Proc. Nail. Acad. Sci. USAVol. 81, pp. 6110-6114, October 1984Genetics

Pilus genes of Neisseria gonorrheae: Chromosomal organizationand DNA sequence

(pathogenesis/phase variation/antigenic variation)

THOMAS F. MEYER*, ELIZABETH BILLYARDt, RAINER HAAS*, SABINE STORZBACH*, AND MAGDALENE SOt*Max-Planck-Institute fur medizinische Forschung, Abteilung Molekulare Biologie and Zentrum fur Molekulare Biologie der Universitat, Jahnstrasse 29, 6900Heidelberg, Federal Republic of Germany; and fDepartment of Molecular Biology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road,La Jolla, CA 92037

Communicated by Ernest Beutler, June 20, 1984

ABSTRACT We have mapped two regions of the Neisseriagonorrheae genome, pilEI and pilE2, which are involved inpilus expression. When the cells are in the piliated P+ state,these two loci carry sequences necessary for pilin production.A silent locus, piSI, also maps near pilEI and pUE2. pilSIcontains structural gene information but lacks pilus promotersequences. The pilus gene sequences in pilEI and piLE2 areidentical in strain MS1l.

The pilus is a predominant surface antigen of Neisseria gon-orrheae and allows adhesion of the bacterium to various hostcells (1-4). Pilus expression can be turned on and off at highfrequency (phase variation), a condition that can be detectedeasily by differences in colonial morphology; piliated (P+)cells are significantly more infective than nonpiliated (P-)cells (5-7). Many pilin serotypes exist, and some gonococcalisolates can express more than one biochemically distinct pi-lin type (8-10). Recently, we have obtained evidence that asingle clone expressing one pilin serotype can give rise tolines that express many antigenically variant pilin (unpub-lished data).The fimbriate pilus structure is composed of identical sub-

units of -"18,000 daltons. Pilin of different serotype all sharea highly conserved strongly hydrophobic amino-terminal re-gion, while their hydrophilic carboxyl-terminal domains arevariable (11, 12). This latter region is the major target for thehost immune response.We have reported the isolation of two pilus gene copies

from N. gonorrheae strain MS11 (13). A single gonococcalgenome contains several pilus-related sequences, and transi-tion from the P+ to the P- state, and vice versa, involvesreversible reorganization of chromosomal elements (13). Wereport here the alignment of some of the pilus gene copies ina chromosomal map and an analysis of their physical andfunctional nature.

MATERIALS AND METHODSBacterial Strains. N. gonorrheae strain MS11 (13) was

used throughout these studies. Recombinant plasmids werefirst transformed (14) into Escherichia coli strain GC1 (13)and subsequently transferred to strain DHI (14) for furtheranalysis.DNA Manipulations. Recombinant DNA technology and

high-stringency Southern blotting were as described by Man-iatis et al. (15). Restriction enzyme digestions and BAL-31digestions from the Hpa I site of pNG1100 were done underconditions recommended by the vendor (New England Bio-labs). HindIII linkers (Collaborative Research) were placedat the ends of the deleted DNA by the technique describedby Maniatis et al. (15).

DNA Sequencing. The 5' ends of the HindIII site from theBAL-31-generated clones and from the Sma I and Cla I sitesof pNG1100 were sequenced as described by Maxam andGilbert (16).DNA Probes. DNA probes were labeled by using T4 poly-

nucleotide kinase (New England Biolabs) as described byMaxam and Gilbert (16).

Plasmid Isolation. Plasmids were isolated by the procedureof Kupersztock and Helinski (17) and purified by passagethrough a Sepharose CL-6B column (Pharmacia).

