Effects of Reca Mutations on Pilus Antigenic Variation and Phase Transitions in Neisseria Gonorrhoeae

Effects of recA Mutations on Pilus Antigenic Variation and Phase Transitions in Neisseria gonorrhoeae

Michael Koomey,” Emil C. Gotschlich,* Ken Robbins,? Sven Bergstrom?’*and John Swansont “Laboratory of Bacteriology and Immunology, The Rockefeller University, New York, New York 10021, ?Laboratory ofMicrobia1 Structure and Function, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana 59840 and $Department ofMicrobiology, University of Umei, S-901 Umei, Sweden Manuscript received February 10, 1987 Revised copy accepted July 18, 1987

ABSTRACT Intragenic recombination between the single complete pilin gene (expression locus) and multiple, distinct, partial pilin gene copies (silent, storage loci) is thought to account for the generation of pilus antigenic diversity and piliation phase (on-off) changes exhibited by Neisseria gonorrhoeae. The mechanisms operating in the genomic rearrangements associated with these forms of pilus variation were investigated through the study of isogenic strains of gonococci bearing either wild-type or altered recA alleles. Examination of the rates of pilus phase variation and the genetic basis for changes in piliation status displayed by these strains show that recA mediated homologous recombination is required for these high frequency events and confirm that the nonpiliated state results from mutations in the expressed pilin gene. In a strain that is deficient in recA mediated homologous recombination, pilus phase variation occurs at a 100-1000-fold reduced rate and results predominantly from one class of spontaneous frameshift mutations within the pilin structural gene.

HE presence of pili (or fimbriae) can be corre- of the gene’s primary translational product (HERMOD- T lated with virulence in many gram-negative SON et al. 1978; MEYERet al. 1984). The pilin molecule pathogenic bacteria that inhabit mucosal surfaces of displays a conserved amino-terminal domain (a.a. 1- mammals. These proteinaceous appendages have Sl), a “semivariable”central region in which moderate been implicated in bacterial adherence and coloniza- amino acid substitutions are found (a.a. 62-1 2 1 with tion of the human host by Neisseria gonorrhoeae, the nonvariable residues at a.a. 79-85, 93-97 and 113- causative agent of gonorrhea (SWANSON1973; PEARCE 116) and a “hypervariable” segment in which contig- and BUCHANAN1978; MCGEEet al. 1978). This par- uous stretches of residues change (a.a. 135-1 50 and ticular species is remarkable in that intrastrain variants 159-166) (SCHOOLNIKet al. 1984; HAGBLOMet al. displaying biochemically and antigenically distinct pili 1985; BERGSTROMet al. 1986; SWANSONet al. 1986). arise at readily detectable although undetermined fre- The regions that flank the hypervariable region (a.a. quencies (VIRJIand HECKELS1983; HAGBLOMet al. 122-134 and 151-158) are conserved and contain 1985; BERGSTROMet al. 1986; SWANSONet al. 1987a, cysteine residues thought to be disulfide bonded to b). The expression of multiple pilin serotypes might one another. The pilin hypervariable domain is the enable the microorganism to maintain essential pilus- immunodominant portion of purified pili and changes mediated properties in an immunocompetent host. In in this region account for some of the demonstrable addition to this form of heterogeneous pilus expres- antigenic differences among gonococcal pili (VIRJI sion, piliated (pilusf) gonococci cultivated in vitro give and HECKELS1983; ROTHBARD,FERNANDEZ and rise to nonpiliated and some, but not all, pilus- var- SCHOOLNIK1984; SWANSONet al. 1987a,b). The gon- iants are capable of spawning pilus+ revertants (SWAN- ococcal chromosome usually contains a single intact SON et al. 1985). Such phase transitions between pi- pilin gene (although in one strain, two complete genes liated and nonpiliated states are easily recognized by have been documented) and multiple copies of variant attendant changes in colonial morphology (SWANSON, partial pilin genes that lack transcriptional promoters KRAUS and GOTSCHLICH1971) and occur with fre- and amino terminal encoding sequences (MEYERet al. quencies that are approximately 1,000- 10,000-fold 1984; SWANSONet al. 1985; HAASand MEYER 1986; higher than the rates of spontaneous mutation. SWANSONet al. 1986). It has been shown that pilus Biochemical and genetic studies have shown that antigenic variation and phase transition both correlate gonococcal pilins are synthesized as polypeptides ap- with DNA rearrangements within the pilin structural proximately 165-1 70 amino acid (a.a.) residues in gene and that these events appear to result from length and that their mature polymerized form pres- genetic exchanges between the complete pilin gene ent in pili lacks the first seven amino terminal residues and nonexpressed partial genes (HAGBLOMet al. 1985;

Genetics 117: 391-398 (November, 1987) 392 M. Koomey et al.

