Vol. 176, No. 15JOURNAL OF BACTERIOLOGY, Aug. 1994, p. 4627-46340021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

vacC, a Virulence-Associated Chromosomal Locus of Shigellaflexneri, Is Homologous to tgt, a Gene Encoding tRNA-Guanine

Transglycosylase (Tgt) of Escherichia coli K-12JEROME M. DURAND,' NOBUHIKO OKADA,' TORU TOBE,1 MASAHISA WATARAI,'ICHIRO FUKUDA,' TOSHIHIKO SUZUKI,1 NOBORU NAKATA,2 KEIKO KOMATSU,'

MASANOSUKE YOSHIKAWA,' AND CHIHIRO SASAKAWA1*

Department of Bacteriology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo108,1 and National Institute for Leprosy Research, 4-2-1, Aobacho, Higashi-murayama, Tokyo 189,2 Japan

Received 8 March 1994/Accepted 19 May 1994

The genetic determinants required for invasion of epithelial cells by Shigellaflexneri and for the subsequentbacterial spreading are encoded by the large virulence plasmid. Expression of the virulence genes is under thecontrol of various genes on the large plasmid as well as on the chromosome. We previously identified one ofthe virulence-associated loci nearphoBR in the NotI-C fragment of the chromosome of S. flexneri 2a YSH6000and designated the locus vacC. The vacC mutant showed decreased levels of IpaB, IpaC, and IpaD proteins as

well as transcription of ipa, an operon essential for bacterial invasion (N. Okada, C. Sasakawa, T. Tobe, M.Yamada, S. Nagai, K. A. Talukder, K. Komatsu, S. Kanegasaki, and M. Yoshikawa, Mol. Microbiol. 5:187-195,1991). To elucidate the molecular nature of the vacC locus, we cloned the vacC region from YSH6000 on a 1.8-kbSalI-BamHI DNA fragment. The nucleotide sequence of the 1,822-bp vacC clone was highly (>98%) homologousto the tgt region of Escherichia coli K-12, which is located at 9.3 min on the linkage map. Complementation testsindicated that the vacC function was encoded by an open reading frame expressing a 42.5-kDa protein, whichcorresponded to the tgt gene of E. coli K-12, coding for tRNA-guanine transglycosylase (Tgt) (K. Reuter, R.Slany, F. Ullrich, and H. Kersten, J. Bacteriol. 173:2256-2264, 1991). The cloned tgt gene from E. coli K-12restored the virulence phenotype to the vacC mutant of YSH6000. Characterization of the vacC mutantindicated that levels of VirG, a protein essential for bacterial spreading, and VirF, the positive regulator forthe expression of the virG and ipaBCD operons, decreased significantly compared with those of the wild type.Similar phenotypic changes occurred in vacC mutants constructed by insertion of a neomycin resistance gene

in shigellae and enteroinvasive E. coli strains, consistent with the hypothesis that the vacC (tgt) gene

contributes to the pathogenicity of Shigella flexneri.

Shigellae causes bacillary dysentery in humans and primates.The early essential steps in pathogenesis comprise invasioninto colonic epithelial cells and subsequent bacterial multipli-cation and spread within the cytoplasm. Consequently, thepathogenicity of shigellae is multifactorial, involving manycomponents of the bacteria, each of which may be under thecontrol of composite regulatory systems.

Eight virulence-associated (vir) loci have been identified onthe large 230-kb plasmid of Shigella flexneri 2a YSH6000. In a31-kb DNA segment, five contiguous virulence segments,designated virB, ipaBCD (ipa), region 3, region 4, and region 5,have been characterized (1, 8, 14, 36, 38, 46, 47). The virB geneacts as a positive regulator for the expression of the other fourvirulence regions (1). The ipa operon encodes three invasion-associated antigens, IpaB, IpaC and IpaD, and IcsB, a proteinessential for intercellular spreading of the bacteria (3), whileregions 3, 4 (mxi), and 5 (spa) are required for secretion of theIpa proteins (4-7, 40, 47). The virG gene, outside the cluster,encodes a 116-kDa surface-exposed outer membrane proteinand is essential for intra- and intercellular spreading of theinvading bacteria (1, 12, 23, 24). The virK gene encodes a36-kDa protein involved in VirG production (27). The virFgene encodes a 30-kDa protein (31, 32) which regulatespositively the expression of the virG and virB genes (33). Thus,expression of the invasion-associated genes on the large plas-

* Corresponding author. Fax: 81-3-5449-5405.

mid is under the control of a dual activation system directed bythe virF and virB genes (1).

Various classes of vir loci have been identified on thechromosome of S. flexneri. Interestingly, some of them areinvolved in expression of the vir genes encoded by the largeplasmid. For example, a locus near the trp gene designated virR(osmZ or hns) controls the temperature-dependent expressionof the invasion-associated genes such as the ipa and region 3,4, and 5 operons (16, 21, 25). The envZ and ompR genes, nearthe malA gene, have also been shown to be involved in theexpression of virulence through the regulation of vir genes onthe large plasmid (11). The vacB locus near the purA gene isrequired for the production of the virulence-associated anti-gens IpaB, IpaC, IpaD, and VirG (44). These findings indicatethat the full virulence phenotype of S. flexneri requires variousgenes dispersed around the chromosome as well as on the largeplasmid and that efficient expression of vir genes encoded bythe large plasmid is controlled by various regulatory systems.