RESULTS

Cloning and Chromosomal Mapping of Pilus Gene Se-quences. The large number of pilus-related sequences in thegonococcal genome (13) complicated the alignment of pilusgene loci in a linear chromosomal map when Southern hy-bridizations alone were used. Applying a different strategy,we obtained recombinant clones containing pilus-related se-quences by subcloning, from a gonococcal chromosomal li-brary from strain MS11 (P+), with pBR322 as a vector and a700-base-pair (bp) Hpa I/Bgl I fragment of pNG1100 asprobe (see the legend to Fig. 1). Of 60 independent clones,we concentrated on 3-pNG1312, pNG1711, and pNG1721-because they showed a particularly strong cross-hybridiza-tion to the probe under highly stringent conditions. Theseplasmids, which carry HindIII or Bcl I inserts of 5-11 kilo-bases (kb) (Fig. 1 c and d), were then deleted for the pilus-related sequences, resulting in subclones pNG1704,pNG1705, and pNG1706 (Fig. 1 c and d) that contain onlythe flanking sequences. pNG1704, pNG1705, pNG1706, andcloned pilus sequences then were used as probes in Southernblotting experiments with chromosomal MS11 DNA that hadbeen digested with various combinations of restriction endo-nucleases. The hybridizations allowed us to map the regionssurrounding the pilus genes within a range of 20-30 kb in thechromosome. Certain restriction fragments hybridized tomore than one probe (Fig. la). This, together with restrictionmaps of the inserts, allowed us to draw a physical map thatlinks three major chromosomal pilus gene loci within a dis-tance of ""80 kb (Fig. lb). This preliminary map was recentlyconfirmed by analysis of larger chromosomal inserts cover-ing this region (Fig. lb).The 80-kb mapped region represents "''5% of the total

chromosome of N. gonorrheae, whose size is 1600 kb (18).This mapped region could only account for three of the eightCla I fragments that had been shown to hybridize strongly topilus sequences (13). Therefore, pilus-related sequences arenot localized to one region but must occur over a wide areaon the gonococcal genome.

Abbreviations: kb, kilobase(s); bp, base pair(s); P+, piliated; P-,nonpiliated.

6110

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. NatL Acad. Sci. USA 81 (1984) 6111

0 10 20 30 40 50 60 70109 I I I I, Kb(a/b)

-

,,0

I k\ I, -ir v r _I - r I

--A

M- -

X2

a Ei

X

pNG1005

pNG1flI2 pNGI7

PG 1003

11

pNG tx0

pNG 1711win

o,

pNG 1721

pNG rAW0

cJuwSi

am _ --N

jf .32 &32 FA,

pNG172

pNG 1704

El

I1IIk;~~~~~II/N~~~~~~~~~~~~4ii 0i i_ _

a

PNG 1705

A B C D

01'ml 1"soI

) r, I -- _ bok . f'at pM S1

-A-

pNC3 I70

0 1 2 3 4 5 6 7 8 9 10 11 Kb (i

pil E;

FIG. 1. Physical analysis ofthree major pilus gene loci andchromosome mapping of pilus

a gene loci. Mapping was accom-plished by Southern blotting with

_~ unique gene sequences from vari-NN- ous pilus clones (which map at the

positions indicated by thickenedlines at corresponding positions ofa and b as hybridization probes ofchromosomal DNA digested witha combination of restriction en-

-rr---- -F--zymes. Fragments that cross-hy-bridize can be aligned in a fashion

IL 2x as shown (a) and provide the basisfor the restriction map (b). Some

b of the indicated fragments hybrid-ize to more than one probe. (b)Chromosomal map of three majorpilus gene loci (pilE), pilE2, andpiS)) and of an opacity gene lo-cus (opal; ref. 18). The relativepositions of these loci in thegenome as determined by South-ern hybridization experimentswere confirmed by restrictionanalysis of cloned fragments fromthis region (indicated by lines be-neath the map). *, Areas thatcross-hybridize to pilus gene

C probes; E, areas that hybridize toopacity genes. (c) Analysis of twoexpression loci. The Bcl I insertsof plasmids pNG1711 andpNG1721 representing pilEl and

2 pilE2, respectively, share a regionof complete homology (a). Thisregion contains the gene for pilin(arrow). Plasmid pNG1711 alsoharbors the opaEl locus (X). Theoriginal pilus gene clonespNG1100 and pNG1200 (13) aswell as subclones pNG1704 andpNG1705 of pNG1711 andpNG1721, respectively, whichcarry unique genome sequences,are aligned at the correspondingpositions. *, Positions of 65-bp

d Sma I/Cia I fragments, which ap-pear to be a characteristic compo-nent of pilus gene loci. (d) Analy-sis of a silent pilus gene locus.Plasmid pNG1312 carries a Hin-dIII fragment containing the pilSIlocus. Besides a 65-bp Sma I/ClaI fragment (P.), no apparent ho-mology between this plasmid andthe expression loci (c) is revealedby coarse restriction analysis.Subclone pNG1706 carries unique

c/d) genome sequences.

