-3 '. PCR was performed according to the protocol given byNew England Biolabs for Vent polymerase. ChromosomalDNA was isolated as previously described by Mekalanos(1983) as modified by Stone et al. (1992). Southern blot hybri-dization analysis was performed on restriction endonuclease-digested chromosomal DNA or plasmid DNA separated byelectrophoresis in a 1% agarose gel and transferred to anylon membrane (NEN–DuPont) according to standard pro-cedures. DNA probes were labelled with digoxigenin andhybridization was performed under high-stringency condi-tions according to the GENIUS protocol (Boehringer Man-nheim). The chromosomal location of cloned plasmid DNAwas determined by Southern blot hybridization of bacterioph-age DNA isolated from a set of Mud-P22 insertions as pre-viously described (Benson and Goldman, 1992). PhageDNA was isolated by polyethylene glycol precipitation frommitomycin-induced lysates. Mud-P22 DNA was blotted ontoa nylon membrane using a DotBlot apparatus (Bio-Rad).

Tn10d mutagenesis

Mutagenesis of ATCC 14028s was performed using Tn10d(Hughes and Roth, 1988). Plasmid pNK972 was used as adonor of transposase after transformation into ATCC14028s (Bender and Kleckner, 1986). TT10604 (Hughesand Roth, 1988) was used as the Tn10d donor. After muta-genesis, approx. 50 000 tetracycline-resistant colonies werepooled and infected with P22 bacteriophage at a multiplicityof infection (m.o.i.) of 1.0 for 2 h. These lysates were crossedwith IB040, a strain containing a prgH ::TnphoA fusion, andcolonies were selected for both tetracycline- and kanamycinresistance. Loss of fusion protein activity was screened forby the loss of blue colony phenotype on XP plates.

Alkaline phosphatase and b-galactosidase assays

Alkaline phosphatase and b-galactosidase assays were per-formed as previously described (Michaelis et al., 1983). Unitswere calculated as defined by the method of Miller (1972).



RNA techniques

RNA was isolated from late-logarithmic-phase cultures usingthe hot-phenol method (Case et al., 1988). Primer-extensionanalysis was performed as previously described (Miller et al.,1986), using SuperScriptII RNase–Reverse Transcriptase(Gibco BRL) and oligonucleotide primers corresponding tonucleotides 1094–1072 of the prgHIJK locus (Universal DNA).

Luciferase assays

Luciferase assays were performed by mixing 90ml of bacterialculture with 10ml of 1M K2HPO4 pH 7.8, 20 mM EDTA pH 8.0,and subjecting the whole to freeze–thaw cycles. Next, 900mlof 1� luciferase buffer (25 mM Tris-phosphate, pH 7.8; 2 mMdithiothreitol (DTT); 2 mM EDTA; 2.5 mg ml71 BSA; 10%glycerol; 1% Triton X-100) was added and the cell suspension

was briefly sonicated to lyse all remaining cells. Cell debriswere pelleted by brief centrifugation at high speed in a micro-fuge, and 10ml of this lysate was assayed for 10 s in a BertholdLB9501 luminometer (100ml injector) using the luciferaseassay reagents from Promega.

Secreted-protein isolation and analysis

Salmonella secreted proteins were isolated by trichloroaceticacid (TCA) precipitation from stationary-phase cultures andanalysed by SDS–PAGE in Tris–Tricine buffer according topreviously described techniques (Hueck et al., 1995; Pegueset al., 1995).

Invasion assays

HEp-2 epithelial cells were grown in Dulbecco’s Modified


Table 3. Bacterial strains, plasmids, and libraries used in this work.

