Proc. Nad. Acad. Sci. USA Vol. 89, pp. 3204-3208, April 1992 Microbiology Neisseria gonorrhoeae PilC expression provides a selective mechanism for structural diversity of pili (phase variation/virulence/biogenesis) ANN-BETH JONSSON*t, JOHN PFEIFERt, AND STAFFAN NORMARKt *Department of Microbiology, University of Ume&, S-90187 Umei, Sweden; and *Department of Molecular Microbiology, Washington University, 660 South Euclid Avenue, St. Louis, MO 63110 Communicated by Emil C. Gotschlich, December 24, 1991

ABSTRACT Pili of Neisseria gonorrhoeae undergo both tical in size to the S-pilin previously described (9), but do not phase and structural variation. Phase variation of gonococcal assemble the pilin subunits into pili. pili can be caused by a RecA-independent on/off switch in Here we provide evidence for a selective mechanism of PiIC, a protein involved in pilus biogenesis. We show here that structural variation of the gonococcal pilus in which the spontaneous nonpiliated PilC- derivatives as well as PilC- selective pressure is exerted by an on/off switch in PilC insertional mutants have also acquired sequence alterations in expression. It is hypothesized that an impaired pilus assem- pilE relative to the piE gene of the piliated MS11,k(P+)-u bly results in accumulation of toxic, unassembled pilin. parent, so that the pilin produced is processed to soluble S-pin Unless the pilin structure is modified so that it can be and can be released into the medium. It is proposed that pilin proteolytically degraded to S-pilin and released into the alterations are selected for in PilC- bacteria if the parental medium, this accumulation is detrimental to the gonococci. nonassembled pilin is toxic to the cells-i.e., is not degradable to S-pilin at rates sufficient to allow viability of the cells. Toxicity is indicated by the extreme instability of certain MATERIALS AND METHODS unassembled pilin sequences and by the low frequency of Bacterial Strains, Mutants, and Growth Conditions. N. nonpillated, pilin+, PilC- variants that emerge from pillated gonorrhoeae MSllmk and VD302, a recA mutant of recA- cells. The presence of a point mutation changing leu- MS11mk, were kindly provided by M. Koomey and were cine-39 to phenylalanine at the cleavage site for S-pilin in one maintained as described (10-12). The particular MS11mk(P+) nonpiliated, PiIC-, recA variant relative to its pillated parent strain used in our studies is designated MSllmk(P+)-u to is a further argument for a selective mechanism of structural distinguish it from other MS11mk(P+) strain samples. Con- diversity of the gonococcal pilin. struction and characterization ofthepilC mutants (8) used the shuttle mutagenesis system of Seifert et al. (13) with a Pili ofNeisseria gonorrhoeae are a major virulence factor and miniTnCm (mTnCm) transposon carrying the chloramphen- promote attachment ofgonococci to eukaryotic cells (1), and icol acetyltransferase (CAT) gene. Transmission electron the pilus subunit protein (pilin) undergoes both phase and microscopy of gonococci used the method of negative stain- antigenic variation. Most strains of N. gonorrhoeae contain ing (8). one copy ofthe expressed major pilus subunit gene, pilE, and DNA Sequencing. The pilE gene from gonococcal variants multiple copies of piS, which are transcriptionally silent, and mutants was PCR-amplified (8) by using the primers incomplete pilin loci carrying variant sequences (2, 3). Re- 5 '-AAATTTAAGGCCTAATTTGCC-3' and 5 '-TTTC- combination events between silent and expressed loci lead to CCCTTTCAATTAGGAGT-3'. Amplified fragments were the formation of diverse pilins either by a transformation- purified from agarose gels and used as templates in asym- mediated recombination of DNA released from lysing gono- metric PCRs. Both strands were amplified in separate reac- cocci or by recombination between a silent locus and an tions using 0.5 pmol of the purified double-stranded PCR expressed locus on the same chromosome (4, 5). The control product. The final concentration of the limiting primer was of phase variation is'complex and appears to involve several 0.005 ,AM and that of the excess primer was 0.5 1uM. DNA mechanisms. Deletion of the 5' end of pilE generates a from the asymmetric PCRs was precipitated and sequenced nonreverting nonpiliated (P-) phenotype, and frameshift directly by dideoxy chain termination (14) with the T7 mutations in pilE can result in production of a truncated pilin sequencing kit (Pharmacia) and internal pilE primers. protein. Another mechanism involves recombination events Immunoblotting and N-Terminal Sequencing of S-pilin. that place a missense gene at the pilE locus. These missense Bacterial cells (10 pug) or precipitated culture supernatants pilins are thought to be assembly-deficient (6, 7). were electrophoresed in SDS/polyacrylamide gels and tested Recently, we demonstrated that reversible phase variation in immunoblots (15). The anti-pili serum was raised in rabbits in pilus expression also results from frequent changes in the against highly purified pili preparations of N. gonorrhoeae number of guanine residues in a tract of guanines located MS11(P+) as described (8). The PilC antiserum was generated within the signal peptide-coding region of pilC, a gene en- in a rabbit against gel-purified PilC from N. gonorrhoeae coding a 110-kDa protein involved in pilus assembly. Strain MS11 (8). Gonococci were grown in Catlin's defined medium MS11,,,k(P+)-u contains two pilC loci, pilCi and pilC2, of (16) for isolation of proteins in the supernatant. S-pilin was which only pilC2 is translationally in frame. Insertional precipitated with 10% trichloroacetic acid and resuspended in inactivation of pilCi did not affect piliation, whereas inacti- sample buffer for SDS/PAGE. The pilin band was transferred vation ofpilC2 abolished expression of pili but not pilin (8). to a poly(vinylidene difluoride) (Immobilon, Millipore) filter P-, PilC- gonococci produce, in addition to the 20-kDa by electroblotting, stained, and used for N-terminal sequenc- mature pilin, a 16-kDa proteolytic degradation product iden- ing (17).