Two Pilus Expression Sites Constitute the P+ State of MS11Gonococci. A detailed restriction analysis of plasmidspNG1711 and pNG1721 (Fig. ic) identified their inserts as

larger equivalents of the original clones expressing pilusgene, pNG1100 and pNG1200 (13). pNG1711 and pNG1721also produced pilin in E. coli (data not shown). The two in-serts, 9.9 and 10.2 kb, share a central homology of 1.7 kbwithin which is the 965-bp Cla I/Hpa I fragment previouslyshown to encode the actively expressing pilus gene (13). Al-though many genomic Cla I fragments hybridize to the pilusgene probes, only the 4.0-kb and 4.1-kb Cla I fragments

show evidence of rearrangement during the P+-to-P- switch(13). Subsequent mapping by Southern hybridizationsshowed that only these 4.0-kb and 4.1-kb Cla I fragmentshybridized to the pilus-gene-flanking sequences of pNG1711and pNG1721 (data not shown). In addition, only these twoCla I fragments were cleaved by Hpa I to generate 965-bpCla I/Hpa I fragments homologous to the pilus gene probe(Fig. 2). Thus, there are two regions on the MS11 genomethat actively express pilin. To distinguish these from regionsthat contain nonexpressed pilin-related sequences, we desig-nate them expression locus 1 (pilE)) and expression locus 2

PNG 1e

o I I I.

4..

As A i

----771I * ;

Genetics: Meyer et aL

CO.2 i

Proc. NatL Acad. Sci. USA 81 (1984)

(pilE2), with pilE2 being the leftmost on the map (Fig. lb).Our conclusion that pNG1711 and pNG1721 contain pilus-

related expression loci is based on two assumptions. First,we assume that gonococcal genes are expressed equally wellin E. coli. Since N. gonorrheae and E. coli are both Gram-negative bacteria, it is not unreasonable to expect gonococ-cal sequences to be recognized efficiently by the E. coli tran-scriptional and translational machinery. Indeed, in additionto the pilin gene, three other gonococcal genes, encoding theIgA protease (19), the opacity (PII) protein (20), and the pro-line biosynthetic pathway (21), also have been cloned andexpressed in E. coli. Second, we assume that the P+-to-P-rearrangement affects actively expressing pilus genes. Blot-hybridization experiments using the 965-bp Cla I/Hpa I frag-ment as a probe against P+ and P- gonococcal RNA showedthat transcription of the pilus-specific mRNA is abolishedduring the switch (unpublished data).Whether pilEl and pilE2 are or can be used simultaneously

is not known at the moment. The inserts covering each ofthese two regions were obtained from the same chromo-somal DNA preparation purified from cells derived from asingle colony with the P+ phenotype. Although it is verylikely that pilE) and pilE2 are both used concurrently, thepossibility exists that the cells from which the DNA prepara-tions were derived are mixtures that alternately express oneor the other but not both.

