Nucleotide Sequence Operon Shigella flexneri 2a …Shigella spp. and enteroinvasive Escherichia coli (45). This plasmid encodes the antigens IpaBCD, which are critical for primarymucosalcell

JOURNAL OF BACrERIOLOGY, Apr. 1994, p. 2362-23730021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

Nucleotide Sequence of the Rhamnose Biosynthetic Operon ofShigella flexneri 2a and Role of Lipopolysaccharide in Virulence

KUMAR RAJAKUMAR,1 B. HELEN JOST,' CHIHIRO SASAKAWA,2 NOBUHIKO OKADA 2MASANOSUKE YOSHIKAWA,2 AND BEN ADLERI*

Department of Microbiology, Monash University, Clayton, Victoria 3168, Australia,1 and Department of Bacteriology,Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan2

Received 21 October 1993/Accepted 3 February 1994

N1308, a chromosomal Tn5 mutant of ShigeUlaflexneri 2a, was described previously as a lipopolysaccharide(LPS) mutant with a short 0 side chain. N1308 formed foci, but not plaques, in LLC-MK2 cell monolayers andwas negative in the Sereny test. In this study, the wild-type locus inactivated in N1308 was cloned and furtherdefined by means of complementation analysis. A 4.3-kb BstEII-XhoI fragment of S. flexneri 2a YSH6200 DNAwas sufficient to restore both normal LPS and virulence phenotype to the mutant. DNA sequencing of thisregion revealed four genes, rjbA, rJbB, rjbC, and rJbD, encoding the enzymes required for the biosynthesis ofactivated rhamnose. The four genes were expressed in Escherichia coli, and the expected protein products werevisualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N1308 was shown to have normallevels of surface IpaC and IpaD, while a Western blot (immunoblot) of whole-cell lysates or outer membranefractions indicated an elevated level of appropriately localized VirG. An in vitro invasion assay revealed thatN1308 had normal primary invasive capacity and was able to multiply and move normally within the initialinfected cell. However, it exhibited a significant reduction in its ability to spread from cell to cell in themonolayer. A double immunofluorescence assay revealed differences between LLC-MK2 cells infected with thewild-type YSH6000 and those infected with N1308. The wild-type bacteria elicited the formation of thecharacteristic F-actin tails, whereas N1308 failed to do so. However, N1308 was capable of inducing depositionof F-actin, which accumulated in a peribacterial fashion with only slight, if any, unipolar accumulation of thecytoskeletal protein.

Bacteria of the genus Shigella are human pathogens thatcause bacillary dysentery, a persisting hazard in much of thedeveloping world. Infection begins with the ingestion of bac-teria, which on reaching the colon invade colonic epithelialcells. Intracellular bacterial multiplication and spread to adja-cent cells eventually lead to inflammation and ulceration of thecolonic mucosa, resulting in pyrexia, abdominal colic, andbloody and mucoid diarrhea (60).The genetic basis of virulence in this genus has been

extensively studied. Much of the work has centered on a large(100- to 140-MDa) plasmid found in all virulent strains ofShigella spp. and enteroinvasive Escherichia coli (45). Thisplasmid encodes the antigens IpaBCD, which are critical forprimary mucosal cell invasion (17, 44), the Mxi proteins, whichare required for normal surface expression of Ipa (2, 48, 56),and VirG, which is essential for intra- and intercellular bacte-rial spread (11, 28). The virulence plasmid also carries theregulatory genes virB (1) and virF (41).

Early intergeneric conjugation between E. coli K-12 andShigella flexneri led to the identification of several chromo-somal virulence-associated loci, including the his-linked locusinvolved in the synthesis of the group-specific 0 3,4 antigen ofS. flexneri (14) and the mtl-linked locus, which mediates thesynthesis of the lipopolysaccharide (LPS) core (51). Otherchromosomal genes identified include the aerobactin genesinvolved in the synthesis of an iron-scavenging siderophore(23, 31) and the regulatory genes ompR-envZ (6), virR (an hns

* Corresponding author. Mailing address: Department of Microbi-ology, Monash University, Wellington Road, Clayton, Melbourne,Victoria 3168, Australia. Phone: 61 3 905 4815. Fax: 61 3 905 4811.

analog) (19), and the recently identified vacB (53), thatmodulate the expression of IpaBCD and VirG.

Recently, Okada et al. (33) used random TnS mutagenesis toidentify nine chromosomal virulence-associated loci whichwere subsequently mapped to various fragments of a NotIphysical map of the S. flexneri 2a chromosome (32). One locuswas responsible for thymine biosynthesis, two loci were in-volved in regulation of Ipa protein expression, and three otherloci were associated with a reduced invasive phenotype, despitethese mutants having normal Ipa expression. The three re-maining loci were associated with LPS biosynthesis and weretentatively designated the mtl-linked rfa locus, the his-linkedrfl locus, and the rfc locus (32, 33). N1308, a TnS mutant of S.flexneri 2a strain YSH6000, was previously described as an LPSmutant with a short 0 side chain and weak agglutination withShigella 0 3,4 antiserum. The Tn5 insert was localized tochromosomal NotI fragment H. N1308 formed foci, but notplaques, and was negative in the Sereny test (33). This provi-sionally designated rfc mutant, which on further characteriza-tion was shown to be an rjb mutant, was the subject of thepresent study.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains andplasmids used in this study are shown in Table 1.Growth media. E. coli K-12 strains were grown on 2 xYT

medium (3), while Shigella strains were grown on LB medium(3). Strains containing pUC18/19-Tp-based plasmids weregrown on Mueller-Hinton medium supplemented with 0.36%(wt/vol) glucose when selection for trimethoprim resistancewas necessary. Antibiotics were added when required at the

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ROLE OF LPS IN VIRULENCE OF S. FLEXNERI 2363

TABLE 1. Bacterial strains and plasmids

Bacterial strain or Relevant characteristic(s) Reference or sourceplasmid

Bacterial strainsS. flexneri 2aYSH6000 Virulent strain 47YSH6200 Avirulent strain of YSH6000 cured of the 230-kb plasmid pMYSH6000 41N1308 YSH6000, LPS' (NotI-H::TnS) 33N1419 YSH6000, LPS' (NotI-H::TnS) 33N1975 YSH6000, LPS' (NotI-H::TnS) 33S7420 YSH6000, LPS' (NotI-H::TnS) 33S7914 YSH6000, LPSa (NotI-H::TnS) 33S7920 YSH6000, LPS' (NotI-H::TnS) 33S6933 YSH6000, LPSb (NotI-H::TnS) 33M94 YSH6000 harboring pMYSH6000(virG::TnS) 28CS2068-1 YSH6000 harboring a plasmid that overexpresses VirG on IPTG induction C. Sasakawa

E. coli DH5a F- 4)80 AlacZ M15 recAl Bethesda ResearchLaboratories

PlasmidspUC18/19 High-copy-number cloning vectors, Apr, AlacZ 58pUC18/19-Tp pUC18/19 derivative with a 0.7-kb Tpr gene inserted at the SspI site B. H. JostpTTQ18/19 Expression vectors, Apr, ptac, lacIq, AlacZ AmershampSBA52 Primary genomic clone containing 6.8- and 6.5-kb EcoRI fragments of YSH6200 This study