The publication costs of this article were defrayed in part by page charge Abbreviations: P', piliated; P-, nonpiliated; mTnCm, miniTnCm. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department of Molecular Microbiology, Washing- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ton University, 660 South Euclid Avenue, St. Louis, MO 63110. 3204 Downloaded by guest on September 29, 2021 Microbiology: Jonsson et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3205

Southern Blotting and Hybridization. Gonococcal DNA 1 1 I'l 4 5 6 7 8 was prepared and Southern blot hybridizations were per- kDa formed as described (8). To identify P-n gonococcal variants (10), a 21-base oligonucleotide complementary to the signal 2- peptide-coding region of pilE (5'-GCCTTTl-lTGAAGGG- 16--- TATTCAT-3') was used as probe. Construction of TnphoA Fusions in pilCI. PilCl-alkaline phosphatase fusions were constructed by using the transpo- FIG. 1. Proteins in culture supernatants of gonococci grown in son TnS derivative TnphoA (18). To allow measurement of liquid GCB medium were precipitated with trichloroacetic acid and switch frequency (identified by blue or white colony pheno- subjected to immunoblot analysis using a polyclonal anti-pili serum. type), the pilC-phoA fusion genes were subcloned into the Variants 5, 8, and M3 are P- derivatives of MS11mk(P+)-u. Lanes: single-copy plasmid pMF3 (19). Escherichia coli CC118 is a 1-4, whole cell lysates; 5-8, precipitated supernatant proteins; 1 and recA- derivative ofstrain CC117 (19). Each switch frequency 5, variant 8 (P-, PilC-, pilin'); 2 and 6, variant 5 (P-, PilC-, pilin'); was determined from at least three separate experiments. 3 and 7, variant M3 (P-, PilC+, pilin'); 4 and 8, MSllmk(P+)-u. pilin'), showed that both contained complete pilE genes RESULTS identical to that ofthe MSllmk(P+)-u parent (data not shown). Consequently, the inability of variant D1 to produce pilin and Characterization of Isogenic Pilus Variants. Forty P- deriv- pili (as revealed by transmission electron atives of piliated MSllmk(P+)-u were isolated on the basis of microscopy) cannot their colony morphology and examined in immunoblots for be explained by alterations in the pilin structural gene. expression of PilC and pilin. This set of P- variants could be Many P- Revertible Gonococcal Derivatives Are the Result of divided in three categories; PilC-, pilin' (15 clones); Pi1C+, Two Genetic Events. The pilE gene sequence of4 ofthe 15 P-, pilin' (4 clones); and PilC+, pilin- (21 clones) (Table 1). The PilC-, pilin+ variants (nos. 3, 5, 6, and 8) was determined. All P- phenotype was confirmed by transmission electron micros- 4 variants contained alterations in thepilE coding frame, which copy. in the deduced amino acid sequence resulted in eight or more In all P-, pilin' derivatives (both PilC+ and PilC-), two amino acid changes compared with pilE of the MSllmk(P+)-u proteins, 20 and 16 kDa, were detected that reacted with a parent (Fig. 2). Each of the 4 PilC- clones was passaged daily polyclonal anti-pili serum in immunoblots. Immunoblot anal- as single colonies for 2 weeks and then resequenced. No ysis of the culture supernatant from two PNC- clones (nos. 5 sequence changes were found, indicating that a stable pilE and 8) and one PilC+ clone (variant M3) confirmed the results sequence had been obtained. The same procedure was per- of Haas et al. (9), that the 16-kDa protein is released into the formed with the MSllmk(P+)-u parent, giving no changes in medium (Fig. 1). N-terminal sequencing ofthe 16-kDa protein the pilE sequence. Further, the obtained pilE sequence of from variant 8 (PiNC-) and of variant M3 (PilC+) showed that MSllmk(P+)-u is identical to the sequence published by Meyer the 16-kDa product is the result of a cleavage between Leu39 et al. (2). These data argue that in PilC- derivatives of and Alae of the mature pilin