Analysis of a Silent Pilus Locus. The third pilus gene locusthat was cloned in plasmid pNG1312 (Fig. ld) hybridizedstrongly with pilus structural gene sequences and much lesswith flanking sequences (data not shown). E. coli containingpNG1312 do not produce pilin, in contrast to those contain-ing pNG1711 and pNG1721 (data not shown). By using sub-clones of the pNG1312 insert as probes in Southern hybrid-ization experiments with gonococcal chromosome, this thirdlocus was mapped to a 5-kb Cla I fragment (ref. 13; Fig. ld).Unlike pilEl and pilE2, which mapped to 4.0-kb and 4.1-kbCla I fragments, respectively (Fig. lb), this third locus didnot undergo rearrangement during pilus phase variation (13).A comparison of the restriction map of this third locus withthe maps ofpilEl and pilE2 (Fig. 1 c and d) suggests that thislocus does not contain an intact pilus structural gene. Thesedata lead us to believe that this site is a silent locus, and wehave designated it pilSI. A 73-bp Sma I/Cla I fragment oc-

A B C

curs in the same orientation in pilSl, pilEl, and pilE2. InpilE) and pilE2 the Sma I/Cla I sites occur 65 bp down-stream from the translated region of the pilus gene (see Fig.4). Whether the sequences in this region play a part in pilusexpression remains to be seen.

Restriction fragments from a series of BAL-31-generateddeletion subclones of the Hpa I/Cla I region of pNG1100(see Fig. 4 and legend) were used as probes in Southern hy-bridization analysis of the pilSl region in pNG1312. Hin-dIII/Mst III fragments from subclones BH2 and BH4, whichare 170 and 240 bp in size, respectively, and contain pilusgene promoter sequences but not the ribosomal binding site(see Fig. 4), did not hybridize to the silent-region clone (Fig.3, lanes c and d). A 250-bp HindIII/Bgl I fragment from BH5(see Fig. 4) containing 5' pilus coding sequences hybridizedto the 2-kb Sma I/Pvu I C fragment and the 1.3-kb PvuI/Sma I D fragment (Fig. ld) within this silent-region insert(Fig. 3). A 350-bp Bgi I/Sma I fragment containing 3' piluscoding sequences (see Fig. 4) also hybridized to the C and Dfragment of pNG1312 (Fig. 3, lane a). DNA sequences ofseveral variant-expressing pilin genes indicate that variabili-ty occurred in the region between the Bgi I site and the stopcodon (unpublished data). Therefore, the latter two probescontain constant and variable pilus gene sequences, respec-tively. Thus, piSl contains structural pilus gene informationbut not pilus promoter sequences found in pilE) and pilE2.Densitometer tracings of hybridization signals indicate thatthe variable region sequences occur 15-20 times more fre-quently than constant region sequences, suggesting that insome regions of pNG1312 the constant and variable regionsequences are not colinear.The variable region sequences detected in these experi-

ments may not be exactly homologous to our 3' pilin geneprobe. Although our hybridization conditions were strin-gent, recent findings (unpublished data) have indicated thatthere are conserved as well as variable regions within the 3'ends of expressed genes encoding variant pilin. Thus, theremay be many copies of different variable regions withinpNG1312.Our previous work (13) has indicated that large sections

of the gonococcal chromosome contain pilus-related se-quences. Since pilE), pilE2, and pilSI account for only aportion of this, many more pilus-related regions remain to be

a b d

2.3C 2.0--

23 -_-9.5 am am.6.7 :4.3 #~am l -

rn ~ FIG. 2. Location of activelyW W expressed pilus genes by South-

ern analysis. Lanes: A, P+ chro-mosomal DNA digested with ClaI; B, P+ chromosomal DNA di-

_ _ gested with Cla I/Hpa I; C, P-chromosomal DNA digested withCla I. The probe is an insert frompNG1100 containing the entire pi-lus gene. HindIII fragments ofDNA served as molecular sizemarkers indicated in kb (arrows).Of all the Cla I genomic fragmentsthat hybridized to pilus gene se-quences, only the 4-kb pilEI and4.1-kb pilE2 fragments were di-gested by Hpa I to generate 3-kband 1-kb fragments.