in pUC18pSBA85 6.8-kb EcoRI fragment of pSBA52 in pUC18-Tpc This studypSBA105 6.8-kb EcoRI fragment of pSBA52 in pUC18-Tpd This studypSBA146 4.8-kb SalI-XhoI fragment of pSBA85 in pUC19-Tpc This studypSBA147 4.8-kb SalI-XhoI fragment of pSBA85 in pUC18-Tpd This studypSBA134 5.9-kb EcoRI-partial HindIII fragment of pSBA85 in pUC18-Tpc This studypSBA117 5.9-kb EcoRI-partial HindIII fragment of pSBA85 in pUC19-Tpd This studypSBA122 3.6-kb SphI-EcoRI fragment of pSBA85 in pUC19-Tpc This studypSBA123 3.6-kb SphI-EcoRI fragment of pSBA85 in pUC18-Tpd This studypSBA133 4.8-kb BstEII-EcoRI fragment of pSBA85 in pUC18-Tpc This studypSBA108 5.1-kb SalI-EcoRI fragment of pSBA85 in pUC19-Tpd This studypSBA54 12.2-kb EcoRI Kmr fragment derived from S6933 in pUC18 This studypSBA55 12.5-kb EcoRI Kmr fragment derived from N1308 in pUC18 This studypSBA95 12.5-kb EcoRI Kmr fragment derived from N1419 in pUC18 This studypSBA97 12.5-kb EcoRI Kmr fragment derived from N1975 in pUC18 This studypSBA99 12.5-kb EcoRI Kr fragment derived from S7420 in pUC18 This studypSBA102 12.5-kb EcoRI KMr fragment derived from S7914 in pUC18-Tp This studypSBA104 12.5-kb EcoRI Kmr fragment derived from S7920 in pUC18-Tp This studypSBA238 0.9-kb NsiI-XhoI fragment of pSBA85 encoding rfbD in pTJQ19 This studypSBA239 1.2-kb NsiI-PstI fragment of pSBA85 encoding rjbB in pTTQ18 This studypSBA251 1.1-kb Asp 700 fragment of pSBA85 encoding nbA in pTTQ18 This studypSBA252 1.5-kb SmaI-HindII fragment of pSBA85 encoding nbC in pTTQ19 This studya LPS with a shortened 0 side chain.b LPS with no 0 side chain.c Insert in the same orientation to the lac promoter of the vector.d Insert in the opposite orientation to the lac promoter of the vector.

following concentrations: ampicillin, 100 ,ug ml 1; kanamycin,50 ,ug ml-l; and trimethoprim, 12 ,ug ml1-.

Molecular biological techniques. Chromosomal DNA wasisolated by the small-scale preparation method described byAusubel et al. (3). Plasmid DNA was isolated by a modificationof the alkaline lysis method (29). Standard cloning procedures(3) were used. Restriction enzymes, alkaline phosphatase,Klenow enzyme, and T4 ligase were obtained from BoehringerMannheim, Bethesda Research Laboratories, or New EnglandBiolabs and were used according to the manufacturer's instruc-tions. Colony hybridization (16) and Southern hybridizationanalysis (52) were performed under conditions of high strin-gency. Hybridization probes were obtained by purifying appro-priate restriction fragments from an agarose gel (Genecleankit; Bio 101, La Jolla, Calif.). DNA was labelled with digoxi-genin-11-dUTP and detected with the Non-Radioactive Label-ling and Detection kit (Boehringer Mannheim), using theprotocol supplied by the manufacturer. Nucleotide sequencing

was performed by the dideoxy-chain termination method (42),using a deaza-nucleotide sequencing kit (Promega), T7 DNApolymerase (Pharmacia), and [35S]dCTP (Amersham). M13universal and reverse sequencing primers (Pharmacia) wereused.

Virulence phenotype assays. The focus-plaque assay wasperformed as described by Sasakawa et al. (46). The invasionassay of LLC-MK2 rhesus monkey kidney epithelial cells wasperformed by the method of Sasakawa et al. (49). The assaywas scored on the basis of three criteria: the ability of shigellaeto spread to adjacent cells from the initial infected index cell(13), the average number of intracellular bacteria per indexcell, and the percentage of cells infected 2 h postinfection(25).

Whole-cell ELISA. The whole-cell enzyme-linked immu-nosorbent assay (ELISA) was performed by dispensing stan-dardized suspensions of bacteria in phosphate-buffered saline,pH 7.2 (PBS), into 96-well microtiter trays (Costar, catalog no.

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2364 RAJAKUMAR ET AL.

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FIG. 1. Restriction endonuclease mapping and deletion analysis of plasmids pSBA52 and pSBA85. Restriction site abbreviations: B, BstEII; E,EcoRI; H, HindIll; S, SphI; Sa, SalI; X, XhoI. Vector sequences are not shown. Vertical bars indicate the sites of Tn5 insertions in each of themutants indicated. (A) Physical map of rhamnose biosynthetic region of S. flexneri 2a and its comparison with that of S. enterica LT2 (20). TheORFs identified by sequencing are shown by arrows. The putative promoter is indicated by the letter P. The hatched bar shows the 1.8-kbEcoRI-SalI probe derived from N1308, used to isolate the primary clone pSBA52. The lines beneath represent pSBA85 and its deletion derivatives.The arrowheads indicate the orientation of the vector-borne lac promoter. The columns on the right show the phenotype of each subclone. Slideagglutination: + + +, normal; +, weak. LPS phenotype: W, wild type; 0, short 0 side chain. Focus-plaque assay (Fpa): P, plaques; F, foci. (B)Comparison between pSBA52 and the rjb locus of S. flexneri 2a cloned by Macpherson et al. (27), showing relative positions of the Tn5 inserts inmutant N1308, with a short 0 side chain, and in mutant S6933, with an absent 0 side chain.

3590; Data Packaging Corp., Cambridge, Mass.). The trayswere left standing for 1 h at room temperature. The cells weredecanted, and the wells were blocked with 1% bovine serumalbumin in PBS for 2 h at 37°C. A primary antibody consistingof a 1/50 dilution of anti-Shigella antibody (Difco) or a 1/100dilution of monoclonal antibodies against IpaC and IpaD(20a), respectively, was then incubated for 2 h at 37°C.Detection was achieved by using a 1/1,000 dilution of either ananti-mouse or an anti-rabbit horseradish peroxidase conjugate(Silenus, Melbourne, Australia) with an o-phenylenediamine-based substrate.

Immunofluorescence. A double immunofluorescence assaywas performed essentially as described by Clerc and Sansonetti(11). Infected LLC-MK2 monolayers were fixed at 2 h postin-fection in 3.7% (wt/vol) formaldehyde-1 mM CaCl2-0.5 mMMgCl2 in PBS at 4°C for 15 min. The cells were permeabilizedwith 0.1% (vol/vol) Triton X-100 in PBS at room temperaturefor 4 min. Bacteria were labelled by indirect immunofluores-cence with a 1/20 to 1/50 dilution of S. flexneri 2a group3,4-specific antiserum (Difco) and a 1/10 to 1/20 dilution ofanti-rabbit fluorescein isothiocyanate-conjugated immuno-globulin (Silenus). F-actin labelling was achieved by using a20-U/ml solution of rhodamine phalloidin (Molecular Probes,Eugene, Ore.) in PBS for 60 min at 37°C.

Slide agglutination tests. Slide agglutination tests wereperformed with stationary-phase bacteria suspended in PBSand S. flexneri 2a group 3,4-specific antiserum (Difco).

Polyacrylamide gels and Western blotting (immunoblot-ting). Whole-cell lysates and outer membrane cell fractionsprepared by sucrose density gradient centrifugation (36) wereelectrophoresed in sodium dodecyl sulfate (SDS) sample

A B C D E F G H I J K L M

FIG. 2. Silver-stained polyacrylamide gel of proteinase K-treatedwhole-cell lysates from YSH6200 (lane C), N1308 (lane B), and N1308harboring plasmids pUC18-Tp (lane A), pSBA85 (lane D), pSBA105(lane E), pSBA146 (lane F), pSBA147 (lane G), pSBA134 (lane H),pSBA117 (lane I), pSBA122 (lane J), pSBA123 (lane K), pSBA133(lane L), and pSBA108 (lane M). The arrowhead indicates lipid A pluscore.

buffer on discontinuous SDS-polyacrylamide gels. Polyacryl-amide gels were either stained with Coomassie blue to visual-ize protein bands or subjected to Western blotting (3). Theprimary antibody was produced by immunizing rabbits with aC-terminal oligopeptide of VirG coupled to keyhole limpethemocyanin (30). Blots were developed with 4-chloro-1-naph-thol. Electrophoresis of proteinase K-treated cell lysates onSDS-free 15% polyacrylamide gels and visualization of LPSbands with silver was performed by the method of Hitchcockand Brown (18).

Nucleotide sequence accession number. The sequence of the4,340-bp BstEII-XhoI fragment shown in Fig. 3 has beendeposited with GenBank under accession number L14842.