BERGSTROMet al. 1986; SWANSONet al. 1986; SWAN- tinguishable from one another. The pilin gene nucleotide sequences and amino acid positions noted here follow the SON et al. 1987a,b). In phase changes, pilus- organisms conventions established in prior studies (BERGSTROMet al. arise from recombinational processes that generate 1986; SWANSONet al. 1986). The nucleotide sequence of nonreverting deletion mutations (SWANSONet al. the VD302 P++ pilin gene determined by oligonucleotide 1985) as well as reverting frameshift or missense mu- primer extension of its pilin mRNA is identical to the DNA tations in the pilin gene (BERGSTROMet al. 1986; sequence of the pilin gene contained in the plasmid pVD203 SWANSONet al. 1986). Thus, the generation of pilus+ (BERGSTROMet al. 1986) with the sole exception of single base difference at nucleotide position 685 that changes an revertants by pilus- organisms and of pilus antigenic alanine (GCC) at amino acid position 1 17 to a glycine (GGC). variants by pilus+ organisms result from identical The VD301 P++pilin gene also contains that same alteration events, that is productive intragenic recombination as the VD302 pilin gene as well as five other nucleotide between the pilin expression locus and incomplete changes between nucleotides 591 and 609. Only two of pilin gene copies. By the use of specific oligonucleotide these result in amino acid alterations so that VD301 pilin has lysine rather than glutamine at amino acid position 89 probes, this genetic exchange has been shown to be and glutamine rather than lysine at amino acid position 9 1. nonreciprocal in character (SWANSONet al. 1986; Nonpiliated variants derived from these strains were iden- SWANSONet al. 1987a) and resembles what has been tified by colonial morphology and in cases where their termed gene conversion. That term may not be en- piliation status was in doubt it was confirmed by scanning tirely appropriate because gonococci are 1) diplococci electron microscopy as described (SWANSONet al. 1985). Piliated revertants were assessed by colonial morphology whose precise chromosomal interactions and consti- alone. tution are not presently well understood and 2) ex- Phenotypic and genotypic analysis of pilus variants: tremely competent for transformation when in a pi- Expression of pilin protein was examined by immunoblot- liated state (SPARLING1966), and since all the prod- ting of solubilized whole gonococci as detailed (SWANSONet ucts of genetic exchange may not be recoverable as al. 1986). The MC02 monoclonal antibody recognizes an epitope that is present on pilin polypeptides of all of the they are following genetic crosses in fungi and yeast. strains examined to date while the 1H5 monoclonal anti- In order to identify the metabolic reactions and body recognizes an epitope encompassed by amino acid mechanisms that are involved in gonococcal pilin in- positions 128-141 of pilins expressed by the parental pilus+ tragenic recombination and to test our current no- variants used here (SWANSONet al. 1987b). The preparation tions about the regulation of pilus expression, we have of genomic DNA and total RNA as well as the conditions for Southern and Northern blotting were performed as studied previously described isogenic gonococcal mu- described (SWANSONet al. 1985). Pilin mRNAs were se- tants that have defective recA alleles with regard to quenced by primer extension using 32P-labeledoligonucle- their relative frequency of pilus phase transitions and otides. The protocols for this technique and the particular the genetic events responsible for the phenotypic primers used have been published previously (BERGSTROM changes. The results confirm that, in wild type gono- et al. 1986; SWANSONet al. 1986). For those pilus- variants that failed to give rise to pilus' revertants at detectable cocci, alterations in the pilin structural gene account frequencies on solid media, organisms were cultivated in for the nonpiliated state and that these high frequency static broth cultures to selectively enrich the recovery of variations require homologous recombination func- rare revertants (FROHOLM,JYSSUM and BOVRE1973). Three- tions that involve the recA gene product. In the ab- milliliter volumes of