in both PilC- and PilC+ MSllmk(P+)-u, sequence changes in pilE are generated to derivatives (data not shown). These results suggest that the obtain a pilin that is degradable to S-pilin. proteolytic cleavage of pilin is not due to changes in PilC Spontaneous P+ revertants ofthe PilC- variants 3, 5, 6, and expression but may be caused by specific alterations in the 8 were isolated at a frequency of 10-4-i0-5 per cell per pilin sequence itself, especially since the S-pilins produced generation and were all PilC+. The pilE sequence in two of from variants 5, 8, and M3 have somewhat different sizes in five revertants contained no further alterations, arguing that immunoblots (Fig. 1). the P- phenotype of the parents (variants 3 and 8) was due to Some P- Revertible Derivatives Result from One Genetic an off switch in PilC expression, since the pilE sequence in Event. The pilE gene was PCR-amplified and sequenced these P- and P+ pairs was identical. Three revertants con- directly from one of the four P-, pilin+, PilC+ variants (M3) tained pilE sequence alterations relative to their P-, PilC- together with its P+ revertant (M3:1). The deduced amino parental clones (Fig. 2). The frequency by which P-, PilC-, acid sequence ofpilE in variant M3 contained 19 amino acid pilin+ variants reverted to P+ was about 100 times lower than replacements relative to that of MSllm(P+)-u, including the rate of revertants from the P-, PilC+, pilin+ variants alterations in MC2 (Fig. 2). The P+ revertant of M3 contained (Table 1). This is as expected, since the latter transition apilE sequence identical to that ofMSll1k(P+)-u. Hence, the requires only rearrangements in pilE, whereas the former P- phenotype of variant M3 may be due to the expression of group of revertants involves translational frameshift muta- a pilin that cannot be assembled into pili. Assembly-defective tions in either pilC locus and, often, rearrangements in pilE. pilins have been suggested as a possible explanation for a Insertional Inactivation of the Expressed pilC2 Gene, But pilus- phenotype (7). Not the SilentpilCI Gene, Results inpiE Sequence Alterations. The P-, PilC+, pilin- group was heterogeneous. Nineteen of The concomitant loss of PilC expression and appearance of 21 were nonreverting (presumably Pn-i.e., lacking the 5' pilE sequence variation in P- variants of MSllmk(P+)-u could end ofpilE), while 2 of 21 reverted at a high frequency (10-3 be due to a coupled switch or due to selection for sequence per cell per generation) to a P+ phenotype. Sequencing ofpilE alterations in pilE as a result of turn-off of PilC production. from one such pair, D1 (P-, PilC+, pilin-) and D1:1 (P+, PilC+, Insertional inactivation ofpilCl by mTnCm (13) did not affect the piliation phenotype, whereas inactivation ofpilC2 gave a Table 1. PilC and pilin transitions in recA+ MS11 P- revertible phenotype that was PilC-, pilin+ (8). A double PilC and pilin in PilC and pilin Rate of P- P+ mutant in pilCI and pilC2 was generated by transforming a 40 P- gonococci variants, % (no.) transition* P+, pilCl::mTnCm mutant with DNA isolated from a P-, pilC2::mTnCm mutant (8). The double mutant was P-, PilC-, PilC-, pilin+ 38 (15) 10-4-_1-5 pilin+ and did not revert to piliation. As with the spontaneous PilC-, pilin- 0 PiIC- derivatives of MSllmk-u, both the pilC2::mTnCm PilC+, pilin+ 10 (4) 10-3 single mutant and the pilCI::mTnCm, pilC2::mTnCm double PilC+, pilin- 47 (19) <10-8 mutant expressed two proteins (20 and 16 kDa) that reacted 5 (2) lo-, with a polyclonal anti-pili serum (data not shown). The pilE Rate of P+ - P- transition: 10-4 per cell per generation. gene in one pilCI:::mTnCm mutant was identical to that ofthe *No. of variants per cell per generation. MS11mk(P+)-u parent, while the pilE sequence of a Downloaded by guest on September 29, 2021 3206 Microbiology: Jonsson et al. Proc. Natl. Acad. Sci. USA 89 (1992)