RESULTS

Cloning of the TnS-bearing fragment of N1308 and of thecorresponding wild-type locus. A digoxigenin-labelled kana-mycin resistance (Kmr) gene probe derived from Tn5 was usedin a Southern hybridization experiment with N1308 DNAdigested with BamHI, Sall, or EcoRI. Sall and BamHI cutonce within Tn5, leaving the Kmr gene intact, while EcoRIdoes not cut within Tn5. The sizes of fragments that hybridizedfollowing digestion with BamHI, EcoRI, and Sall were >23,12.5, and 9.2 kb, respectively. Both the 12.5-kb EcoRI and9.2-kb Sall Kmr fragments were cloned from N1308 into theappropriate sites in pUC18. A 1.8-kb EcoRI-SalI fragment ofShigella DNA flanking the Tn5 insert in N1308 (Fig. 1A) wasused to probe an EcoRI genomic library of S. flexneri 2a strainYSH6200. A clone bearing plasmid pSBA52, with an insertconsisting of 6.8- and 6.5-kb EcoRI fragments of YSH6200DNA, was isolated. The 6.8-kb EcoRI fragment had a restric-tion profile identical to that of the DNA flanking the TnS insertin N1308.Mapping of other Tn5 insertions. Five other Tn5 mutants

that had been assigned to the same class as N1308 by Okada etal. (33) were also investigated. Genomic DNA of N1419,N1975, S7420, S7914, and S7920 was digested with EcoRI andligated into pUC-based vectors (Table 1). Kmr clones wereselected in each case. All five cloned EcoRI fragments were12.5 kb in size. Restriction mapping permitted the approximatelocalization of the Tn5 inserts corresponding to each mutantand showed that the six Tn5 inserts spanned a distance ofabout 2.3 kb (Fig. 1A).Complementation analysis of N1308. Various subclones of

the 6.8-kb EcoRI fragment were constructed by using thevectors pUC18-Tp and pUC19-Tp. These recombinant plas-mids were transformed into N1308 for complementation anal-ysis. A 4.3-kb BstEII-XhoI fragment was sufficient to restore anormal agglutination reaction with 0 3,4 antiserum and torestore virulence as measured by the focus-plaque assay (Fig.1A). The ability of plasmids to restore a virulence phenotypecorrelated with their ability to restore a normal LPS profile toN1308, as assessed from silver-stained polyacrylamide gels

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orf2. 8GGTGACCTCTCTGAATACTCCGTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAAG D L S E Y S V I Q T K E P L D R E G K V S R I V E F I E K

P D Q P Q T L D S D I M A V G R Y V L S A D I W P E L E R T

Q P G A W G R I Q L T D A I A E L A' K K Q S V D A M L M T G

GACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTD S Y D C G K K M G Y M Q A F V K Y G L R N L K E G A K F R

AAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAGATTCGTGK G I E K L L S E *

GCATAAGTTTTGCTCATTGAATTTTCCTGCCGTTGCCGTTTTTATAAAGCCTATAAAAACAAGAAGTTACATAGCTCATATTATTCAAAT

-35 - 10TTTTCCAGGATTTTCCTTGTTTCTACGGCTGTTTGGTAAGACAATTAACGTTTGAATTTTTCGGATTTGGCGCGAGTGGCTAACGCTCGC

NsiICACATCGTTGAGATGCATGCAGTGCACTGGTAGCTGTTAAGCCAGGGGCGGTAGCGTGTATTAATACCTCTATTAATCAAATCTGGAGCA

rfbBGTCTATTTCACAGCATGCTCTATAGCTATATGGAATAAAAAAGTGAAGATACTTGTTACTGGTGGCGCAGGGTTTATTGGTTCTGCTGTT

V K I L V T G G A G F I G S A V

GTTCGTCACATAATAAATAATACGCAAGATAGTGTTGTTAATGTCGATAAATTAACATACGCCGGGAACCTGGAATCACTCGCTGATGTTV R H I I N N T Q D S V V N V D K L T Y A G N L E S L A D V

TCTGATTCTGAACGTTATGCTTTTGAGCATGCAGATATCTGCGATGCTGTAGCCATGTCGCGTATTTTCGCACAGCACCAGCCAGACGCGS D S E R Y A F E H A D I C D A V A M S R I F A Q H Q P D A

HindIIGTGATGCACCTGGCGGCAGAGAGCCACGTTGACCGTTCAATAACGGGCCCTGCGGCATTTATTGAAACCAATATTGTGGGGACTTATGTTV M H L A A E S H V D R S I T G P A A F I E T N I V G T Y V

CTTTTAGAAGCGGCGCGCAATTACTGGTCTGCTCTGAATGATGAAAAGAAAAAAGTTTCCGCTTTCATCATATTTCTACTGATGAAGTTL L E A A R N Y W S A L N D E K K K S F R F H H I S T D E V

TATGGTGACTTACCCCATCCGGATGAAGCAAATAATAACGAAGCGTTACCGCTATTTACGGAAACGACAGCTTACGCGCCAAGTAGCCCGY G D L P H P D E A N N N E A L P L F T E T T A Y A P S S P

HindIIITATTCTGCGTCTAAAGCTTCCAGCGATCATTTGGTACGTGCCTGGAAGCGGACTTATGGTTTACCAACCATTGTGACTAATTGCTCGAACY S A S K A S S D H L V R A W K R T Y G L P T I V T N C S N

AATTATGGTCCTTATCACTTCCCGGAAAAACTTATTCCATTGGTTATTCTGAATGCTCTGGAAGGTAAGGCATTACCTATTTATGGTAAAN Y G P Y H F P E K L I P L V I L N A L E G K A L P I Y G K

GGGGATCAAATTCGCGACTGGTTGTATGTAGAGGATCATGCTCGTGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGTGAAACTTATG D Q I R D W L Y V E D H A R A L Y T V V T E G K A G E T Y

AACATTGGTGGACATAACGAAAAGAAAAACATCGACGTAGTGCTCACTATTTGTGATTTGTTGGATGAGATTGTACCGAAAGAGAAATCTN I G G H N E K K N I P V V L T I C D L L D E I V P K E K S

SmaITATCGTGAGCAAATTACTTATGTTGCCGATCGCCCGGGGCATGACCGCCGTTACGCCATTGATGCCGATAAAATTAGCCGCGAATTGGGCY R E Q I T Y V A D R P G H D R R Y A I D A D K I S R E L G

J. BACrERIOL.

90

180

270

360

450

540

630

720

810

900

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1080

1170

1260

1350

1440

1530

1620

1710

FIG. 3. Nucleotide and deduced amino acid sequences of the rhamnose biosynthetic region of the rib locus of S. flemnei 2a. Presumptiveinitiation codons are underlined, the putative promoter sequence is double underlined, and likely ribosome binding sites are shown in boldface.

(Fig. 2, lanes D to G, L, and M). Partial complementation ofthe LPS profile with restoration of small amounts of 0 sidechains was observed when N1308 was transformed with each ofplasmids pSBA134, pSBA117, pSBA122, and pSBA123 (Fig. 2,lanes H to K).

Nucleotide sequence of the 4.3-kb BstEH-XhoI fragment.

The nucleotide sequence of the 4.3-kb BstEII-XhoI fragmentwas determined (Fig. 3). Analysis revealed four complete openreading frames (ORFs) and indicated transcription of all fourin the, same direction. The deduced molecular masses of theprotein products were 40.5, 32.6, 32.5, and 20.8 kDa. Inaddition, the sequence commenced with an ORF open at the5' terminus and ended with a short ORF open at the 3'terminus.