B

D

A1.(0

amOW

(.8 -t.-

FIG. 3. Presence of pilus gene sequences in piuS]. pNG1312DNA was cleaved with Cla I/Sma I/Pvu I/HindIII and blotted ontonitrocellulose filters for Southern analysis with probes containing 3'pilus gene sequences within a Sma I/Bgl I fragment of pNG1100(lane a), 5' pilus gene sequences within a HindIII/Bgl I fragment ofBH5 (see Fig. 4) (lane b), pilus promoter region sequences within a240-bp HindIII/Mst II fragment of BH2 (see Fig. 4) (lane c), andpilus promoter region sequences within a 120-bp HindIII/Mst IIfragment of BH4 (see Fig. 4) (lane d). Location of restriction frag-ments A-D within the pNG1312 insert is given above the map of thisclone in Fig. ld. The upper two size markers (in kb) are HindIIIfragments; the lower two markers are 4AX174 HincII fragments.

2.3 --1.9

6112 Genetics: Meyer et aL

Genetics: Meyer et aL

examined. These uncharacterized regions also do not under-go rearrangement during the pilus phase switch and maycontain other silent loci. Of 60 recombinant clones, thosecontaining pilEl, pilE2, and pilSI were chosen for study be-cause they showed the strongest hybridization signals to ourpilus gene probe. Since highly stringent hybridization condi-tions were followed in our experiments, the uncharacterizedrecombinant clones giving weak signals most probably con-tain fewer pilin gene sequences. Whether they contain pro-moter sequences is not known at present.

Nucleotide Sequence of the Pilus Structural Gene. pNG1100and pNG1200 inserts were sequenced by using the techniqueofMaxam and Gilbert (16). Because of a lack ofgood restric-tion sites for sequencing, BAL-31 deletions were made fromthe single Hpa I site of both plasmids. HindIII linkers wereinserted at the ends of the deleted DNAs before religation.Deletion endpoints (Fig. 3) within the Cla I-Hpa I region aswell as the Cla I and Sma I ends were labeled for sequencing.

Fig. 4 contains the DNA sequence of the region betweenthe Hpa I and Cla I sites of pNG1100. In this area is a single

Proc. Nat. Acad. Sci. USA 81 (1984) 6113

open-reading frame, 498 bp in length, starting at position 335and ending at position 833 near the Cla I site. A strong ribo-some binding site A-G-G-A-G occurs seven bases upstreamof the start codon. Primer extension studies using a syntheticoligonucleotide complementary to the 3' untranslated regionof pilus-specific mRNA placed the start of transcription atthe guanosine at position 245 (unpublished data); five basesupstream of this putative transcription initiation site is astrong -10 sequence (T-A-T-A-A-T), although a classical-35 sequence is missing.We also have determined a portion of the sequence of the

pilin gene within the pNG1200 insert, covering the 3' end ofthe coding sequence (variable region). From the Sma I site tothe Bgl I site within the gene, the sequences in pNG1100 andpNG1200 are identical. pNG1100 and pNG1200 also showexact homology within a 200-bp region extending from theHpa I site towards the pilin gene. Therefore, it is more thanlikely that both expression loci are expressing the samestructural gene. This has been confirmed by sequence analy-sis of pilus-specific mRNA (unpublished results).