In this context, to further elucidate the roles of chromo-somal virulence genes in the pathogenicity of S. flexneri, weperformed random Tn5 insertion mutagenesis and screenedchromosomal insertion mutants for reduced virulence in afocus plaque-forming test (FP test) (29). One of the mutants,called N1436, whose mutated virulence-associated locus(vacC) was close to thephoBR region, showed reduced contacthemolytic (Chl) activity (29), a property shown to correlatewith invasion ability (35). In addition, the levels of productionof the IpaB, IpaC, and IpaD proteins as well as the level of the

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TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Relevant characteristic(s) Reference orsource

S. fiexneri 2aYSH6000 Virulent strain 39YSH6000T A tetracycline-, chloramphenicol-, ampicillin-, and streptomycin-sensitive derivative of YSH6000 27YSH6200 An avirulent strain of YSH6000 cured of the 230-kb plasmid 38N1436 YSH6000 vacC::TnS 29TC501 A vacC::Tn5 transductant of YSH6000T from N1436 This study

S. dysenteriae 6 Our collectionS. flexneri 5 Our collectionS. boydii 4 Our collectionS. sonnei form I Our collectionEIEC 0152 Our collectionE. coli K-12W3110 22HfrH metB Our collectionK38 HfrCA; a host strain for the T7 RNA polymerase-dependent expression system 43JM109 recAl supE44 endAl hsdRl7 gyrA96 relAl thl A(lac-pro) F'[traD35 proAB lacIq lacZM15] 25aSY327Apir F- araD A(lac-pro) argE(Am) recA56 Rif' nalA 26

PlasmidspBluescriptIIKS+ Apr, phagemid cloning vector StratagenepBluescriptIISK+ Same as pBluescriptIIKS+ but the opposite orientation of the multiple-cloning sitespMW119 NippongenepMW119Tp A pMW119 derivative containing a Tpr gene This studypT7-5 T7 RNA polymerase promoter-dependent expression vector 43pT7-6 Same as pT7-5 but the opposite orientation of the multiple-cloning sites 43pGP1-2 T7 RNA polymerase expression vector 43pY12 pBluescriptIIKS+ containing the 1.8-kb SalI-BamHI segment from YSH6000 encoding the vacC This study

locuspBS145 pBluescriptIIKS+ containing the 1.8-kb SalI-BamHI segment from Kohara phage 145 This studypJD100 pMW119Tp containing the 1.8-kb SalI-BamHI segment encoding the vacC locus This studypJD101 A linker insertion mutation at the SmaI site on pJD100 This studypJD102 A deletion mutation of the 200-bp EcoRV segment on pJD100 This studypJD103 A fill-in mutation at the ApaLI site on pJD100 This studypJD104 Same as pJD102 but with the same fill-in mutation as on pJD103 This studypGP704 A suicide vector 26

ipa mRNA were reduced in N1436 compared with those of thewild type, YSH6000 (29). Furthermore, N1436 showed re-duced capacity in the Sereny test for provoking keratoconjunc-tivitis (41). These data taken together indicated that N1436 wasless efficient in the invasion of epithelial cells and, perhaps, insubsequent bacterial spreading into adjacent cells. Hence, inthis study, we further characterized N1436 and undertook toidentify the genetic determinant responsible for vacC function.The results indicated that the vacC gene of S. flexneri wasequivalent to the tgt gene of Escherichia coli K-12, encodingtRNA-guanine transglycosylase (Tgt) (19, 30).

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacteria and plasmidsused are listed in Table 1.

Media. Bacteria were routinely grown at 37°C in brain heartinfusion broth (Difco, Detroit, Mich.) or LN broth (37).Mueller-Hinton broth (Difco) was used only to grow bacteriacarrying trimethoprim resistance. Solid media were made bythe addition of agar (1.5%) to LN broth. Antibiotics wereadded as appropriate to the following final concentrations:ampicillin, 250 ,g/ml; kanamycin, 50 jig/ml; trimethoprim, 12.5,ug/ml.Mating and transduction procedures. Portions (5 ml) of

overnight cultures of HfrH and N1436 were washed with0.85% saline and suspended in 5 ml of LN broth. HfrH (0.2ml), N1436 (2 ml), and LN broth (2 ml) were mixed and

incubated at 37°C for 30 min. Mating was terminated byvortexing the mixture vigorously, and the bacteria were washedwith saline and plated on M9-lactose (0.2%) agar (25a)containing nicotinic acid (2 ,ug/ml) and kanamycin (50 ,ug/ml).Note that the HfrH strain used was Lac' and metB, whileN1436 was a Kmr (TnS) derivative of YSH6000 which wasLac- and required nicotinic acid. The Km' Lac' transconju-gants of N1436 thus selected on the minimum medium werepicked and purified on the same medium. Preparation ofphage P1 lysate from N1436 and the transduction procedure toYSH6000T were done as described by Formal et al. (18).

Virulence phenotype assays. The FP test (38) and Chl assay(35) have been described previously.