A comparison of the nucleotide sequence with entries in theGenBank data base revealed high levels of similarity with partof the rjb operon of Salmonella entenca (Salm'onella typhi-murium) LT2 (20). Each of the four complete ORFs was highlyhomologous with its counterpart in S. enterica. In addition, theORF open at the 5' end was also highly conserved. Only thesecond incomplete ORF, designated orfl, shared no similarity.Hence, gene designations identical to those in S. entenica wereused, and the five similar ORFs in the 5'-to-3' order weredesignated orJ2.8, rJbB, rjbC, nbA, and rjbD (40). The extent ofthe identity ranged from 65% for rfbD to 79% for rfbA and rfbBat the nucleic acid level and increased to up to 92% for rfbAwhen compared at the amino acid leveL The extent of conser-vation exhibited by rfbD was restricted to the initial 477 bases,


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W K P Q E T F E S G I R K T V E W Y L A N T N W V E N V K S

rfbCGGTACTTATCAGTCGTGGATTGAACAAAACTATGAGGGCCGTCAGTAATGAATATCCTGCTTTTCGGCAAAACAGGGCAGGTGGGTTGGGG T Y Q S W I E Q N Y E G R Q * M N I L L F G K T G Q V G W

PstIAACTGCAGCGTGCTCTGGCTCCTCTGGGTAACTTGATTGCGCTCGATGTTCACTCCACTGATTATTGTGGAGATTTCAGCAACCCCGAAGE L Q R A L A P L G N L I A L D V H S T D Y C G D F S N P E

PstIGTGTGGCTGAAACCGTCAAAAAAATTCGCCCCGATGTTATTGTTAATGCTGCTGCTCACACTGCAGTAGACAAAGCGGAATCAGAACCGAG V A E T V K K I R P D V I V N A A A H T A V D K A E S E P

ATTTCGCGCAATTACTTAACGCGACATGCGTCGAAGCGATTGCAAAAGCAGCTAATGAAGTCGGAGCCTGGGTTATACACTACTCCACTGN F A Q L L N A T C V E A I A K A A N E V G A W V I H Y S T

ATTATGTTTTCCCAGGCAACGGTGACACGCCATGGCTGGAAACGGATGCAACAGCACCGCTAAATGTCTACGGTGAAACCAAGCTAGCTGD Y V F P G N G D T P W L E T D A T A P L N V Y G E T K L A

GGGAAAAAGCGTTACAAGAACATTGCGCAAAGCATCTTATTTTCCGTACCAGCTGGGTATACGCTGGTAAAGGAAATAACTTTGCCAAAAG E K A L Q E H C A K H L I F R T S W V Y A G K G N N F A K

CGATGTTGCGCCTGGCAAAAGAACGCGAAGAGTTGGCTGTAATAAACGATCAATTCGGCGCACCAACGGGGGCTGAATTACTGGCTGATTT M L R L A K E R E E L A V I N D Q F G A P T G A E L L A D

GCACCGCTCATGCCATTCGCGTGGCCGCAAATAAACCAGAAGTTGCTGGTTTGTACCATCTGGTTGCTGGTGGTACAACAACCTGGCACGC T A H A I R V A A N K P E V A G L Y H L V A G G T T T W H

ATTATGCCGCTCTGGTATTTGAAGAAGCGCGCAGAGCGGGGATCAACCTTGCTCTTAACAAACTTAATGCCGTGCCGACAACGGCCTATCD Y A A L V F E E A R R A G I N L A L N K L N A V P T T A Y

AsP700CCACACCAGCACGTCGTCCCCATAATTCTCGCCTCAATACCGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACTGGCAGGTGGP T P A R R P H N S R L N T E K F 0 Q N F A L V L P D W Q V

GCGTGAAACGTATGCTCAACGAATTATTTACGACCACGGCAATTTAACAAATTATTGCATCTCGCTCATGATGCTAGAGCGAGATGAATTG V K R M L N E L F T T T A I *

rfbAAAAAGGAATGGTGAAATGAAAACGCGTAAAGGTATTATTCTGGCTGGTGGTTCCGGCACTCGTCTTTATCCTGTGACAATGGCTGTGAGC

M K T R K G I I L A G G S G T R L Y P V T M A V S

AAACAACTGCTACCGATTTATGATAAACCGATGATTTATTATCCGCTTTCAACGCTTATGTTGGCGGGTATTCGAGATATTCTGATTATCK Q L L P I Y D K P M I Y Y P L S T L M L A G I R D I L I I

AGTACGCCGCAGGATACACCACGTTTTCAACAATTGCTTGGTGATGGAAGCCAGTGGGGGCTTAATCTGCAATATAAAGTACAACCGAGTS T P Q D T P R F Q Q L L G D G S Q W G L N L Q Y K V Q P S

CCGGATGGTCTGGCGCAAGCATTTATCATTGGTGAAGAATTTATAGGTGGTGATGATTGTGCGCTGGTACTTGGTGATAATATCTTCTATP D G L A Q A F I I G E E F I G G D D C A L V L G D N I F Y

HindIIGGTCACGACCTGCCGAAATTAATGGATACCGCTGTTAACAGAGAGAGTGGCGCAACGGTTTTTGCCTATCACGTAAATGATCCTGAGCGCG H D L P K L M D T A V N R E S G A T V F A Y H V N D P E R

TACGGTGTGGTGGAATTTGATGATAACGGTACGGCAATTAGTCTGGAAGAAAAACCGCAGGAACCAAAAAGTAACTATGCGGTTACTGGGY G V V E F D D N G T A I S L E E K P Q E P K S N Y A V T G

CTTTATTTCTATGACAATGACGTTGTAGAAATGGCGAAAAACCTTAAACCATCTGCACGTGGCGAACTGGAAATTACCGATATTAACCGTL Y F Y D N D V V E M A K N L K P S A R G E L E I T D I N R

FIG. 3-Continued.

with no similarity even at an amino acid level over theremaining 69 bases of the gene. Table 2 lists the percentageidentity and the percentage G+C content of each of the genesalong with the deduced molecular masses of their proteinproducts.

Intergenic regions were short, except that between o42.8and rfbB, which was 372 bp in length. This intergenic regionwas also highly similar to that of S. enterica. Three potentialpromoter sequences were identified in the region upstream ofrfbB. The best of these is represented by TTGAATN17TITTAT, While a second sequence, TTGTN7GACAAT(Fig. 3), is identical, even in its position relative to r,fB, to thepresumptive promoter of the S. enterica LT2 operon (20). Atleast one of these promoters appears to function in Shigellaspp. as the introduced DNA complemented in trans, even when

TABLE 2. Similarity between the rhamnose biosynthetic regionsof S. flexneri 2a and S. enterica LT2

Identity betweenS. flexneri and G+C content (%) Deduced molecular

S. enterica genes mass of proteins (kDa)Gene (%)a

Nucleic Amino

acid acid S. flexneri S. enterica S. flexneri S. enterica

rlB 79 88 44.5 44.1 40.5 40.7rbC 73 86 49.5 50.5 32.6 32.6rfbA 79 92 44.1 45.7 32.5 32.4rflD 65 70 37.9 40.4 20.8 20.6

a Calculated over the entire ORF of each gene.

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NsiIATTTATATGGACCAAGGACGTTTGTCTGTCGCTATGATGGGGCGTGGTTATGCATGGCTGGACACGGGGACACATCAAAGTCTTATTGAAI Y M D Q G R L S V A M M G R G Y A W L D T G T H Q S L I E

GCAAGCAACTTCATTGCAACAATTGAAGAGCGTCAGGGGCTGAAAGTTTCCTGCCCGGAAGAAATTGCTCACCGTAAAGGCTTTATTGATA S N F I A T I E E R Q G L K V S C P E E I A H R K G F I D

rfbDGCTGAGCAAGTGAAGGTATTAGCCGAGCCGCTGAAGAAAAATGCTTATGGTCAGTATCTGCTGAAAATGATTAAAGGTTATTAATAAAATA E Q V K V L A E P L K K N A Y G Q Y L L K M I K G Y * M

HindIIIGAACGTAATTAAAACTGAAATTCCTGATGTGCTAATTTTTGAACCAAAAGTATTTGGTGATGAGCGGGGCTTTTTTATGGAAAGCTTCAAN V I K T E I P D V L I F E P K V F G D E R G F F M E S F N

AsP700TCAGAAAGTTTTCGAAGAGGCTGTAGGACGAAAGGTTGAGTTTGTTCAGGATAACCATTCTAAATCAACTAAGGGTGTGTTACGCGGACT

Q K V F E E A V G R K V E F V Q D N H S K S T K G V L R G L

GCACTATCAGCTGGAACCTTATGCTCAAGGAAAACTGGTACGCTGTGTTGTCGGTGAAGTTTTTGACGTAGCTGTTGATATTCGGAAATCH Y Q L E P Y A Q G K L V R C V V G E V F D V A V D I R K S

ATCACCAACATTTGGCAAGTGGGTTGGTGTAAATTTGTCGGCGGAGAATAAGCGTCAATTATGGATACCGGAAGGATTCGCTCATGGGTTS P T F G K W V G V N L S A E N K R Q L W I P E G F A H G F

TTGCGTTCTTAGTGATGAAGCAGAATTTGTCTACAAAACAAATAATTTCTATTCAAAAATGCAAGAGCGTGGGATTTTATGGAGCGACAAC V L S D E A E F V Y K T N N F Y S K M Q E R G I L W S D K

AAGTATAAATATAGAATGGCCGGTTCAAAATCCATTGCTTTCTGATAAAGATATTAATGGTCAAATTTGTAGATGCTGATTATTTTATS I N I E W P V Q N P L L S D K D I N G Q K F V D A D Y F I

orflATGATAAAGAAATGTAATAATGAGCATAATAAAAAATAGTGTCTGGAACCTTTTTGGCTATGCAATACCAACTTTAATTGCTATCCCCTC

* M S I I K N S V W N L F G Y A I P T L I A I P S

XhoIGCTAGGATTTCTCGCTCGAG 4340

L G F L A R

FIG. 3-Continued.

present in an orientation opposite that of the lac promoter ofthe vector. Putative ribosome binding sites were identifiedwithin 8 to 11 bases upstream of the ATG initiation codons ofrfbA, rfbC, rfbD, and orfl and the GTG initiation codon of rfbB.No characteristic transcription termination sequences werefound within the sequenced region.