tenfold dilutions of these organisms sence of functional recA gene product, pilus- variants (starting at 10' colony-forming units (cfu) per ml) in clear media lacking agar were cultivated without shaking in 12 X arise at rates that are at least thirty-fold reduced as 75 mm polystyrene round bottom tubes (Falcon 2054, Bec- compared to the recA+ strain and result from one ton Dickinson) and incubated at 37" in the presence of 5% major class of spontaneous frameshift mutations COs for at least 36 hr. The pellicle of bacterial growth at within the pilin gene. The generation of pilus+ re- the air-liquid interface was carefully subcultured onto solid vertants by these recA pilin mutants occurs at rates media and the colonies examined. Variants that failed to show evidence of reversion under these conditions were that are a 1000-fold lower than that found in wild- examined for the loss of the 5' end of the pilin gene by type strains and results from true reversions at the colony hybridization (GRUNSTEINand HOGNESS1975) using same mutational hotspot. the oligonucleotide GC06 (SWANSONet al. 1985) that is complementary to the sequences that encode the first seven MATERIALS AND METHODS amino acids of the pilin polypeptide. Measurement of the rates of piliation transitions: Rates Bacterial strains: Strains VD300 (pilus+,ApilEn), VD30 1 of piliation transitions were calculated by modification of a (pilus+, ApilE2, recA3OI) and VD302 (pilus+, ApilE2, published protocol (ENOMOTOand STOCKER,1975). Single ArecA302) (KOOMEYand FALKOW1987) were grown at 37" whole colonies of gonococci were excised by stabbing the in 5% COS on clear medium as described before (SWANSON agar with the broad end of a sterile Pasteur pipette and et al. 1985). These organisms have a single pilin expression transferred to 1 ml of broth media lacking supplements. locus designated pilEI and have a deletion within the pilE2 After vigorous vortexing and exposure to brief (5-1 0 sec) locus that encompasses the complete 4.3-kb ClaI fragment pulses of disruption in a sonicating water bath (Astroson previously characterized (MEYER et al. 1984). The pilin model 9, Heat Systems Ultrasonics) to disrupt bacterial subunits elaborated by organisms displaying P++ colony pi- aggregates, dilutions were spread so that each plate con- liation phenotypes (SWANSONand BARRERA1983) of each tained between 50 and 100 well separated colonies. This of the strains are biochemically and immunologically indis- treatment did not result in decreased viability and in the Pilus Variation in N. gonorrhoeae 393 case of piliated organisms increased the number of cfu five- TABLE 1 to tenfold. The term colony forming unit is used rather than cell since gonococci exist as diplococcal units. After Rates of pilus transitions” varying time lengths of incubation, at least six colonies of a given variant at a given time point were treated as above P+ + P- P- * P+ and dilutions were plated to calculate viable cfu and to VD300 (recA’) 2.2 x 10-5 6.5 x 10-4 determine the ratio of piliated or nonpiliated colonies (as VD301 (recA301) 4.1 X 4.5 X (8/18 P-r) determined by colonial morphology) to total cfu. Rates of VD302 (ArecA302) C6.0 X lo-‘ <5.0 X lO-’(12/12 P-r) piliation transitions were calculated by the formula (M/N)/ g where M/N is the ratio of variants to total cfu and g is the a Results are expressed as variants/cfu/generation and are averages with actual values varying &20%.P+, pilus+; P-, pilus-. Num- number of generations of growth from a single cfu to the bers within parentheses indicate the ratio of nonpiliated variants number of cfu within a colony (log2of total cfu). The results capable of spawning piliated revertants to the total number of presented are the averages of these results. All colonies nonpiliated variants studied. displaying P++ or P+ morphology contain pilus+ organisms but not all colonies of pilus+ organisms display P++ or P+ analysis strains VD300 and VD30 1 were propagated morphotypes (M. KOOMEY, unpublished data). That is to for 18 hr or less while strain VD302, with a slightly say that some colonies displaying a pilus- morphology ac- tually consist of pilus+ organisms. Therefore, the rates of longer generation time, was propagated for 