Preparation of vacC probe. The DNA region flanking theTnS insertion in N1436 was cloned to be utilized as a vacC-specific probe. A 4.4-kb BamHI segment, consisting of the Kmrgene of TnS, ISSOL, and its flanking DNA sequence, wascloned from the N1436 chromosome into pBluescriptIIKS'.An internal 1.6-kb DNA segment of the 4.4-kb BamHI seg-ment containing the TnS-flanking DNA sequence was used asthe vacC probe.

Immunoblotting. Bacteria were grown at 37°C in brain heartinfusion broth to mid-log phase (A60, approximately 0.5) andharvested. The bacteria were washed and then boiled for 3 minin sample buffer (0.1 M Tris [pH 6.8], 3.2% sodium dodecylsulfate (SDS), 16% glycerol, 8% 2-mercaptoethanol) prior toSDS-polyacrylamide gel electrophoresis (SDS-PAGE). Immu-

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noblotting of SDS-PAGE-resolved antigens was performed as

described previously (27). The VirG-specific antiserumVRG-C was prepared in our previous study (27). A VirF-specific antiserum, VRF-C, was obtained by immunization ofrabbits with peptides encompassing residues 250 to 262 (ITP-KKFYLYHKKF) of the VirF polypeptide (32) by coupling tokeyhole limpet hemocyanin with benzidine.RNA extraction and dot blotting. RNA was extracted from

bacteria as described previously (2) with slight modification(45); briefly, bacteria grown in LN broth were chilled quickly,harvested, and suspended in ASE buffer (20 mM sodiumacetate [pH 4.8], 0.5% SDS, 1 mM EDTA). After addition ofphenol (equilibrated in 20 mM sodium acetate, pH 4.8), themixture was incubated at 60°C for 5 min with gentle shaking.After centrifugation, the RNA was precipitated by addition of2 volumes of ethanol to the aqueous phase. The precipitatedRNA was then collected by centrifugation, redissolved in ASEbuffer, and precipitated with ethanol two more times. Afterbeing washed with 70% ethanol, the final precipitate was

dissolved in ASE buffer, and the RNA concentration was

determined by measuring the A260. Northern (RNA) dothybridization analysis was described previously (44), as were

the DNA probes specific for virG and virF (27, 44). The relativeradioactivity count was measured with a FUJI BAS 2000apparatus (Fuji Film, Co., Ltd.).

Nucleotide sequence. The sequences of both DNA strandswere determined by the chain termination method of Sanger etal. (34), with a Sequenase 7-deaza-dGTP kit (United StatesBiochemical Corp.), following cloning into pBluescriptIISK+and KS'.

Protein product analysis. Protein products specific for thecloned fragment were analyzed by the T7 RNA polymerase-promoter system (43) with the K38 strain harboring pGP1-2and pT7-5 (or pT7-6) containing the 1.8-kb vacC SalI-BamHIsegment.

Construction of vacC mutated strains. To inactivate thevacC gene in shigellae and enteroinvasive E. coli (EIEC)strains, an internal 207-bp EcoRV fragment of a truncatedvacC segment was replaced with a neomycin resistance gene

(neo) cassette and cloned into pGP704, a suicide vector (26).The neo cassette constructed consisted of a 1,375-bp neo gene

segment from pBR322::TnStacl (15) and a 177-bp rnBT1segment from pKK232-8 (Pharmacia), in which the rmBTl, a

transcriptional terminator, was placed upstream of the neopromoter. The resulting pGP704 plasmid containing vacC::neowas introduced from SY327Apir into shigellae and EIECstrains, and Kmr Aps transformants selected were used for thevirulence assay.

Nucleotide sequence accession number. vacC nucleotidesequence data will appear in the DDBJ, EMBL, and GenBankdatabases with accession number D26469.

RESULTS

Identification of the vacC locus. We previously showed thatthe Tn5 in N1436 was inserted into the NotI-C fragment of thechromosome, which consisted of a DNA segment that hybrid-ized with DNA probes obtained from the phoBR (9.1 min),purE (12.2 min), and galK (17.0 min) genes of E. coli K-12 (29).In that study, the 33- and 337-kb NotI-C subfragments gener-

ated as a result of the NotI site on TnS in N1436 hybridizedwith the phoBR probes and the purE and galK probes, respec-

tively, indicating that the vacC locus was located between thephoBR and purE genes (29). Thus, we first asked if the vacClocus was also present on the chromosome of E. coli K-12. Totest this, a conjugational cross of N1436 (Lac- Kmr) with E.

coli K-12 HfrH (Lac' Kms) was carried out, and the twelveLac' KmS intergeneric transconjugants of N1436 constructedwere examined for the ability to form plaques (Vir+) in the FPtest. The results showed that all of the transconjugants wererestored to the Vir+ phenotype in the FP test, suggesting thatthe chromosome of E. coli K-12 contains vacC.To clone the vacC locus from the chromosome of E. coli