Expression of the individual protein products. The fourgenes, rflB, rJbC, rJbA, and rfbD, were cloned individually intoeither pTTQ18 or pTTQ19 on 1.2-kb NsiI-PstI, 1.5-kb SmaI-Hindll, 1.1-kb Asp 700, and 0.9-kb NsiI-XhoI fragments, re-spectively. The plasmids were introduced into E. coli DH5ao. Ineach case, a unique protein band was observed when whole-cell lysates of the 5 mM isopropyl thiogalactopyranoside(IPTG)-induced cultures were analyzed on Coomassie blue-stained SDS-polyacrylamide gels (Fig. 4). The apparent mo-lecular masses of 43, 31.5, 32, and 20.8 kDa were close to thosededuced from their respective nucleotide sequences.

Virulence-associated phenotype of the LPS mutant N1308.Whole-cell ELISA experiments using anti-IpaC and anti-IpaDmonoclonal antibodies indicated normal levels of surfaceexpression of these antigens. Western blotting with anti-VirGantiserum revealed that VirG was present in increasedamounts in the mutant N1308 compared with the wild-typeYSH6000, which in the Western blot showed only subdetect-able levels of VirG. Furthermore, Western blot analysis ofouter membrane cell fractions of N1975 and S7420 confirmedthat VirG localized appropriately to the outer membrane inthis class of mutants (Fig. 5). The minor band seen in lanes Eand F of Fig. 5 is believed to represent either a breakdownproduct of VirG or an internally translated product of virG(25).The virulence phenotype of N1308 was further characterized

by an invasion assay. At 2 h postinfection, monolayers infectedwith equal inocula of YSH6000 and N1308 revealed no defect

in the primary invasive capacity of the mutant. A time courseexperiment revealed that N1308 was able to invade the mono-layers and spread normally within the cytoplasm of the infectedLLC-MK2 cells. Intracellular N1308 isolated from LLC-MK2cells had the abnormal LPS phenotype, and Southern analysisfurther confirmed the stability of the TnS mutation duringpassage through the cell line (results not shown). By 5 hpostinfection, N1308 exhibited a tendency to assume a coccoid

kDa

94-67-

43-

30-

20-

14-

A B C D E F G H I J K L

_+ -_+ -_+ + -_+ -_ +

FIG. 4. Coomassie blue-stained SDS-polyacrylamide gel showingexpression of the four rhamnose biosynthetic enzymes in E. coli DH5Saharboring plasmids pSBA239 (rjbB) (lanes A and B), pTTQ18 (lanes Cand D), pSBA251 (nbA) (lanes E and F), pSBA238 (rfbD) (lanes Gand H), pTTQ19 (lanes I and J), and pSBA252 (rflC) (lanes K and L).The presence (+) or absence (-) of IPTG in the bacterial cultures isindicated. Unique plasmid-encoded proteins are marked by arrow-heads. The positions of standard molecular mass markers are shown onthe left.

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kDa A B C D E F G

94-

67- |

30l

FIG. 5. Western blot analysis with VirG-specific antiserum. Whole-cell lysates are shown in lanes A (YSH6000), B (N1308), and C(CS2068-1). Outer membrane-specific cell fractions are shown in lanesD (YSH6000), E (N1975), F (S7420), and G (CS2068-1). VirGexpression in CS2068-1 cultures was induced with 5 mM IPTG. The116-kDa VirG protein is indicated by the arrowhead. The positions ofstandard molecular mass markers are shown on the left.

shape, unlike YSH6000, which retained normal bacilliformmorphology even at 8 h postinfection. The most apparentdifference was the delayed and probably impaired ability ofN1308 to spread to adjacent cells. However, unlike VirGmutant M94, which was unable to spread, N1308 did showsome degree of cell-to-cell spread (Table 3; Fig. 6). Further-more, N1308 did not exhibit an increased rate of intracellularbacterial multiplication during the early stages of infection, aphenomenon often observed with M94 (Fig. 6G). By 8 hpostinfection, division of infected LLC-MK2 cells resulted in aslightly accentuated apparent spreading ability. This was alsoevident for M94, which is known to be incapable of intercel-lular spread (7, 28).The intracellular behavior of mutant N1308 with respect to

F-actin deposition revealed a significant difference from that ofYSH6000. Intracellular N1308 bacteria at 2 h postinfectionwere clearly outlined with peribacterial F-actin, with only slightpolar accumulation of the actin. No F-actin tails were present.In contrast, YSH6000 was only faintly outlined with peribac-terial actin and exhibited significant unipolar accumulation ofthe actin. F-actin tails that were often many times the length of

TABLE 3. Cell-to-cell spread and intracellular multiplicationof S. fiexneri in infected LLC-MK2 monolayers

YSH6000 N1308 M94

Time No. of No. of No. of No. of No. of

(h) bacterial adjacent bacteria/ djacent bacteria/ MK2index MKell index cells index cellscell' ifctedbs cell ifctedl cell cel

infected' infected ~~~infected

1 1.9 0.1 1.2 0.1 11.4 0.02 7.1 1.9 2.6 0.8 12.9 0.03 15.4 4.7 9.5 2.1 >20 0.45 > 20 6.0 > 20 4.0 NDC ND8 >20 6.0 >20 4.3 >20 2.3

a Data obtained by averaging counts from a minimum of 10 infected indexcells.

b Average number of adjacent cells infected in an islet defined as the six MK2cells nearest the index cell.

c ND, not done.

the bacterium were frequently observed. M94, as expected, didnot induce F-actin deposition (Fig. 7).

DISCUSSION

LPS is the major surface molecule of gram-negative bacte-ria. LPS is anchored in the bacterial outer membrane by thelipid A moiety, with an intermediate and relatively conservedcore oligosaccharide and finally a distal, serotype-specific0-antigen polysaccharide side chain. With the exception ofserotype 6, the S. flexneri 0 side chain is composed of atetramer of rhamnose-rhamnose-rhamnose-N-acetylglucos-amine with serotype specificity resulting from variations de-rived by acetylation and/or glucosylation of the sugars compris-ing the repeat unit (26). Genetic mapping studies in S. flexnerihave revealed that the rjb locus encoding the synthesis of thesubunit tetramer is his linked (14), while the locus responsiblefor serotype specificity is derived from a bacteriophage and isintegrated near the pro-lac region on the chromosome (14, 38,57). Okada et al. (32, 33) defined a third LPS locus involved inO side chain synthesis, the putative rfc locus, characterized bymutant N1308. They identified this locus as being approxi-mately 10 kb from the rfb locus. Mutations in this new locusresulted in bacteria exhibiting LPS with a markedly shortenedO side chain, similar to rfc mutants of Salmonella typhimuriumwhich have, at most, one 0-antigen tetrasaccharide unit at-tached to each core oligosaccharide (61). The rfc locus of S.typhimunium, which is distant from its rib locus, has beencloned and found to encode a single protein believed tofunction as an 0-subunit polymerase (12). However, the locusdefined by S. flexneri mutant N1308 spanned a region of about3.3 kb, as judged from complementation studies, and wasunlikely to encode only one protein. Nucleotide sequencing ofthe region confirmed this locus as part of the rJb region of S.flexneri and not of the postulated rfc locus. Full complementa-tion of the mutant required the presence of all four rhamnosebiosynthetic genes. However, silver-stained polyacrylamidegels revealed a partial complementation of the LPS phenotypein N1308 transformed with plasmids lacking either the 5' endof rfbB or the 3' end of rJbD. The result seen with pSBA122and pSBA123 may have been a consequence of a truncatedrjbB product expressed from the chromosome of N1308 beingcomplemented by the remaining genes in trans, while thecomplementation seen with pSBA134 and pSBA117 possiblyresulted from transcription of rJbD from a weak promoterupstream of the chromosomal gene.