___ ) + 10 20 30 40 50 60 70 MS11 (p+PilC MNTLQKGWTLIEMIVIAIVILAAVALPAYQDYTARAQVSEAIAYQDQTI TYL T*&VAS 3 tPPilC) ......

3:1 (P+PiiC+) ......

...... 5 (P PIIC ) . . M ......

5:1 ...... (P+PiIc+) ......

6 (p PiiC ) ......

LE6:1 (P+Pilc+) ...... N.N...I A 8 (PPiIC ) ......

8:1 (P+PiIC+) ...... K....c^... 8:6 (P+Pilc+) ...... -1 ......

M3 (P Pi1C) ......

...... C;7WM:l (P+PiIC ) ......

+ pllCl::m=Ca (P+PiIC ...... -plC2: :=Tnc (P PiIC.) ...... KE double mutant (P PiIC ......

2A (PPRIC+) ...... 2B (P+RIC+ ...... I ...... D

FMSIIrecA (P PilC ) ......

R12 ...... (P PiIC ) . F ...... M6

80 90 100 110 120 130 140 150 MS1I IPPSDT8D3pQJITvYTv ISGVZI KLS S SV RDDeGE sTCRDCA* 3 S.T......

3:1 8S.T ...... 5 .ADK...... AK E .... K .1 K.QD ......

:5:1 S .T ......

6 S.T .Q. K......

L:6:1 SA .S.T.A.K...... K. QD ...... _ 8 S.T...... A.T......

8:1 8S.T...... I .A.T......

8:6 ......

M3 . ADX ...... E. A.T ...X......

M3:1 ......

pllCl: :TnCm ...... C2: :UTnc ...... Kp I...... I...... T...X ...... double mutant .ADK ...... EA . A.T ...X...... K.QID ..A. ... DTFrAG . .CN.IN > 2A ...... > 2B S...... R.R.A . .... E...T .. K......