*BH1 16 31 46 61TTA ACG CGT AAA TTC AAA AAT CTC AAA TTC CGA CCC AAT CAA CAC ACC CGA TAC CCC ATG

*BH2 76 91 106 121CCA ATA AAA AAG TAA CGA AAA TCG GCA CTA AAA CTG ACA ATT TTC GAC ACT GCC GCC CCC

BH4 o1.36 151 166 161

CTA CTT CCG CAA ACC ACA CCC ACC TAA AAG AAA ATA CAA AAT AAA AAC AAT TAT ATA GAG

196 211 226 241ATA AAC GCA TAA AAT TTC ACC TCA AAA CAT AAA ATC GGC ACG AAT CTT GCT Tl JTAA_AC

1 256 271 286 301GCA GTT GTC GCA ACA AAA AAC CGA TGG TTA AAT ACA TTG CAT GAT GCC GAT GGC AAG C

Mst I *BH5 -. 1 6 3731ITGAGlt 7TT -LLC L7 TCA ATl AGG AGI AAT TTT

376CTir ATC GAG CTG A rLeu lIe GlU Lei. Met

436GtL TAG CAA GAC TACAla Tyr GIn Asp Tyr

496:AA tAAA TCA GCC GTCGin Lys Ser Ala Val

291ATT G1G ATC GCT ATC GTCile Val Ile Ala Ile Val

451AGG OCC CGC GCG CAA GTT TGCThr Ala Arg Ala GIn Val Ser

51 1ACC GAG TAT TAC CTG AAT CAC GGCThr GItU Tyr Tyr Leu Asn His Gly

346 36 1ATG AAT ACC LTT CAA AAA GGC T7r ACCIMet Asn Thr Leu GIn Lys G1y Phe Thr

406 *BH6 421GGC ATT TTG GCG GCA GTC GCC CTT CCCGly lie Leu Ala Ala Val Ala Leu Pro

466GAA 6CC ATCGBlu Ala Ile

526AAA TGGLys Trp

481CTT TTG GCC GAA GGTLeu, Leou Ala Glu Gly

541CCG GAA AAC AAC ACTPro Glu Asn Asn Thr

BgfI 556 5 -7 1TCT MCC GGC GTG GtC TCC CCC' CCC TCC GACSer Ala Gly Val Ala 5cr Pro Fro Ser (:Asp

GTT AAA AAC GGCVa1 Lys Asn Gly

616GTCVa1

ATC AAA GGC AAAIle Lys Gly Lys

TATTyr

6 1 646GTT AC'(' CC AlA ATB CTT TCA AGC GGC GTAVal lhr Ala Thr Met Leu Ser S6cr- Gly Val

676 69I1GG3C AAA AAA CTC TCC CTG TGG GCC AGG CGTI1 y L ys Lyy LeLu 5er LeU Trp Ala Arr Arg

736 751

CAG CCG GTT ACG CGC AGC GAC GAC GAC ACC

Gin Pro Val Thr Arg Thr Asp Asp Asp Thr

GAC ACC AAG CACAsp Thr L ye His

TTA GGC CTT AAA

796 811

CTG CCG TCA ACC TGC CGCLeu Pro Ser 7hr Cys Argr

856 871

TTT TAA ATA AAT C;AA GCG

706

GAA AAC GGT TCG GTAGLu Asn Gly Ser Val I

766GTT GCC GAC GCC AAA IVal Ala Asp Ala Lys

826GAT AAG GCA TCT BAT (Asp Lys A.la Ser Asp

GTA

Sma I 916 931CCG GGC TTG TCT TTT AAG GGT TTG CAA GGC

886AGT GAT TTT CCA

946GG6 CGG GGT CGTI

Cla ITAA TAT ATC GAT]

G6T AAA GAG GTT GAA FIG. 4. DNA sequence of theVal Lys Gl L Val Gu1 Hpa I/Cia I fragment of

661 pNG1100. The deduced aminoAAC AAT GAA ATC AAA acid sequence of MS11 pilin ap-Asn Asn G5u Ile Lys pears below the coding sequence

starting at position 335. The -10

AAA TGG TTC TGC GG7 sequence and the ribosomal bind-Lys Trp Phe Cys 61 y ing site are underlined. The arrow

at position 244 indicates the start781 of pilus mRNA synthesis. Key re-

GAC GC AAA GAA ATC striction sites used for obtainingAsp 61 y Lys G6 lU I 1 e fragments for Southern hybridiza-

841 tion probes are boxed. DotsBCC AAA T13G GGC AAA above the various bases indicateAl a Lys the endpoint of BAL-31 deletions,

901 where HindIII linkers were insert-CCC: GCC CGG ATC A[ ed. The designation for each of

these deletion-generated sub-clones appears next to each end-

CCG TTC CGG TGG AA9 point. The boxed amino acids in-dicate residues predicted by theDNA sequence not present in thereported NH2-terminal end ofMS11 pilin.