Further restriction analysis of the primary clone pSBA52,derived from YSH6200, and its comparison with the restrictionmaps of both the 9.2-kb Kmr Sall fragment of N1308 and the12.2-kb Kmr EcoRI fragment of the rjb mutant S6933 (33)revealed that the two EcoRI fragments of pSBA52 werecontiguous (data not shown). S6933 had LPS totally lacking an0 side chain. The TnS inserts in mutants N1308 and S6933were 5.5 kb apart (Fig. 1B). Given the ability to fully comple-ment mutant N1308 with only part of the rjb locus, it seemshighly probable that this locus in S. flexneri 2a consists of atleast two distinct operons.

Recently Macpherson et al. (27) cloned and mapped the riblocus of S. flexneri 2a. Using the synthesis of immunologicallyreactive 0 antigen as an assay, they defined an rjb locus ofabout 11 kb (Fig. 1B). Yao et al. (59) also cloned and mappedthis locus from S. flexneri 2a and noted slight differences in therestriction map of their clone compared with that of Macpher-son et al. (27), who also identified a region contiguous with therfb locus involved in the regulation of the 0 side chain length.

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FIG. 6. Intracellular movement and cell-to-cell spread of S. flexneri in an infected LLC-MK2 monolayer. (A to C) YSH6000 at 1, 3, and 8 h;(D to F) N1308 at 1, 3, and 8 h; (G to I) M94 at 1, 3, and 8 h. Bar: 10 pum.

This is homologous with the rol gene of E. coli and S.typhimurium (4, 5). The rjb loci of S. flexneri 3a (59) and S.flexneri 6 (10) have also been cloned. On the basis of Southernanalysis, the locus present in serotype 6 shares little similaritywith any of the other S. flexneri serotypes (10), a fact consistentwith it encoding a unique 0 side chain subunit. There are nosequence data available for any of these cloned loci. Klena andSchnaitman (21) have recently cloned and partially sequencedthe rjb gene cluster of Shigella dysenteriae 1. They haveidentified eight functional genes within this locus, at the 5' endof which lie the rfbBCAD genes that are organized as in S.flexneni 2a and S. entenica LT2. The only sequence dataavailable are of the distal two-thirds of rJbD, and as expected,this region is highly similar to that of S. flexneri 2a. Interest-ingly, though, the point of divergence between the rJbD genesof S. flexneni 2a and S. dysentenae 1 occurs 58 bases prior tothe corresponding point between S. flexneni 2a and S. entenicaLT2.The sequenced region of the rJb locus of S. flexnen 2a shared

a marked level of similarity with that of S. enterica LT2 (20).

The 4.3-kb BstEII-XhoI fragment encodes 387 bp of orJ2.8 andthe four rhamnose biosynthetic genes rJbB, rfbC, rJbA, andrfbD. The entire sequence, including the 372-bp intergenicregion, showed high levels of similarity at the nucleotide level,with only slightly reduced homology at the 3' end. Theconservation of gene order and the extensive homology suggesta relatively recent acquisition of this region by some recombi-national event from a common ancestral source (39). Assum-ing an organization similar to that of Salmonella spp., the rjblocus of S. flexneri, in addition to the four rhamnose biosyn-thetic genes, probably encodes three different transferases,each being responsible for one of the linkages of the 0-antigentetramer (24). An additional gene that may also be encodedwithin the locus is the putative rfc gene. The presence of rfcwithin the rjb region has been shown in S. dysenteriae 1 (21)and S. entenica group C2 (8, 9), with a similar organizationbeing likely in several other Salmonella species (24). Usingminicell and maxicell analysis of the cloned rjb of S. flexneri 2a,Macpherson et al. (27) have identified and partially mapped sixproteins in the region involved in 0-antigen biosynthesis.


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FIG. 7. Double immunofluorescence assay showing the same fieldof LLC-MK2-infected monolayers in each horizontal pair of panels.Association of F-actin with YSH6000 (A and B), N1308 (C and D), andM94 (E and F) is shown. Bacteria were visualized by immunofluores-

cence, using rabbit anti-O 3,4 antiserum and fluorescein isothiocya-nate-conjugated goat anti-rabbit immunoglobulin G (A, C, and E).F-actin was labelled with rhodamine-labelled phalloidin (B, D, and F).Arrowheads indicate the formation of F-actin tails behind YSH6000

(B) and of peribacterial actin, without tails, around N1308 (D). Bar: 10jim.

Proteins G and F probably correspond to the products of rjbDand either r-fA or rJbC, respectively. However, they haveaccounted for only about 5 kb out of a total of 11 kb of DNA,hence supporting the proposal that the rjb region may encodeeight genes. Sequencing the remainder of the rjb locus of S.flexneri 2a would confirm these hypotheses.

The endotoxic effects of LPS and its ability to inducenonspecific resistance to infection have been known for sometime (26). A more specific role for LPS, largely determined bythe 0 antigen, in the pathogenesis of bacillary dysentery isbecoming increasingly apparent. Early studies with spontane-ous rough mutants of S. flexneri showed that these LPS mutantscould invade HeLa cells but were avirulent and could notinduce a positive Sereny test (34, 35).Recent work by Okada et al. (33) has further clarified the

role of LPS in virulence. An LPS rfa mutant with a defectivecore structure (N1876) and an rjb mutant with an absent 0 sidechain (S6933) were able to invade LLC-MK2 cells with an

efficiency equal to that of the wild-type strain YSH6000.However, they exhibited a reduced capacity to spread toadjacent LLC-MK2 cells. Unlike the VirG mutant (7, 28), theycould spread within the cytoplasm. At 4 h postinfection, theLPS mutants showed a tendency to swell, with this featurebecoming more evident with time (33). These observations ledOkada et al. (33) to propose that the smooth surface ofinvading shigellae is required for spread to adjacent cells andfor protection of the intracellular bacteria from the hostdefense mechanisms. Paradoxically, Okada et al. (33) alsoobserved that the LPS mutant S2687 (rfa::TnS), with a defec-tive core structure but an apparently normal 0 side chainprofile and hence presumably a smooth bacterial surface,shared this virulence phenotype.

Whole-cell ELISA experiments confirmed that N1308 hadnormal levels of surface-expressed IpaC and IpaD. On thebasis of studies of the surface coexpression of Ipa antigens(2, 48, 56), normal levels of surface IpaB would also beexpected. This result was consistent with the finding that themutant had no defect in the primary invasion of LLC-MK2cells. N1308 could spread and multiply normally within thecytoplasm of infected cells, but it showed a significant delayand probably a defect in its ability to spread to adjacent cells.Like the other LPS mutants described by Okada et al. (33),intracellular N1308 exhibited morphological changes by 5 hpostinfection.The limited ability of N1308 to spread to adjacent cells

suggested a possible link with the VirG protein, but unlike theVirG- mutant M94, N1308 appeared to move normally withinan infected cell. Recent studies by Vasselon et al. (54, 55) havedemonstrated the existence of a second type of intracellularmovement, designated the Olm phenotype, which remainsintact in M94 and explains the accumulation of the VirG -strain near the cell nucleus. It is possible that N1308 lacks thisability to track along the actin stress filaments and conse-quently is randomly distributed throughout the cell cytoplasm.N1308 and two other mutants in this class (N1975 and S7420)were shown to have enhanced levels of immunologically reac-tive VirG compared with the wild-type YSH6000. Examinationof outer membrane fractions of N1975 and S7420 confirmed anormal cellular localization of VirG.These findings led us to speculate about the functional

characteristics of the VirG protein in the mutant. A doubleimmunofluorescence assay on infected LLC-MK2 monolayersshowed that N1308, unlike the VirG - mutant (7), induced thepolymerization of F-actin, but there was a defect in theorganization of F-actin around the intracellular bacteria. Theinability of the mutant to induce the formation of a normalF-actin tail and its tendency to accumulate greater amounts ofperibacterial actin with only slight polar accumulation maylargely explain the impaired ability of the mutant to spreadfrom cell to cell. Sanger et al. (43) showed that host cell actinassembly was a necessary prerequisite for intracellular move-ment of Listeria monocytogenes. The formation of F-actin tailsin infected cells correlated directly with bacterial movement.An avirulent mutant of L. monocytogenes with an impairmentin intracellular and intercellular spread has been isolated byTn916 mutagenesis (22). Interestingly this mutant, like S.flexneri mutant N1308, still induced polymerization of actin,which also accumulated as a dense peribacterial cloud ofF-actin (22). There are several possible explanations for thecontribution of the abnormal LPS in N1308 to this defect. Itmay result in abnormal surface presentation or posttransla-tional modification of VirG, as is the case with the requirementfor LPS in the normal trimerization of the OmpF porin of E.coli (50). The abnormal LPS may result in excessive amounts of

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surface VirG, as was seen on Western blot analysis, or in an

abnormal distribution of VirG on the cell surface. A recentreport by Goldberg et al. (15) demonstrated that VirG was

usually localized at one pole of the bacterium, a feature thatmay well be disrupted in N1308. Finally, the change in LPStype may have wide-ranging effects on the surface characteris-tics of the mutant (37). Any of these changes may affect thenormal organization of polymerized actin and consequentlyimpair bacterial movement.