...... i M I ...... s1recA .I..I. .I. .C R12 ...... MC5 MA4 MC3 MC2 FIG. 2. Deduced amino acid sequence of the pilin structural gene, pilE, of N. gonorrhoeae MS11mk(P+)-u and its derivatives and mutants. pilE was PCR-amplified and sequenced directly. Amino acids of the propilin are given in one-letter code. Arrow after amino acid 7 marks the cleavage site for the signal peptidase; arrow after amino acid 46 marks the cleavage site for generation of S-pilin. Dots indicate amino acids identical with those ofthe MS11,k(P+)-u parental pilin. The six variable mini-cassettes ofpilE are boxed and marked MC1-MC6. P- spontaneous PilC- variant R12 was derived from a recA- mutant, VD302, of MS11mk(P+) (11, 12).

pilC2::mTnCm mutant was altered and contained in the with loss ofpilin expression. It may therefore be that this pilin deduced amino acid sequence 3 amino acid replacements variant is toxic but not completely lethal to the bacterial cells. relative to the MS11mk(P+)-u parental pilin (Fig. 2). The P-, The pilE sequences expressed by the P-, PilC- variant 8 pilCl::mTnCm, pilC2::mTnCm double mutant carried an and its P+, PilC+ revertant 8:1 (Fig. 2) are identical and must extensively changed pilE sequence, with a deduced amino encode a pilin that is nontoxic in its unassembled state and acid sequence containing 29 amino acid replacements and a assembly-proficient in PilC+ cells. We therefore predicted 5-amino acid insertion relative to the pilin of strain that PilC turn-off in variant 8:1 might not select for pilin MS11k(P+)-u. These extensive pilE sequence changes may variation. Consequently, we expected a number of P-, PilC- be the result of the extra handling required to inactivate both derivatives from 8:1 to have an unaltered pilE sequence. pilC copies. Thus, both spontaneous P- PilC- derivatives Three independent P-, PilC-, pilin+ variants were isolated and PilC- insertion mutants contain alterations in the pilE from variant 8:1. The pilE sequence of two of the three was sequence. identical to that of the parental clone, whereas the third Evidence for Toxic and Nontoxic Pilins. The pilCI, pilC2 contained sequence alterations (Fig. 3). Thus, certain pilE double mutant clone described above formed small sequences seem to code for nontoxic pilins and can be unusually conserved in pilus' to pilus- switches, while other pilins colonies, and a large fraction of cells to have appeared lysed seem to be very unstable. when these small colonies were examined electron mi- - by Characterization ofP1C1- Derivatives from a rcA Mutant. croscopy (data not shown). Fast-growing clones appeared at To determine the frequency of frameshift mutations in pilC, high frequency (10-2 per cell per generation); they remained plasmid pABJ04 (which contains a full-length copy ofpilCI) PilC- but had ceased to express pilin as determined by was mutagenized with TnphoA. No difference in switch immunoblot analysis (data not shown). DNA hybridization frequency was seen between recA+ and recA- E. coli (Table using a probe specific for the signal peptide-coding region of 2). Thus, frameshift mutations in pilC occur by a RecA- pilE was negative, suggesting that the fast-growing pilin- independent mechanism in E. coli. variants carried deletions in the 5' end of pilE (data not We observed, as previously reported by Koomey et al. (11, shown). Thus, resumption of normal growth rate correlated 20), a 100-fold reduction in the frequency ofP- offspring from Downloaded by guest on September 29, 2021 Microbiology: Jonsson et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3207

+ 10 20 30 40 + 50 60 70 8:1 (P PilC ) INTLQKGFTLIELMIVIAIVGILAAVALPAYQDYTARAQVSEAILEGQ VTEYYLNGEWPENNTh l*8:1/1 (P PilC ) ...... -.l...... l -*8:1/4 (P-PilC-) ...... 8:1/10 (P PilC+) ...... -- 8:1/2 (P PilC) ...... MC6

80 90 100 110 120 130 140 150 160 811 SPTDIi . ATGV S R S FCGQPaRTDDDTVADA KD- sTcRrKSA

...... 8 :1/1 0 .AR...... K...... Q ...... ||......