K-12 W3110, the vacC probe constructed (see Materials andMethods) was hybridized with membranes containing theordered set of Kohara A phage clones (Takara Shuzo Co., Ltd.)(22). Only one of the X phage clones, number 145, reacted,indicating that the vacC locus lies near 9.3 min. Furtherhybridization analysis of restriction segments of X phage clone145 with the vacC probe located the hybridizing DNA se-quence to a 7.8-kb KpnI segment (Fig. 1). The 7.8-kb KpnIsegment cloned into pBluescriptIIKS+ (pEVC2) was tested bythe FP test and the Chl test for its ability to restore the Vir+phenotype to a vacC::TnS transductant of YSH6000T (TC501).YSH6000T is a strain sensitive to ampicillin, tetracycline,chloramphenicol, and streptomycin derived from YSH6000(reference 27 and Table 1). The results showed that TC501carrying pEVC2 gave rise to plaques in the FP test and showedan Inv' phenotype in the Chl test, indicating that the KpnIfragment encodes the vacC function. To localize the vacCregion, various deletion derivatives were constructed frompEVC2 and tested for the ability to complement TC501 in theFP test. The DNA region encoding the vacC function wasthereby localized to a 1.8-kb SalI-BamHI segment (Fig. 1).We then cloned the DNA segment from YSH6000 capable

of restoring the Vir+ phenotype to TC501. DNA fragments ofabout 2 kb generated by SalI-BamHI digestion of chromosomeDNA were excised from an agarose gel, ligated to pBluescriptIIKS+, and transformed into JM109. Plasmid DNA extractedfrom each of the resulting 80 transformants was then subjectedto DNA dot hybridization with the vacC probe, therebyidentifying four positive clones. The four resultant plasmidscould all restore the Vir+ phenotype to TC501 in the FP test,and all contained a 1.8-kb SalI-BamHI segment. One of theclones, designated pY12, was further characterized.

Nucleotide sequence of the vacC region and analysis of itsprotein products. The nucleotide sequence of the 1,822-bpSalI-BamHI segment of pY12 was determined and found tocontain two open reading frames (ORFs), from nucleotides215 to 1339 (ORF-1) and from nucleotides 1365 to 1694(ORF-2) (Fig. 2). The 5' end of each ORF was oriented towardthe Sall site. ORF-1 was terminated by a TAA stop codon,although this was followed in frame by the three additionalstop codons TAA TAA TGA. The deduced sequences of the375 and the 110 amino acids encoded by ORF-1 and ORF-2,respectively, indicated proteins of 42.5 and 12 kDa. The site ofTnS insertion in N1436 was found to be at nucleotide 717 fromthe 5' end of ORF-1.

Protein products expressed from the 1,822-bp SalI-BamHIsegment were analyzed with the phage T7 RNA polymerase-dependent expression system. The results showed that 45- and13-kDa proteins were expressed from the 1,822-bp Sall-BamHI segment in the correct orientation to the phage T7RNA polymerase-directed promoter 010 on the vector pT7-6(Fig. 3).A search of the GenBank (R73.0) and EMBL (R32.0)

sequence databases with the 1,822-bp sequence revealed sig-nificant (98%) homology of the DNA sequence with a reportedDNA sequence from the tgt region, which consisted of a 3'portion of the queA gene, tgt, and ofl2 located at 9.2 to 9.3 minon the map of E. coli K-12 (30). The tgt gene has been shownto encode tRNA-guanine transglycosylase, while the function

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FIG. 1. Identification of the vacC locus. The top line represents the NotI-C fragment and portions of the adjacent NotI-F and NotI-Q fragmentsof the YSH6000 chromosome (28). The arrowhead over the line indicates the site of TnS insertion in N1436. The restriction map under the NotImap indicates the allocation of a part of Kohara's map of E. coli K-12 containing the phoBR and tgt regions (22). The numbers over and beneaththe map indicate positions in kilobases and minutes, respectively. The thick bars under the map represent Kohara's A clones 144, 145, 146, and147. The enlarged restriction map under X clone 145 shows the 7.8-kb KpnI segment cloned into pBluescriptIIKS+. The black box in the KInIsegment represents a portion of the A vector, EMBL4 (22). The bars under the Kjpnl map indicate the subclones in pBluescriptIIKS+. Fpa, plaqueforming ability of subclones introduced into TC501 as measured by the FP test; P, positive plaque formation; P/F, small plaque formation; Bm,BamHI; Bg, BglII; E, EcoRI; Ev, EcoRV; H, HindIII; Kp, KpnI; Pt, PstI; Pv, PvuII; S, Sal.

of orfl2 remains unknown (30). The comparison of the 375-amino-acid sequence deduced from ORF-1 in the vacC regionwith that from the tgt gene in E. coli K-12 indicated that the twoproteins were identical except for one amino acid, and that the110 amino acids encoded by ORF-2 downstream of ORF-1were identical to those of the 12-kDa protein encoded by orfl2of E. coli K-12 (Fig. 3). These results indicated that the vacClocus on the chromosome of YSH6000 was equivalent to the tgtregion on the chromosome of E. coli K-12 (30).