ACKNOWLEDGMENTS

We are grateful to Ian McPherson for skilled technical assistance.This work was supported by project grants from the National Health

and Medical Research Council, Canberra, Australia, and the Ministryof Science, Education and Culture, Tokyo, Japan. K. Rajakumar was

the recipient of a Macfarlane Burnet Biomedical Research Scholar-ship.

REFERENCES1. Adler, B., C. Sasakawa, T. Tobe, S. Makino, K. Komatsu, and M.

Yoshikawa. 1989. A dual transcriptional activation system for the230 kb plasmid genes coding for virulence-associated antigens ofShigella flexneri. Mol. Microbiol. 3:627-635.

2. Andrews, G. P., A. E. Hromockyj, C. Coker, and A. T. Maurelli.1991. Two novel virulence loci, mxiA and mxiB, in Shigella flexneri2a facilitate excretion of invasion plasmid antigens. Infect. Immun.59:1997-2005.

3. Ausubel, F. M., R. Brent, R. E. Kingston, D. M. Moore, J. G.Seidman, J. A. Smith, and K. Struhl (ed.). 1991. Current protocolsin molecular biology. Greene Publishing Associates and Wiley-Interscience, New York.

4. Bastin, D. A., G. Stevenson, P. K. Brown, A. Haase, and P. R.Reeves. 1993. Repeat unit polysaccharides of bacteria: a model forpolymerization resembling that of ribosomes and fatty acid syn-thetase, with a novel mechanism for determining chain length.Mol. Microbiol. 7:725-734.

5. Batchelor, R. A., P. Alifano, E. Biffali, S. I. Hull, and R. A. Hull.1992. Nucleotide sequences of the genes regulating 0-polysaccha-ride antigen chain length (rol) from Escherichia coli and Salmo-nella typhimurium: protein homology and functional complemen-tation. J. Bacteriol. 174:5228-5236.

6. Bernadini, M. L., A. Fontaine, and P. J. Sansonetti. 1990. Thetwo-component regulatory system OmpR-EnvZ controls the viru-lence of Shigella flexneri. J. Bacteriol. 172:6274-6281.

7. Bernardini, M. L., J. Mounier, H. d'Hauteville, M. Coquis-Rondon, and P. J. Sansonetti. 1989. Identification of icsA, a

plasmid locus of Shigella flexneri that governs bacterial intra- andintercellular spread through interaction with F-actin. Proc. Natl.Acad. Sci. USA 86:3867-3871.

8. Brown, P. K., L. K. Romana, and P. R. Reeves. 1991. Cloning ofthe rib gene cluster of a group C2 Salmonella strain: comparisonwith the rib regions of groups B and D. Mol. Microbiol. 5:1873-1881.

9. Brown, P. K., L. K. Romana, and P. R. Reeves. 1992. Molecularanalysis of the rjb gene cluster of Salmonella serovar muenchen(strain M67): the genetic basis of the polymorphism betweengroups C2 and B. Mol. Microbiol. 6:1385-1394.

10. Cheah, K.-C., D. W. Beger, and P. A. Manning. 1991. Molecularcloning and genetic analysis of the rib region from Shigella flexneritype 6 in Escherichia coli K-12. FEMS Microbiol. Lett. 83:213-218.

11. Clerc, P., and P. J. Sansonetti. 1987. Entry of Shigella flexneri intoHeLa cells: evidence for directed phagocytosis involving actinpolymerization and myosin accumulation. Infect. Immun. 55:2681-2688.

12. Collins, L. V., and J. Hackett. 1991. Molecular cloning, character-ization, and nucleotide sequence of the rfc gene, which encodes an

0-antigen polymerase of Salmonella typhimurium. J. Bacteriol.173:2521-2529.

13. d'Hauteville, H., and P. J. Sansonetti. 1992. Phosphorylation ofIcsA by cAMP-dependent protein kinase and its effect on inter-cellular spread of Shigella flexneri. Mol. Microbiol. 6:833-841.

14. Formal, S. B., P. Gemski, Jr., L. S. Baron, and E. H. Labrec. 1970.Genetic transfer of Shigella flexneri antigens to Escherichia coliK-12. Infect. Immun. 1:279-287.

15. Goldberg, M. B., 0. Bfirzu, C. Parsot, and P. J. Sansonetti. 1993.Unipolar localization and ATPase activity of IcsA, a Shigellaflexneri protein involved in intracellular movement. J. Bacteriol.175:2189-2196.

16. Grunstein, M., and D. S. Hogness. 1975. Colony hybridization: amethod for the isolation of cloned DNAs that contain a specificgene. Proc. Natl. Acad. Sci. USA 72:3961-3965.

17. High, N., J. Mounier, M. C. Prevost, and P. J. Sansonetti. 1992.IpaB of Shigella flexneri causes entry into epithelial cells andescape from the phagocytic vacuole. EMBO J. 11:1991-1999.

18. Hitchcock, P. J., and T. M. Brown. 1983. Morphological hetero-geneity among Salmonella lipopolysaccharide chemotypes in sil-ver-stained polyacrylamide gels. J. Bacteriol. 154:269-277.

19. Hromockyj, A. E., S. C. Tucker, and A. T. Maurelli. 1992.Temperature regulation of Shigella virulence: identification of therepressor gene virR, an analogue of hns, and partial complemen-tation by tyrosyl transfer RNA (tRNAJTYr). Mol. Microbiol.6:2113-2124.

20. Jiang, X.-M., B. Neal, F. Santiago, S. J. Lee, L. K. Romana, andP. R. Reeves. 1991. Structure and sequence of the rfb (O antigen)gene cluster of Salmonella serovar typhimurium (strain LT2). Mol.Microbiol. 5:695-713.

20a.jost, B. H. Unpublished data.21. Klena, J. D., and C. A. Schnaitman. 1993. Function of the rib gene

cluster and the rfe gene in the synthesis of 0 antigen by Shigelladysenteriae 1. Mol. Microbiol. 9:393-402.

22. Kuhn, M., M. Prevost, J. Mounier, and P. J. Sansonetti. 1990. Anonvirulent mutant of Listeria monocytogenes does not moveintracellularly but still induces polymerization of actin. Infect.Immun. 58:3477-3486.

23. Lawlor, K. M., P. A. Daskaleros, R. E. Robinson, and S. M. Payne.1987. Virulence of iron transport mutants of Shigella flexneri andutilization of host iron compounds. Infect. Immun. 55:594-599.

24. Lee, S. J., L. K. Romana, and P. R. Reeves. 1992. Sequence andstructural analysis of the rfb (O antigen) gene cluster from a groupCl Salmonella enterica strain. J. Gen. Microbiol. 138:1843-1855.

25. Lett, M., C. Sasakawa, N. Okada, T. Sakai, S. Makino, M.Yamada, K. Komatsu, and M. Yoshikawa. 1989. virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virGprotein and determination of the complete coding sequence. J.Bacteriol. 171:353-359.

26. Lindberg, A. A., A. Karnell, and A. Weintraub. 1991. The lipopoly-saccharide of Shigella bacteria as a virulence factor. Rev. Infect.Dis. 13:S279-S284.