L> 8:1/2 ...... QD...... M:5 MC4 MC3 MC2 MM

FIG. 3. Deduced amino acid sequence ofpilE of the P+ variant 8:1 and four of its P- derivatives. Amino acids identical to the 8:1 parental pilin are marked with dots. The six variable mini-cassettes (MC1-MC6) are boxed.

the recA- mutant VD302 compared with its recA' parent DISCUSSION MSllmk(P+). Seventeen P- derivatives of VD302 were tested This study demonstrates that spontaneous P- PiIC- deriva- in immunoblots for the production of PilC and pilin (Table 3). tives, as well as PilC- insertional mutants, carry sequence Four of 7 PilC- clones were pilin- and nonreverting, while alterations in the pilin structural gene, so that the pilin the remaining three PilC- variants expressed the pilin. The expressed can be processed to a shorter, soluble form (S- three PilC-, pilin' variants reverted to a P+ phenotype at a pilin) that is released into the medium. Structural diversity in high frequency (10-4-10-` per cell per generation), and the the pilin is mediated by RecA-dependent recombinations revertants were all PilC+ when examined in immunoblots. Of between the pilin structural gene (pilE) and silent partial pil the remaining P- variants, all 10 were PilC+, pilin- (Table 3). gene sequences (3, 11), while translational frameshifting in One additional variant had a colony morphology typical ofP- pilC is independent of recA, when pilC-phoA fusions are gonococci, but still expressed pili. This clone contained no examined in E. coli. Nevertheless, P-, PilC- clones arise sequence changes in pilE relative to the parent (data not much less frequently in a recA- mutant. The pilE sequence shown). The pilE gene from one PilC-, pilin' variant was of one such P-, PilC-, recA- clone carried a point mutation sequenced and found to contain a base-pair substitution in the region encoding the cleavage site for S-pilin. Thus, lack changing Leu39 in the deduced amino acid sequence to Phe; ofPilC expression is suggested to select for a pilin variant that i.e., the mutation had occurred at the cleavage site for S-pilin can be cleaved into the soluble S-pilin and be released from (Fig. 2). Production of S-pilin in the PilC- derivative was the cells. Sequence alterations of pilE relative to the parental confirmed by immunoblotting with polyclonal anti-pili serum pilE sequence are proposed to occur in PilC- derivatives, if (Fig. 4). These results support a selective model for structural the parental pilin is toxic-i.e., not degradable to S-pilin at variation and provide further evidence that the P- phenotype rates sufficient to allow viability of the cells. of PilC- cells is primarily due to the absence of PilC and that All spontaneous P-, pilin' derivatives (both PilC- and pilin alteration is a secondary phenomenon. PiKC+) and the pilC2: :mTnCm mutant contain either a Lys -* PilC1 and PilC2 Can Separately Promote Assembly of an Glu replacement at position 63 or an Ile -- Met change at Identical Pilin Sequence. To examine whether pilus assembly position 17 in the propilin. The only exception to this rule is dependent on PilC1 could affect the pilE sequence, two P+ the pilin' but unstable pilCl, pilC2 double mutant; its pilE is PilC+ revertants (2A and 2B) from a P-, pilC2::mTnCm identical to that of the parent MS11mk(P+)-u up to position 83 mutant were analyzed. Southern blot hybridizations using a of the propilin. It may be that certain amino acid changes pilC-specific probe and a probe specific for mTnCm revealed close to the proteolytic cleavage site after amino acid 46 of no alterations, implying that variants 2A and 2B express propilin (no. 39 of mature pilin) might influence the folding of PilC1. The pilE sequence of variant 2A (P', PilC+) was the pilin molecule so that it is more easily cleaved. All identical to that of the MS11mk(P+)-u parent and the reported pilS sequences lack the nucleotides coding for at pilCi::mTnCm mutant (P', PilC2+). Variant 2B contained least the first 37 amino acids of propilin (3). Substitution