Identification of the genetic determinant encoding the vacCfunction. To confirm the ability of the 1,822-bp SalI-BamHIsegment from YSH6000 to restore the Vir+ phenotype toN1436 and to determine which ORFs were responsible for thevacC phenotype, the 1,822-bp SalI-BamHI segment was sub-cloned into a trimethoprim-resistant derivative of pMW19(pMW119Tp), a pSC101-based low-copy-number vector. Aseries of mutants, with mutations such as a linker insertionmutation in ORF-1 (pJD101), an in-frame deletion mutation

in ORF-1 (pJD102), a fill-in mutation in ORF-2 (pJD103), anda fill-in mutation in ORF-2 on pJD102 (pJD104), was con-structed (Fig. 4). Each of the resulting plasmids was introducedinto N1436, which was then tested for restoration of the Vir+phenotype by the FP test. pJD100 and pJD103 could restorethe Vir+ phenotype, but the others, pJD101, pJD102, andpJD104, could not, indicating that ORF-1 was the vacCdeterminant.

Characterization of the vacC mutant. N1436 was initiallyisolated as a mutant of reduced virulence which was unable toform large plaques in the FP test. Other invasion-relatedphenotypes, such as Chl and the expression of the ipaBCDoperon, were decreased to less than half that of the wild type(29). Since the ability of the invading bacteria to spread fromone epithelial cell to another requires virG expression (11, 23,24), we investigated N1436 for the level of VirG production byimmunoblotting with a VirG-specific antiserum, VRG-C. Thelevel of VirG in the whole-cell lysate of N1436 was significantly