Strain/Plasmid Genotype/Phenotype Source/Reference

Strain

E. coli DH5a F7 (f80dlacZDM15 )lacZYA–argF U169 endA1recA1 hsdR17 deoR thi-1supE44 (l gyrA96 relA1 )

BRL

S. typhimurium14028s Wild type ATCCCS019 phoN2 zxx ::6251Tn10d-Cm Miller et al. (1989)TT10604 Tn10d donor proAB47/F '

pro + lac + zzf1831 Tn10dHughes and Roth

(1988)IB040 CS019 with prgH1::TnphoA Behlau and Miller

(1993)DAP3 IB040 with sirA::Tn10d This workCJ001 DAP3 with pWKS30 This workCJ002 DAP3 with pCJ13 This workCJ003 DAP3 with pCJ20 This workCJ004 DAP3 with pCJ22 This workCJ005 DAP3 with pCJ13a This workCJ006 DAP3 with pCJ13c This workCJ007 DAP3 with pCJ13b This workCJ010 CS019 with sirA::Tn10d This workCJ011 CJ010 with pCJ13 This workCJ012 CJ010 with pCJ13b This workCJ018 DAP3 with pCJ13d This workCJ019 CJ010 with pCJ13d This workCL87 hilA–iagB ::lacZY C. Lee (HMS)CJ020 CL87 with sirA::Tn10d This workCJ021 CJ020 with pCJ13b This workCJ022 CJ020 with pCJ20 This workCJ023 CJ020 with pCJ22 This workCJ025 CJ020 with pCJ13d This workCJ034 CS019 with prgH ::luc

(chromosomal integrationof pCJ31 into prgH )

This work

CJ035 CJ034 with sirA::Tn10d This work

Plasmid

pWSK29 AmpR Fu and Kushner(1991)

pWKS30 AmpR Fu and Kushner(1991)

pNK972 AmpR Bender andKleckner (1986)

pCJ13 AmpR, 3 kb of sirA region This work

Table 3. Continued.

Strain/Plasmid Genotype/Phenotype Source/Reference

pCJ13a AmpR, 2.1 kb Pst I fragmentof sirA region (contains allof sirA and uvrC )

This work

pCJ13b AmpR, 1.5 kb Pst I–SmaIfragment (containsupstream portions of sirA,all of sirA and part ofuvrC )

This work

pCJ13c AmpR, 1 kb EcoRV fragment(contains upstream portionof sirA and N-terminus ofsirA)

This work

pCJ13d AmpR, 1.1 kb PCR fragment(contains upstreamportions of sirA and theentire sirA gene)

This work

pCJ20 AmpR, contains 4.4 kb ofsirC region

This work

pCJ22 AmpR, contains 4.6 kb ofsirB region

This work

pWKSH5 AmpR, contains 5 kb ofprgHIJK region

Pegues et al.(1995)

pGPL01 AmpR, luciferase suicidevector

Gunn and Miller(1996)

pKAS32 AmpR, StrepS, pGP704-based suicide vector

Skorupski andTaylor (1996)

pCJ30 AmpR, StrepS, 5 kb insertfrom pWKSH5 cloned intopKAS32

This work

pCJ31 1.5 kb fragment of pGPL01,containing the luciferasegene, cloned into prgKregion of pCJ30

This work

Library

JS82 8–10 kb fragments of S.typhimurium chromosomalDNA generated by partialdigestion with Sau 31Aand ligated into pWKS30

Jim Slauch (U of I)

ATCC, American type culture collection; HMS, Harvard MedicalSchool; DMS, Dartmouth Medical School; U of I, University of Illinois;AmpR, ampicillin resistant; StrepS, streptomycin sensitive.


Eagle Medium (DMEM) and 10% heat-inactivated fetal bovineserum (FBS) and passagedevery 2–3 d. Invasion assays wereperformed as previously described (Hueck et al., 1995; Leeand Falkow, 1990).

Acknowledgements

We thank Jim Slauch for generously providing the S. typhi-murium genomic library JS82, and Kelly Hughes for assis-tance with the fine chromosomal mapping of sirA. We alsothank members of our laboratory for helpful discussions andtechnical advice. This work was supported by GrantAI30479 from the National Institutes of Allergy and InfectiousDisease (NIAID) to S.I.M., and Grant AI33444 from the NIAIDto C.A.L. D.A.P. was supported by an individual NationalResearch Service Award from the NIAID (Grant AI08873).

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