21 hr pilus+ to pilus- transitions determined here represent max- before harvesting and plating. To minimize the en- imal values while the rates of pilus- to pilus+ transitions richment of pilus- organisms during the subsequent represent minimal values. growth on solid media required to assess piliation, colonial morphology was scored at 17 and 20 hr, RESULTS respectively. The wild-type strain (VD300) generated Effects of altered recA alleles on the rates of pilus+ pilus- variants at a rate of 2.2 x 10-5/cfu/generation to pilus- transitions: The recA gene from the gono- while the rate of appearance of such variants in the coccal strain MS 1 1mk has been cloned in Escherichia recA insertion mutant (VD301) was reduced to 4.1 X coli and derivatives of this cloned gene were used to 1O-‘/cfu/generation (Table 1). Under these condi- construct, by transformation-mediated marker rescue, tions, no pilus- variants were found among 120,000 an isogenic set of piliated MSll derivatives bearing colonies examined of the recA deletion mutant, strain mutated recA alleles. The wild-type parent strain, a VD302 (C6 X 1O-’/cfu/generation). However, by mutant that displays a reduced proficiency for recA prolonged growth (42 hr or more) rare pilus- variants associated functions due to an insertion of DNA near could be recovered from that strain. the recA structural gene (the recA301 allele) and a Pilus- variants can be classified into two distinct mutant that displays an absolute deficiency in recA phenotypes, one is incapable of spawning piliated re- functions due to a deletion that encompasses part of vertants (P-n) while the other retains this capability the gene and its flanking sequences (the ArecA302 (P-r); organisms displaying a P-r phenotype can be allele) have been designated strains VD300, VD301 further categorized by their synthesis of pilin (P-rp’) and VD302, respectively (KOOMEYand FALKOW or lack thereof (P-rp-). The P-n phenotype results 1987). A modified version of the protocol developed from loss of the single genomic copy of the transcrip- for the study of flagellar phase variation in Salmonella tional promoter and 5’ end of the pilin gene (SWAN- was used to determine the frequency of pilus+ to pilus- SON et al. 1985). Organisms of the P-r phenotype have transitions. With strain VD300, it became clear that an intact pilin gene and express pilin mRNA that colonies harvested after 21 hr of growth yielded in- either contains a premature translation termination creased values for the rates of pilus+ to pilus- transi- signal (P-rp-) or encodes full-length, antigenically de- tions when compared to colonies picked before that tectable pilin polypeptide that is not polymerized into time (data not shown), a phenomenon that has been pili (P-rp’) (BERGSTROMet al. 1986; SWANSONet al. previously documented (NORLANDERet aE. 1979). 1986). In strain MSl lmk, the P-rp+ phenotype is most Since there was little increase in the total cfu in a often associated with the expression of an “assembly colony after 21 hr of growth, the increase in propor- missense” pilin polypeptide bearing amino acid resi- tion of pilus- variants appears to reflect the selective dues 136-148 derived from pilin gene copy 5 of the growth advantage of pilus- variants generated pre- pilSl locus (BERGSTROMet al. 1986; SWANSONet al. vious to this period of nonexponential growth. This 1986; HAASand MEYER1986). The phenotypes and was not surprising considering that pilus+ organisms genotypes of independently derived pilus- variants form small domed colonies with well defined edges from strains VD301 and VD302 but not VD300 were due to their autoagglutinability (KELLOGGet al. 1968; investigated since the latter is virtually identical to the SWANSONet al. 197 1) while pilus- organisms form flat, MSl lmk strain used in previous studies of this kind spreading colonies and since nonselective subculture (BERGSTROMet al. 1986; SWANSONet al. 1986; on solid media has long been known to result in a loss KOOMEYand FALKOW1987). of piliation and virulence within the gonococcal pop- Characterization of pilus- variants of the recA ulation (KELLOGG et al. 1963). Therefore, in this insertion mutant VDSOL: Ten of 18 VD301 pilus- 394 M. Koomey et al.