586 C0 1

Proc. NatL. Acad. Sci. USA 81 (1984)

The amino acid sequence of MS11 pilin, predicted fromthe DNA sequence, is shown in Fig. 4. Comparison of thissequence with that of purified pilin from the same strain (22)reveals that the primary translational product in MS11 con-tains an additional seven amino acid residues at the amino-terminal end (Fig. 4). These extra amino acid residues arelikely to be the leader peptide needed for transport of thesubunit across the bacterial membrane. All bacterial leaderpeptide sequences studied so far are at least 20 amino acidslong and are distinctly hydrophobic (23). In contrast, theseven amino acids of the gonococcal "prepilin" contain amixture of uncharged and nonpolar side chains. Since this isthe first report of a gonococcal prepilin sequence, it is notclear whether this sequence is characteristic of all gonococ-cal leader sequences or whether it has a function specific forpilin export and assembly. Aside from these seven aminoacids, the two sequences are identical.

DISCUSSIONWe mapped two chromosomal loci in N. gonorrheae strainMS11, pilE) and pilE2, that are involved in pilin expression.Within these loci is the sequence information needed for pi-lin production. In MS11, at least the pilin gene is the same inboth loci, and the direction of transcription is also the same.Both pilE) and pilE2 undergo rearrangement during pilusphase variation (13). This rearrangement in many cases in-volves deletion of pilus sequences (unpublished data). Athird region, a silent locus (pi1S1), contains pilin structuralgene sequences but not promoter sequences. piS) maps to a5-kb chromosomal Cla I fragment that does not undergo re-arrangement during the phase switch. pilE), pilE2, and piS)are located within a 40-kb region of the gonococcal chromo-some. In this context, note that we have recently mapped aregion (opal) close to pilE) that is important in the expres-sion of the gonococcal opacity (Op or PII) protein (Fig. 3,lane b; ref. 21). Op, an outer-membrane protein with viru-lence properties, also undergoes phase and antigenic varia-tion (24-27). The proximity of opal to pilE) may not be en-tirely fortuitous, although there is as yet no indication thatthe Op and pilus variation systems are related.

pilEl and pilE2 also share some homology both upstreamand downstream of the pilin gene sequence. The down-stream homology is limited to a 65-bp sequence located be-tween Cla I and Sma I sites. Southern-blot analysis and re-striction-site distribution within pilS) give no indication ofan intact structural pilin gene in this locus. However a 65-bpCla I/Sma I fragment does occur in the extreme right-handportion of this locus, in the same orientation as the ClaI/Sma I fragments in the pilEl and pilE2 loci (Fig. 1 c and d).The role that this 65-bp region plays in pilin gene expressionis not known at present. However, examination of the pilE)and pilE2 loci in P- cells shows that, in many cases, a dele-tion of pilin gene sequences occurs close to this 65-bp se-quence (unpublished data). Nor do we know at presentwhether the majority of variant pilin gene sequences are de-rived from the pilSi locus.

This arrangement of expression and silent loci for pilusexpression is reminiscent of the yeast mating-type intercon-version system, where the MAT locus functions in theexpression of the a or a gene, while silent copies of a and aare located elsewhere (28). However, unlike the silent a anda genes, the pilSi locus does not express pilin because itlacks pilin promoter sequences and not because of the pres-ence of a repressor. Moreover, there appear to be morecopies of the variable region pilus sequences than constantregion sequences in pilS). In this, piS) is more analogous tothe arrangement of immunoglobulin structural gene se-quences, where tandem copies of variable regions of the Iggene are separated from copies of the constant region se-quences. To generate an intact, Ig-expressing gene with a

particular specificity, intervening sequences in many casesare irreversibly deleted during B-cell maturation (29). We be-lieve that no such irreversible deletion occurs during pilusphase and antigenic variation, since a single gonococcal cellexpressing one pilus idiotype can express a different idio-type and subsequently switch back to express the originalpilus idiptype (unpublished data).The exact nature of the gonococcal pilus phase switch and

antigenic variation remains to be determined. Both phenom-ena occur in a variety of prokaryotic and eukaryotic systems(30, 31). Antigenic variation is thought to comprise a seriouschallenge to the immune surveillance of the host. Since N.gonorrheae is amenable to genetic studies, it offers an excit-ing model system for studying the molecular basis of thesetwo phenomena.We thank Elizabeth Simpson for her help in preparing the manu-

script. This work was supported by National Institutes of HealthGrant Al 20845 and National Science Foundation Grant PCM823174 and by Deutsche Forschungsineinschaft Grant Mc 705/2-1.