27. Macpherson, D. F., R. Morona, D. W. Beger, K.-C. Cheah, andP. A. Manning. 1991. Genetic analysis of the rfb region of Shigellaflexneri encoding the Y serotype 0-antigen specificity. Mol. Mi-crobiol. 5:1491-1499.

28. Makino, S., C. Sasakawa, K. Kamata, T. Kurata, and M. Yo-shikawa. 1986. A genetic determinant required for continuousreinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell46:551-555.

29. Morelle, G. 1989. A plasmid extraction procedure on a miniprepscale. Focus 11:7-8.

30. Nakata, N., C. Sasakawa, N. Okada, T. Tobe, I. Fukuda, T. Suzuki,K. Komatsu, and M. Yoshikawa. 1992. Identification and charac-terization of virK, a virulence-associated large plasmid gene essen-

tial for intercellular spreading of Shigella flexneri. Mol. Microbiol.6:2387-2395.

31. Nassif, X., M. C. Mazert, J. Mounier, and P. J. Sansonetti. 1987.Evaluation with an iuc::TnJO mutant of the role of aerobactinproduction in the virulence of Shigella flexneri. Infect. Immun.55:1963-1969.

32. Okada, N., C. Sasakawa, T. Tobe, K. A. Talukder, K. Komatsu,and M. Yoshikawa. 1991. Construction of a physical map of thechromosome of Shigella flexneri 2a and the direct assignment ofnine virulence-associated loci identified by Tn5 insertions. Mol.Microbiol. 5:2171-2180.

33. Okada, N., C. Sasakawa, T. Tobe, M. Yamada, S. Nagai, K. A.Talukder, K. Komatsu, S. Kanegasaki, and M. Yoshikawa. 1991.

J. BAc-rERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


Virulence associated chromosomal loci of Shigella flexneri identi-fied by random TnS insertion mutagenesis. Mol. Microbiol. 5:187-195.

34. Okamura, N., T. Nagai, R. Nakaya, S. Kondo, M. Murakami, andK. Hisatsune. 1983. HeLa cell invasiveness and 0 antigen ofShigella flexneri as separate and prerequisite attributes of virulenceto evoke keratoconjunctivitis in guinea pigs. Infect. Immun. 39:505-513.

35. Okamura, N., and R. Nakaya. 1977. Rough mutant of Shigellaflexneri 2a that penetrates tissue culture cells but does not evokekeratoconjunctivitis in guinea pigs. Infect. Immun. 17:4-8.

36. Osborne, M. J., J. E. Gander, E. Parisi, and J. Carson. 1972.Mechanism of assembly of the outer membrane of Salmonellatyphimurium-isolation and characterization of cytoplasmic andouter membrane. J. Biol. Chem. 247:3962-3972.

37. Parker, C. T., A. W. Kloser, C. A. Schnaitman, M. A. Stein, S.Gottesman, and B. W. Gibson. 1992. Role of the rfaG and rfaPgenes in determining the lipopolysaccharide core structure andcell surface properties of Escherichia coli K-12. J. Bacteriol.174:2525-2538.

38. Petrovskaya, V. G., and T. A. Licheva. 1982. A provisionalchromosome map of Shigella and the regions related to pathoge-nicity. Acta Microbiol. Acad. Sci. Hung. 29:41-53.

39. Reeves, P. 1993. Evolution of Salmonella 0 antigen variation byinterspecific gene transfer on a large scale. Trends Genet. 9:17-22.

40. Reeves, P. Personal communication.41. Sakai, T., C. Sasakawa, and M. Yoshikawa. 1988. Expression of

four virulence antigens of Shigella flexneri is positively regulated atthe transcriptional level by the 30 kilodalton virF protein. Mol.Microbiol. 2:589-597.

42. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencingwith chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467.

43. Sanger, J. M., J. W. Sanger, and F. S. Southwick 1992. Host cellactin assembly is necessary and likely to provide the propulsiveforce for intracellular movement of Listeria monocytogenes. Infect.Immun. 60:3609-3619.

44. Sasakawa, C., B. Adler, T. Tobe, N. Okada, S. Nagai, K. Komatsu,and M. Yoshikawa. 1989. Functional organization and nucleotidesequence of virulence Region-2 on the large virulence plasmid inShigella flexneri 2a. Mol. Microbiol. 3:1191-1201.

45. Sasakawa, C., J. M. Buysse, and H. Watanabe. 1992. The largevirulence plasmid of Shigella. Curr. Top. Microbiol. Immunol.180:21-44.

46. Sasakawa, C., K. Kamata, T. Sakai, S. Makino, M. Yamada, N.Okada, and M. Yoshikawa. 1988. Virulence-associated geneticregions comprising 31 kilobases of the 230-kilobase plasmid inShigella flexneri 2a. J. Bacteriol. 170:2480-2484.

47. Sasakawa, C., K. Kamata, T. Sakai, S. Y. Murayama, S. Makino,and M. Yoshikawa. 1986. Molecular alteration of the 140-megadal-ton plasmid associated with loss of virulence and Congo red

binding activity in Shigella flexneri. Infect. Immun. 51:470-475.48. Sasakawa, C., K. Komatsu, T. Tobe, T. Suzuki, and M. Yoshikawa.

1993. Eight genes in region 5 that form an operon are essential forinvasion of epithelial cells by Shigella flexneri 2a. J. Bacteriol.175:2334-2346.

49. Sasakawa, C., S. Makino, K. Kamata, and M. Yoshikawa. 1986.Isolation, characterization, and mapping of Tn5 insertions into the140-megadalton invasion plasmid defective in the mouse Serenytest in Shigella flexneni 2a. Infect. Immun. 54:32-36.

50. Sen, K., and H. Nikaido. 1991. Lipopolysaccharide structurerequired for in vitro trimerization of Escherichia coli OmpF porin.J. Bacteriol. 173:926-928.

51. Simmons, D. A. R., and E. Romanowska. 1987. Structure andbiology of Shigella flexneri 0 antigens. J. Med. Microbiol. 23:289-302.

52. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

53. Tobe, T., C. Sasakawa, N. Okada, Y. Honma, and M. Yoshikawa.1992. vacB, a novel chromosomal gene required for expression ofvirulence genes on the large plasmid of Shigella flexneri. J. Bacte-riol. 174:6359-6367.

54. Vasselon, T., J. Mounier, R. Hellio, and P. J. Sansonetti. 1992.Movement along actin filaments of the perijunctional area and denovo polymerization of cellular actin are required for Shigellaflexneri colonization of epithelial Caco-2 cell monolayers. Infect.Immun. 60:1031-1040.

55. Vasselon, T., J. Mounier, M. C. Prevost, R. Hellio, and P. J.Sansonetti. 1991. Stress fiber-based movement of Shigella flexneriwithin cells. Infect. Immun. 59:1723-1732.

56. Venkatesan, M. M., J. M. Buysse, and E. V. Oaks. 1992. Surfacepresentation of Shigella flexneni invasion plasmid antigens requiresthe products of the spa locus. J. Bacteriol. 174:1990-2001.

57. Verma, N. K., J. M. Brandt, D. J. Verma, and A. A. Lindberg. 1991.Molecular characterization of the 0-acetyl transferase gene ofconverting bacteriophage SF6 that adds group antigen 6 to Shigellaflexneri. Mol. Microbiol. 5:71-75.

58. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. ImprovedM13 phage cloning vectors and host strains: nucleotide sequencesof the M13mpl8 and pUC19 vectors. Gene 33:103-119.

59. Yao, Z., H. Liu, and M. A. Valvano. 1992. Acetylation of 0-specificlipopolysaccharides from Shigella flexneri 3a and 2a occurs inEschenichia coli K-12 carrying cloned S. flexneri 3a and 2a rfbgenes. J. Bacteriol. 174:7500-7508.

60. Yoshikawa, M., and C. Sasakawa. 1991. Molecular pathogenesis ofshigellosis: a review. Microbiol. Immunol. 35:809-824.

61. Yuasa, R., K. Nakane, and H. Nikaido. 1970. Structure of cell walllipopolysaccharide from Salmonella typhimunum-structure oflipopolysaccharide from a semirough mutant. Eur. J. Biochem.15:63-71.

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Nucleotide Sequence Operon Shigella flexneri 2a …Shigella spp. and enteroinvasive Escherichia coli (45). This plasmid encodes the antigens IpaBCD, which are critical for primarymucosalcell