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VOL. 176, 1994

SalI1 GTCGACGCTGATTATGCTGGTTTCGGCCTTTGCCGGTTATCAACACACCATGAATGCCTATAAA

65 GCGGCGGTAGAAGAGAAATATCGCTTTTTTAGTTACGGTGATGCGATGTTTATCACGTACAATC

129 CGCAGGCAATTAATGAGCGCGTCGGGGAGTAATTCCGCGGCGCTGGTTTAAACGTTGGACTGTT

1931

vacC LOCUS OF S. FLEXNERI 4631

1 2 3

ORF-1 M K F E L D T T D G R A R R

257 GGCCGCCTGGTCTTTGATCGTGGCGTAGTGGAAACGCCTTGTTTTATGCCTGTTGGCACCTAC15 G R L V F D R G V V E T P C F M P V G T Y

32036

38357

G T V K G M T P E E V E A T G A Q I I L GSmaI

AACACCTTCCACCTGTGGCTGCGCCCGGCCAGGAAATCATGAAACTGCACGGCGATCTGCACN T F H L W L R P G Q E I M K L H G D L H

446 GATTTTATGCAGTGGAAAGGACCGATTCTTACCGACTCCGGCGGCTTCCAGGTCTTCAGCCTT78 D F M Q W K G P I L T D S G G F Q V F S L

509 GGTGATATTCGTAAAATCACCGAACAGGGCGTTCACTTCCGTAACCCGATCAACGGCGACCCG99 G D I R K I T E Q G V H F R N P I N G D P

EcoRV572 ATTTTCCTCGACCCGGAAAAGTCGATGGAGATTCAGTACGATCTTGGTTCGGATAT_CGTCATG120 I F L D P E K S M E I Q Y D L G S D I V M

635 ATCTTTGATGAGTGTACGCCGTATCCTGCTGACTGGGATTACGCAAAACGCTCTATGGAGATG141 I F D E C T P Y P A D W D Y A K R S M E M

698 TCTCTGCGTTGGGCGAAGCGTAGCCGTGAGCGTTTTGACAGTCTTGGTAACAAAAATGCGTTA162 S L R W A K R S R E R F D S L G N K N A L

97-

67-

43-

30-

761183

824204

TTTGGTATAATTCAGGGCAGTATTTACGAAGATTTACGTGATATATCTGTTAAAGGTCTGGTAF G I I Q G S I Y E D L R D I S V K G L V

EcoRVGATATACGGCTTTGATGGCTACGCTGTCGGCGGTCTGGCTGTGGGTGAGCCGAAAGCAGATATGD I G F D G Y A V G G L A V G E P K A D M

887 CACCGTATTCTGGAGCATGTGTGTCCGCAAATTCCGGCAGACAAACCGCGTTACCTGATGGGC225 H R I L E H V C P Q I P A D K P R Y L M G

950 GTTGGTAAAccGGAAGACCTGGTTGAAGGCGTACGTCGCGGTATCGATATGTTTGACTGCGTA246 V G K P E D L V E G V R R G I D M F D C V

1013 ATGCCAACCCGCAACGCCCGAAATGGTCATTTGTTCGTGACCGATGGCGTGGTGAAAATCCGC267 M P T R N A R N G H L F V T D G V V K I R

1076 AATGCGAAATATAAGAGCGATACTGGCCCACTCGATCCTGAGTGTGATTGCTACACCTGTCGC288 N A K Y K S D T G P L D P E C D C Y T C R

1139 AATTATTCACGCGCTTACTTGCATCATCTCGACCGTTGCAACGAAATATTAGGCGCGCGACTC309 N Y S R A Y L H H L D R C N E I L G A R L

1202 AACACCATTCATAACCTTCGTTACTACCAGCGTTTGATGGCGGGTTTACGCAAGGCTATTGAA330 N T I H N L R Y Y Q R L M A G L R K A I E

1265 GAGGGTAAATTAGAGAGCTTCGTAACTGATTTTTACCAGCGTCAGGGGCGAGAAGTACCACCT351 E G K L E S F V T D F Y Q R Q G R E V P P

1328 TTGAACGTTGATTAATATTAATAATGAGGGAAATTTAATGAGCTTTTTTATTTCTGATGCGGTA372 L N V D * ORF-2 M S F F I S D A V

ApaLI1392 GCGGCAACGGGTGCACCGGCGCAAGGTAGCCCGATGTCTTTGATTTTGATGCTGGTGGTATTC

iG A A T G A P A Q G S P M S L I L M L V V F

1455 GGTCTGATTTTCTATTTCATGATCCTGCGTCCACAGCAGAAGCGCACCAAAGAACACAAAAAG31 G L I F Y F M I L R P Q Q K R T K E H K K

1518 CTGATGGACTCCATTGCCAAAGGTGATGAAGTTCTGACGAACGGTGGCCTGGTTGGTCGCGTAL M D S I A K G D E V L T N G G L V G R V

158173

164494

ACCAAAGTAGCGGAAAACGGCTACATTGCTATCGCGCTGAATGACACCACTGAAGTAGTTATTT K V A E N G Y I A I A L N D T T E V V I

AAACGTGACTTCGTAGCTGCCGTCCTGCCGAAAGGCACCATGAAGGCGCTGTAATTAAAATTTTK R D F V A A V L P K G T M K A L *

1708 TCCCTAAGGGAATTGCCGTGTTAAACCGTTATCCTTTGTGGAAGTACGTCATGCTGATCGTGGTBamHI

1772 GATTGTCATCGGTCTGCTGTATGCGCTTCCCAACCTGTTTGGTGAggATC

FIG. 2. Nucleotide sequence of the vacC region. The sequence ofthe sense strand is shown as well as the deduced amino acid sequencesfor two ORFs, ORF-1 and ORF-2. Positions 208 to 1335 correspond tothe vacC gene (ORF-1). The arrow position between 716 and 717shows the site of Tn5 insertion in N1436. Restriction sites areunderlined.

decreased (Fig. SA, lane 2) compared with that of the wild type(lane 1). The levels of virG mRNA expressed from N1436 andYSH6000 were examined by Northern dot blotting with a

virG-specific probe, which showed that the level of virG mRNAin N1436 was decreased compared with that of YSH6000 (Fig.6). Since expression of both the virG and the ipa operonsrequires the functioning positive regulator VirF (33), thepossibility that the reduced expression of those two operonsresulted from reduced VirF expression existed. To test this,N1436 and YSH6000 were examined for their levels of VirFprotein by immunoblotting with a VirF-specific antibody,

20-

14-

FIG. 3. Protein products expressed from the vacC region with theT7 RNA polymerase-dependent promoter system. Lane 1, SalI-BamHI segment (see Results) in the incorrect orientation with respectto the +10 promoter on pT7-6; lane 2, same as lane 1 but in the correctorientation on pT7-5; lane 3, pT7-5 vector control. The arrowheadsindicate the VacC (top) and ORF-2 (bottom) proteins. Molecularmasses (in kilodaltons) are indicated at the left.

VRF-C. The results showed that the level of VirF in N1436 wasdecreased compared with that of the wild type (Fig. SB). Thelevels of virF mRNA expressed from N1436 and YSH6000examined by Northern dot blotting with a virF-specific probe,however, showed no significant difference (Fig. 6). To furtherconfirm the effect of the vacC mutation on the production ofVirG and VirF proteins, N1436 carrying pJD100 or its deriv--atives was used (Fig. 4) to perform immunoblot analysis withVRG-C or VRF-C antiserum. The results showed that the twocomplementation-positive plasmids, pJD100 and pJD103,could restore the production of VirG and VirF to N1436, butpJD102 and pJD104 could not (Fig. 5A and B). These dataindicated that vacC function affected the production of theVirG and VirF proteins in addition to that of the IpaB, IpaC,and IpaD proteins (28) and that the low levels of expression ofthe virG and ipa operons seen in N1436 (this study andreference 28) were probably due to the low production of VirFprotein.

Presence of vacC in other shigellae and in EIEC. Southernhybridization with the vacC probe against SalI-BamHI-di-gested chromosomal DNA from the S. dysenteriae 6, S. flexneri5, S. boydii 4, S. sonnei form I, and EIEC 0152 strains revealedthat they all contained a DNA segment homologous to thevacC region (data not shown). To confirm that vacC functionwas present, the vacC genes of each of the shigellae and EIECstrains were disrupted by allelic replacement with a vacC::neocassette introduced on a suicide plasmid vector, pGP704 (seeMaterials and Methods). The mutants constructed all hadreduced levels of VirF compared with their respective parental

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4632 DURAND ET AL.

Tn5

S Sm Ev |EvI I I

(%)

1 00Ap B

PTle.IORF-i fvacC) @ ORF-2:

........................... . ........................... ..... _......