VD301 P** 1234567 120 125 .130 Glu Ian Gly S.r Vml Lya Trp Phe Cyr Gly Gln m- GAA AAC CGT TCG GTA AAA TOO TTC TGC GGA CAG - ti0 Ti0

VD301 P' I3 120 125 Glu Aan Cly Sar Val Lys GAA AAC CGT TCG GTA AAA TAG TTC TGC FA CAG I ti0 120 FIGURE2.-Pilin mRNA sequences of strain VD3Ol P" and P- #3 (P-rp' phenotype) variants. Pilin gene nucleotide (underneath) FIGURE1 .-Pilin polypeptides of P" and P- variants of strains and encoded amino acid positions (above) are according to BERGS VD3Ol and VD302 detected by immunoblotting.Whole cell lysates TR~Met al. (1 986). Sequences start with the codon for amino acid to of pilus variants (equivalent to 10' cfu) were subjected to SDS 120 and extend through the codon for amino acid 130. The polyacrylamide gel electrophoresis and were immunoblotted with arrow denotes the location of a single base change (a G to A antipilus monoclonal antibody MC02. Lane 1, VD302 P**; 2. transition) within the codon for amino acid 126 of the VD3Ol P- VD301 P++;3. VD301 P- #I (P-rp+ phenotype); 4, VD301 P- #3 #3 transcript that results in translational terminationand expression (P-rp' phenotype); 5, VD301 P- #5 (P-n phenotype): 6, VD302 P- of a truncated pilin polypeptide. #1 (P-rp- phenotype); 7, VD302 P- #2 (P-rp- phenotype). Num- bers on left represent the migration of M, standards (X IO-'): form is posttranslationally derived from the M, = carbonic anhydrase, 31,000; soybean trypsin inhibitor, 21,000; 21,000 polypeptide. The properties of seven of the lysozyme, 14.000. eight pilus- derivatives expressing pilin in this strain bearing the recA301 allele were identical to those of variants displayed P-n phenotypes as deduced from analogous variants characterized in recA+ strains and their failure to revert at detectable frequency, and to it seems most likely that they arose by recombinational express either pilin mRNA or pilin antigen. Southern events similar to those previously described. However, blotting studies were used to show that the 4.1-kb in one of the P-rp+ variants of this strain (P-rp' #3), Cia1 fragment carrying the pilin gene had undergone a single pilin species of M,= 15,000 was found (Figure a gross rearrangement characteristic of this genotype 1, lane 4). The basis for the unique phenotype of this (SWANSONet al. 1985)and confirmed that the genome variant became apparent when its pilin mRNA and of these organisms had lost the unique copy of the 5' that of its piliated parent were sequenced by oligonu- end of their pilin gene as seen by their failure to react cleotide primer extension and compared (Figure 2). with an oligonucleotide probe complementary to the The entire sequences of these two mRNAs were iden- region encoding the first seven amino acids of the tical except for a single nucleotide difference at the pilin gene product (data not shown). second position of the codon specifying amino acid The eight remaining pilus- variants were of the 126 that creates an amber translational stop codon in P-rp+ class in that they expressed pilin protein detect- the pilin transcript of the pilus- organism. The relative able by immunoblotting with the broadly cross-reac- mobility of the pilin detected by immunoblotting cor- tive monoclonal antibody, MC02 (SWANSONand BAR- related with the truncated 125 amino acid polypeptide RERA 1983). The pilins expressed by these pilus- predicted by premature translational termination. variants were heterogeneous in electrophoretic mo- Frameshift mutation accounts for phase transi- bilities and antigenically distinct from the pilin ex- tion in the absence of homologous recombination pressed by their piliated progenitor as seen by their functions: Twelve independent pilus- variants de- failure to react following immunoblotting with the rived from the recA deletion mutant (strain VD302) 1 H5 monoclonal antibody, that recognizes a defined were selected for study. These variants failed to show linear epitope between amino acids 128-141 (SWAN- any evidence of spawning pilus+ revertants despite SON et al. 1987b) as well as other selected monoclonal rigorous examination during numerous daily subcul- antibodies (data not shown). In strain MSll and its tures nor did they express pilin antigen detectable by