1. Swanson, J. (1973) J. Exp. Med. 127, 571-589.2. Ward, M. E., Watt, P. J. & Robertson, J. N. (1974) J. Infect.

Dis. 129, 650-659.3. lames-Holrnquest, A. N., Swanson, J., Buchanan, T. M.,

Wende, R. D. & Williams, R. P. (1975) Infect. Immun. 9, 897-902.

4. Buchanan, T. M. & Pearce, W. A. (1976) Infect. Immun. 13,1483-1489.

5. Kellogg, D. S., Jr., Peacock, W. L., Jr., Deacon, W. E.,Brown, L. & Pirkle, C. L. (1963) J. Bacteriol. 85, 1274-1279.

6. Kellogg, D. S., Jr., Cohen, I. R., Norins, L. C., Schroeter,A. L. & Reising, G. (1968) J. Bacteriol. 96, 596-605.

7. Jephcott, A. E., Reyn, A. & Birch-Andersen, A. (1971) ActaPathol. Microbiol. Scand. Sect. B 79, 437-439.

8. Robertson, J. N., Vincent, P. & Ward, M. E. (1977) J. Gen.Microbiol. 102, 169-177.

9. Lambden, P. R., Robertson, J. N. & Watt, P. J. (1980) J. Bac-teriol. 141, 393-395.

10. Lambden, P. R. (1982) J. Gen. Microbiol. 128, 2105-2111.11. Hermodson, M. A., Chen, K. C. S. & Buchanan, T. M. (1978)

Biochemistry 17, 442-445.12. Schoolnik, G. K., Tai, J. Y. & Gotschlich, E. C. (1983) Prog.

Allergy 33, 314-331.13. Meyer, T. F., Mlawer, N. & So, M. (1982) Cell 30, 45-52.14. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580.15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular

Cloning: A Laboratory Manual (Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY).

16. Maxam, A. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560.

17. Kupersztock, Y. M. & Helinski, D. (1973) Biochem. Biophys.Res. Commun. 54, 1451-1459.

18. Kingsbury, D. T. (1969) J. Bacteriol. 98, 870-874.19. Koomey, J. M., Gill, E. & Falkow, S. (1982) Proc. Natl. Acad.

Sci. USA 79, 1881-1885.20. Stein, D. C., Silver, L. E., Clark, V. L. & Young, F. E. (1984)

J. Bacteriol., in press.21. Stern, A., Nickel, P., Meyer, T. F. & So, M. (1984) Cell 37,

447-456.22. Schoolnik, G. K., Fernandez, R., Tai, J. Y., Rothbard, J. &

Gotschlich, E. C. (1984) J. Exp. Med. 159, 1351-1370.23. Silhavy, T. J., Benson, S. A. & Emr, S. D. (1983) Microbiol.

Rev. 47, 313-344.24. Swanson, J. (1978) Infect. Immun. 19, 320-331.25. Lambden, P. R. & Heckels, J. E. (1978) Fed. Eur. Microbiol.

Soc. Lett. 5, 263-265.26. Swanson, J. (1982) Infect. Immun. 37, 359-368.27. Virgis, M. & Everson, J. S. (1981) Infect. Immun. 31, 965-970.28. Herskowitz, I. (1983) Curr. Top. Dev. Biol. 18, 1-14.29. Joho, R., Nottenburg, C., Coffman, R. L. & Weissman, I. L.

(1983) Curr. Top. Dev. Biol. 18, 15-31.30. Szekeley, E., Silverman, M. & Simon, M. (1983) in Biological

Consequences ofDNA Structure and Genome Arrangement,Symposium of the Fifth John Innis Society, eds. Hopwood,D. A. & Chater, K. (Croom Helm, London), pp. 117-129.

31. Bloom, B. R. (1979) Nature (London) 279, 21-26.

6114 Genetics: Meyer et aL