(tgt) (orf12)

pJD100pJD101pJD102 EJpJD103pJD104 AV

80 _

Fpa

p

P/F

P/F

p

P/F

FIG. 4. Identification of the vacC gene. The top line represents a1.8-kb SalI-BamHI segment cloned from the YSH6000 chromosomeinto pBluescriptIIKS+ and designated pY12. The boxes with dottedlines indicate the placement of the two ORFs deduced from thenucleotide sequence (Fig. 3). The parentheses beneath the boxesindicate the corresponding tgt and orfl2 from E. coli K-12. pJD100 wasconstructed by subcloning the 1.8-kb SalI-BamHI segment intopMW119Tp. pJD101 to pJD104 were derived from pJD100 followingeither linker or fill-in mutation (V) or deletion (EZ1). Fpa, plaqueforming ability of subclones introduced into N1436 as measured by theFP test; P, positive plaque formation; P/F, small plaque formation.Designations for restriction sites are abbreviated as in Fig. 1.

strains (Fig. 7). Expression of the IpaB, IpaC, and IpaDproteins as well as the VirG protein in the mutants was alsoreduced compared with that of each of the parental shigellastrains (data not shown). These results indicated that the vacC(tgt) gene played a similar important role in the virulence ofother shigellae and EIEC.

DISCUSSION

This and our previous studies (29) have confirmed that thevacC locus on the chromosome of S. flexneri YSH6000 is an

A 1 2 3 4 5 6 7 8

B 1 2 3 4 5 6 7 8

FIG. 5. Effect of vacC mutation on VirG and VirF expression. (A)Immunoblot analysis of VirG production. Equal amounts of totalprotein (20 ,ug) from each strain grown at 37°C were electrophoresedin an SDS-polyacrylamide gel and transferred to nitrocellulose mem-branes. VirG production was detected in the immunoblots by use of apolyclonal antibody, VRG-C. Lanes: 1, YSH6000; 2, N1436; 3, N1436carrying pJD100; 4, N1436 carrying pJD101; 5, N1436 carryingpJD102; 6, N1436 carrying pJD103; 7, N1436 carrying pJD104; 8,N1436 carrying pMW119Tp. The arrowhead indicates the VirG pro-tein. (B) Immunoblot analysis of VirF production. The procedure andlane contents were the same as for panel A except that immunostain-ing was performed with the antibody VRF-C. The arrowhead indicatesthe 30-kDa VirF protein, while the 32-kDa protein band over VirFrepresents a cross-reacting protein present in all strains including thevector control.

6 0

40O

20

0

1 08

FIG. 6. Expression of virG and virF mRNAs. RNA was extractedfrom N1436 and YSH6000 at 1, 2, 3, and 4 h postinoculation (1:100dilution) at 37°C, at which the CFU per 50 ,ul of the bacterial culturewere measured as shown on the horizontal axis. Aliquots of total RNA(5, 1.25, and 0.6 p.g per dot) were dotted onto nitrocellulose mem-branes, and virG and virF mRNAs were detected by hybridization witha specific 32P-labeled DNA probe. The circles and squares representthe levels of virG mRNA or virF mRNA from YSH6000 and N1436,respectively, standardized by use of three different RNA dots. Thevertical axis presents the percentage of each virG mRNA (solid lines)or virF mRNA (dotted lines) relative to that expressed from YSH6000at the maximum level (100%).

essential requirement for full expression of vir genes encodedby the large plasmid. The genetic and nucleotide sequenceanalysis of the vacC region indicated that the virulence-associated determinant was equivalent to the tgt gene of E. coliK-12, which encodes the tRNA-guanine transglycosylase (Tgt)(19, 30). Thus, vacC is similar to other chromosomal virulence-associated loci such as virR (hns), emvZ and ompR (11), andvacB (44), which affect expression of vir genes on, the largeplasmid, but through quite different mechanisms.

Direct assignment of the vacC locus to the NotI-C fragmentof the YSH6000 chromosome with a vacC probe constructedfrom N1436 indicated that vacC was located near the phoBRregion at 9.1 min on the genetic map of the E. coli K-12chromosome (Fig. 1). DNA hybridization analysis of theKohara X library with the vacC probe revealed that thehomologous DNA region was located at 9.2 to 9.3 min on theE. coli K-12 chromosome. In agreement with this, the nucle-otide sequence of the 1.8-kb SalI-BamHI vacC clone fromYSH6000 revealed highly significant (>98%) homology to the

1 2 3 4 5 6 7 8 9 10

FIG. 7. VirF expression from constructed vacC mutants. Immuno-blots with VRF-C antibody of the whole-cell protein extracts of eachwild type and of its constructed vacC mutant are shown in odd- andeven-numbered lanes, respectively. Lanes: 1 and 2, S. dysenteriae 6; 3and 4, S. flexneri 5; 5 and 6, S. boydii 4; 7 and 8, S. sonnei form I; 9 and10, EIEC 0152.