of pilE sequence alterations relative to the P- parent (Fig. 2). Met for Ile at position 17 of propilin in variant 5 must then be Variant 2A (P', PilC1N) contained -50% fewer pili per cell caused by an independent point mutation (C -- G) and not than the pilCi::mTnCm mutant (P', PilC2+), although the result from transfer of a pilS sequence to the pilE gene. The two pilins were identical. However, variant 2B, expressing an finding of such a low-frequency genetic event in a PilC- altered pilin, had the same degree of piliation as the derivative provides further evidence for a selective mecha- pilCi: :mTnCm mutant. This argues that the same pilin can be nism for pilin alterations in these P- variants. The to was more assembled into pili whether either PilC1 or PilC2 is ex- transition from P+ P-, PilC-, pilin' commonly found (38%) than that of P+ to P-, PilC+, pilin' pressed. However, we cannot exclude the possibility that (10%), even though the former switch must involve at least additional changes have occurred in one or both of the pilC two genetic events: frameshifting in pilC and recombinations mutants, since the respective mutation was introduced in independent reactions. Table 3. PilC and pilin transitions in recA- MS11 PilC and pilin in PilC and pilin Rate of P- -> P Table 2. Switch frequency of PilC expression in E. coli 17 P- gonococci variants, % (no.) transition* Strain Transition Rate* x 105 PilC-, pilin+ 18 (3) 10-4_10-5 CC117 (recA+) PilC+- PilC- 0.23 ± 0.09 PilC-, pilin- 23 (4) <10-8 PilC- PilC+ 0.71 ± 0.24 PilC+, pilin+ 0 CC118 (recA-) PilC+- PilC- 0.38 ± 0.09 PilC+, pilin- 59 (10) <10-8 PilC-*PilC+ 0.58 ± 0.38 Rate of P+ -> P- transition: 10-6 per cell per generation. *Rate of phase transition per cell per generation (mean ± SD). *No. of variants per cell per generation. Downloaded by guest on September 29, 2021 3208 Microbiology: Jonsson et al. Proc. Natl. Acad Sci. USA 89 (1992) degraded, which we propose causes toxic effects on the kDa bacterial cells if the pilin is produced in sufficient quantities. 110- Am ... anti-PilC- Surviving cells are those in which no or less pilin is produced serum or those that contain alterations in the pilin such that it can be proteolytically cleaved to yield S-pilin that can be released from the membrane. Since the latter variants can be gener- ated by homologous recombination between pitS and pilE sequences, it dominates in wild-type recA' gonococci. The 20- _ anti-pili- pilE sequences selected in P-, PilC- derivatives may or may serum not be altered again in the pilus' revertants; it is possible that 16- specific pilE sequence alterations are required to regenerate P+ revertants depending on which of the two pilC loci are turned on. It is not known which specific role PilC plays in FIG. 4. Immunoblot of gonococcal recA variants with polyclo- the biogenesis ofgonococcal pili and whether PilC affects the nal anti-pili and anti-PilC sera. Lane 1: P-, Pi1C- derivative R12 (see biological properties of pili. The finding that PilC is enriched Fig. 2) of the piliated recA- parent. Lane 2: P', recA mutant of in pili preparations, however, suggests that it is associated MS11ll,(P+)- with the polymerized pilus fiber. in pilE. Transition from P+ to P-, PilC+ occurs at a 10-fold We thank Lenore Johansson for skillful assistance with electron lower frequency (10-4-10-5 per cell per generation) than the microscopy and Robert Tolan, Jr., for manuscript reading. This work reversion from P-, PilC+ to P+ (10-3 per cell per generation). was supported by the Swedish Medical Research Council (Dnr04769) If the P- phenotype of PilC+, pilin+ cells is explained by the and by grants from Washington University Medical School, St. expression of an assembly-deficient pilin variant, the differ- Louis. ence in transition rate implies that pilE sequence combina- 1. Swanson, J. (1973) J. Exp. Med. 137, 571-589. tions encoding assembly-deficient pilin variants are much 2. Meyer, T. F., Billyard, E., Haas, R., Storzbach, S. & So, M. less common than are pilE sequences encoding assembly- (1984) Proc. Natl. Acad. Sci. USA 81, 6110-6114. proficient pilins. P+ revertants of P-, PilC-, pilin+ variants 3. Haas, R. & Meyer, T. F. (1986) Cell 44, 107-115. at a of per cell per generation. 