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vacC LOCUS OF S. FLEXNERI 4633

tgt region of E. coli K-12 (30), which mapped at 9.2 to 9.3 minnear the phoBR genes (Fig. 1). Indeed, a 1.8-kb SalI-BamHIsegment from E. coli K-12 obtained as a portion of Kohara Aclone 145 was confirmed as containing the tgt sequence (17)and restored the virulence phenotype to TC501, a vacC::TnStransductant of YSH6000T, as assessed by the FP test. Subse-quent analysis of the vacC region from YSH6000 by geneticcomplementation and nucleotide sequencing revealed that oneof the ORFs, comprising 1,108 bp and called ORF-1, was thevacC determinant. The other ORF downstream of the vacCgene, ORF-2, was identical to the orfl2 gene in the tgt regionof E. coli K-12 and was not directly related to vacC function(Fig. 4). These results taken together strongly suggested thatthe function encoded by ORF-1 in the vacC locus of S. flexneriis identical to that encoded by the tgt gene of E. coli K-12 (30).

Reuter et al. (30) indicated that the tgt gene in E. coli K-12was part of a complex putative operon comprising five genes,queA, tgt, orfl2, secD, and secF. Recently, Slany and Kerstenproposed that these genes form an operon, designated thetgt/sec operon (42), in which the upstream activation sequencefor the entire operon contained a potential binding site for thefactor of inversion stimulation (FIS). In this regard, ourcomplementation tests of N1436 with the vacC clone indicateda different organization, as pJD103 could complement N1436(Fig. 4). Since a TnS insertion into an operon is known to causea polar mutation which affects the expression of downstreamgenes (9, 10), the complementation of N1436 with pJD103 maybe due to the presence of a cryptic promoter for ORF-2 in thevacC region in S. flexneri. Indeed, orfl2 (corresponding toORF-2) in the tgt region in E. coli K-12 was shown to possessits own promoter (30). We observed that the level of ORF-2mRNA in N1436 was the same as that in YSH6000 as judgedby Northern dot blotting with an ORF-2-specific probe (17).The function of tgt has been studied extensively in E. coli

K-12 (30, 42), in which the Tgt and QueA proteins participatein the biosynthesis of the hypermodified tRNA-nucleosidequeuosine [Q; 7-(((4,5-cis-dihydroxy-2-cyclopentene-1-yl)-ami-no)-methyl)-7-deazaguanosine] (13, 19, 30). Q is usuallypresent in the first position of the anticodon in tRNAs specificfor Asn, Asp, His, and Tyr of eukaryotes and bacteria (13).Although the action of the Tgt enzyme has been elucidated interms of the biosynthesis of tRNA, no clear biological defect isobserved in tgt mutants of E. coli. The only notable change sofar reported was a twofold increase in the frequency ofincorrect read-through of the UAG codon depending on thecodon context (19). Whether this slight increase in errorfrequency is of any biological significance is still obscure (19,30). In this respect, the vacC mutant of S. flexneri (N1436)produced levels of IpaB, IpaC, IpaD, VirG, and VirF lowerthan those of the wild type, thus accounting for the less virulentphenotypes in the FP test, the Chl assay, and the Sereny test(28). Interestingly, levels of mRNAs for the ipaBCD and virGoperons, but not the virF operon (17), were shown to bedecreased in N1436 compared with those of YSH6000. Thus, itis unlikely that vacC directly affected the production of IpaB,IpaC, IpaD, and VirG but rather that the effect of vacC onVirF caused a decrease of mRNAs for ipaBCD and the virGoperon (reference 28 and Fig. 6), consistent with the role ofVirF as a positive transcriptional regulator for the virG andipaBCD operons (33). However, it is not clear whether theeffect of the vacC mutation on VirF expression is direct orindirect. The possibility that some other unidentified genespecifically involved in VirF production may be affected byvacC (tgt) function in shigellae exists. In any case, elucidationof the mechanisms responsible for the less efficient production

of VirF found in the vacC mutant requires further studies onvirF expression as well as on VacC (Tgt) function.

Interestingly, a similar class of virulence locus has recentlybeen found in the plant pathogenic bacterium Agrobacteriumtumefaciens (20). A less virulent mutant of A. tumefaciensisolated by TnS insertion mutagenesis was found to be mutatedin an miaA homolog known in E. coli K-12, which encodes atRNA isopentenyltransferase (20) essential for the modifica-tion of position A37 of tRNA (13). The miaA gene of A.tumefaciens was shown to be involved in expression of variousvir genes. Moreover, the cloned miaA gene fromA. tumefacienscould complement an E. coli miaA mutant.

Despite the fact that mechanisms of VacC function affectingthe production of various virulence proteins in N1436 remainto be elucidated, the vacC gene clearly plays an important rolein full expression of the virulence phenotype in shigellae.Indeed, when the vacC locus was mutated in shigellae andEIEC strains, the expression of VirF (Fig. 7) and VirG andthat of IpaB, IpaC, and IpaD were all decreased (17), givingrise to less-virulent phenotypes in Chl assays and in FP tests.These results thus strongly support the notion that vacC (tgt) isa prerequisite for bacterial pathogenesis in bacillary dysentery.

ACKNOWLEDGMENTS

We thank B. Adler for critical reading of the manuscript and Y.Nakamura and G. R. Bjork for valuable discussion.

This work was supported by grants 01440031, 03304030, 03557022,and 04454194 from the Ministry of Education, Science, and Culture ofthe Japanese Government. J.M.D. was supported by research fellow-ships from the Japan Society for the Promotion of Science and theEuropean Communities S&T Fellowship Program in Japan. C.S. wasthe recipient of a grant from the Naito Foundation.

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