4. Gibbs, C. P., Reimann, B. Y., Schultz, E., Kaufman, A., occur frequency 10-4_10-5 Haas, R. & Meyer, T. F. (1989) Nature (London) 44, 682-685. This must reflect the rate of frameshift mutations in the 5. Seifert, H. S., Aijoka, R. D., Marchal, C., Sparling, P. F. & stretch of guanine residues in pilC and of a selection for an So, M. (1988) Nature (London) 336, 392-395. assembly-proficient pilin, in cases where an assembly- 6. Bergstrom, S., Robbins, K., Koomey, J. M. & Swanson, J. deficient pilin is expressed. Generation of assembly- (1986) Proc. Nat!. Acad. Sci. USA 83, 3890-3894. proficient pilin variants can occur at a rate of 10-3 per cell per 7. Swanson, J., Bergstrom, S., Robbins, K., Barrera, O., Corwin, D. & Koomey, J. M. (1986) Cell 47, 267-276. generation, as observed in the P-, PilC+, pilin+ to P', PilC+, 8. Jonsson, A.-B., Nyberg, G. & Normark, S. (1991) EMBO J. 10, pilin+ transition. 477-488. The Leu39 Phe39 mutation occurring in the sequenced 9. Haas, R., Schwartz, H. & Meyer, T. F. (1987) Proc. Nat!. recA- P-, PilC-, pilin+ variant supports the selection model. Acad. Sci. USA 84, 9079-9083. First, this alteration is a base-pair substitution in the unique 10. Swanson, J., Bergstrom, S., Barrera, O., Robbins, K. & 5' end of pilE and cannot have been generated by a recom- Corwin, D. (1985) J. Exp. Med. 162, 729-744. bination event but must have been caused by a spontaneous 11. Koomey, M., Gotschlich, E., Robbins, K., Bergstrom, S. & Swanson, J. (1987) Genetics 117, 391-398. mutation. The frequency of spontaneous base-pair substitu- 12. Koomey, J. M. & Falkow, S. (1987) J. Bacteriol. 169, 790-795. tions in N. gonorrhoeae is probably not much higher than in 13. Seifert, H. S., Ajioka, R. S., Parachuri, D., Heffron, F. & So, E. coli, about 10-8 per cell per generation per gene. In the M. (1990) J. Bacteriol. 172, 40-46. absence ofselection, double mutants in bothpilC andpilE are 14. Sanger, F., Coulson, A. R., Barrel, B. G., Smith, A. J. H. & therefore not expected to be found. Second, the amino acid Roe, B. A. (1980) J. Mol. Biol. 143, 161-178. change has occurred very near to the peptide bond cleaved by 15. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Nat!. the protease(s) generating S-pilin. It seems likely that the Acad. Sci. USA 76, 4350-4354. 16. Catlin, B. W. (1973) J. Infect. Dis. 128, 178-194. presence of Phe instead of Leu here increases the suscepti- 17. Matsudaira, D. (1987) J. Biol. Chem. 262, 10035-10038. bility ofthis peptide bond to proteolysis. The PilEL39F mutant 18. Maniol, C. & Beckwith, J. (1985) Proc. Nat!. Acad. Sci. USA pilin is most likely assembly-proficient, since P+ revertants 82, 8129-8133. occur at a high frequency (10-4-10-5). This frequency must 19. Manis, J. J. & Kline, B. C. (1977) Mol. Gen. Genet. 152, solely reflect the frequency offrameshifting in eitherpilCl or 175-182. pilC2. 20. Koomey, M., Bergstrom, S., Blake, M. & Swanson, J. (1991) In E. coli, hybrid proteins stalled in the cytoplasmic Mol. Microbiol. 5, 279-287. 21. Boyd, D. & Beckwith, J. (1989) Proc. Nat!. Acad. Sci. USA 86, membrane are toxic to the cell, probably due to saturation of 9446-9450. all available sites for protein secretion (21). Further, entero- 22. Hultgren, S. J., Lindberg, F., Magnusson, G., Kihlberg, J., bacterial pilus subunit proteins are trapped in the cytoplasmic Tennent, J. M. & Normark, S. (1989) Proc. Nat!. Acad. Sci. membrane and subsequently proteolytically degraded in the USA 86, 4357-4361. absence of the periplasmic chaperone protein (22, 23). In N. 23. Lindberg, F. P., Tennent, J., Hultgren, S. & Normark, S. gonorrhoeae MSllmk(P+)-u, the nonassembled pilin is not (1989) J. Bacteriol. 171, 6052-6058. Downloaded by guest on September 29, 2021