derivatives, P-rp' gonococci elaborate two major spe- standard immunoblotting techniques. They did not, cies of pilin antigen (M,= 21,000 and M, = 16,000- however, correspond to the previously described P-n 17,000;see Figure 1, lane 3, for example) while pilus+ phenotype that results from 5' deletions of the pilin gonococci produce a single predominant species of gene since they showed no evidence of gross rear- the slower migrating (M,= 21,000) form (SWANSON rangement of the pilin expression locus by Southern et al. 1985, 1986). Although the biochemical basis for blotting and they expressed typical levels of full size the M, = 16,000-17,000 class of pilin polypeptide pilin mRNA as seen by Northern blotting (data not associated with the P-rp' phenotype is not clear, the shown). In light of this data, immunoblotting was shared reactivity pattern of both forms with pilin repeated using increased amounts of whole cell lysates monoclonal antibodies and the facts that these variants and the MC02 monoclonal antibodies (Figure 1). have a single active pilin gene and a homogeneous Under these conditions, a faint reactive band of M,= population of pilin mRNA suggest that the lower M, 2 1,000 was detected in these samples but not in iden- Pilus Variation in N. gonorrhoeae 395 tically prepared lysates of VD301 P-n variants with VD302 PM 70 80 the nonreverting pilin gene deletion mutations. The Ser Ala Gly Val Ala Ser Fro Pro Ser Asp Ile Lys Gly Lys Tyr Val Lys Clu Val pilin antigen found in the VD302 pilus- variants rep- TCT CCC GGC GTG CCA TCC CCC CCC TCC CAC ATC AAh 0% MA TAT GTT AAA GAG CTT 5M 5i)o resented either an extremely low level of expression 90 100 by all organisms (a leaky phenotype) or pilin synthe- Glu Val Lys Asn Cly Val Val Thr Ala Ihr Met Leu Ser Ser Cly Val Asn Asn Glu GAA CTT AMAAC GGC GTC W ACC CCC ACA AX3 CTT TCA GGC GTA AAC AAT GM sised by pilus" revertants too rare to be detected by 6iO MO our standard visual assay. Piliated strains of Neisseria meningitidis and other gram-negative species form a pellicle when cultured in static broth media and such VD32 P- 70 4 80 conditions can be used to selectively enrich for pilus+ Ser Ald Gly Val Ala Ser Pro Pro Pro "r Ser Lys Ala Asn Met Leu Lys Arg Leu organisms and facilitate their recovery (FROHOLM, TCT CCC CCC GTG CCA XCCCC CCT CCG ACA TCA AAC CCA AAT ATG TTA AAG ACC TTG 5<0 5iO JYSSUM and BOVRE 1973). When grown in this man- 90 100 ner, all twelve VD302 pilus- variants formed surface Lys Leu Lys "r Ala Ser Leu Pro Pro Cln Cys F'he Cln Ala Ala *** AAG TTA AAA ACG GCG TCG TTA CCC CCA CAA TGC TTT CAA CCG CCC TAA ACA ATG AAA pellicles, which when subcultured onto solid media, 6io 640 regularly yielded pilus+ revertants while identical cul- FIGURE3.-Location and determination of the frameshift mu- tures of VD300 and VD301 variants with pilin gene tation of the pilin structural gene associated with phase variation in deletion mutations failed to do so. The pilus- variants strain VD302. Pilin gene nucleotide (underneath) and encoded of the recA deletion mutant therefore displayed a amino acid positions (above) are according to BERGSTROMet al. (1986). Sequences start with the codon for amino acid 70 and P-rp- phenotype. extend through to the codon for amino acid 107. On the top is Under the immunoblotting conditions used above shown the mRNA sequence determined for the VD302 P++ pro- it was also noted that samples of piliated organisms genitor and piliated revertants derived from independent VD302 demonstrated multiple discrete reactive species be- P- variants. On the bottom is shown the mRNA sequences found tween M, = 21,000 and 14,000 that were absent in in three independent VD302 P- variants. The arrow denotes the frameshift in translational reading frame that results from the samples derived from organisms of the P-rp+ pheno- deletion of any one of eight cytidines between nucleotides 559 and type and that the patterns of VD301 P++and VD302 566. P++ lysates were indistinguishable (Figure 1). The correlation of the expression of these minor pilin gene conversion that results in the synthesis and forms of altered mobility and piliation has been con- expression of a novel functional pilin protein. Since firmed repeatedly by analysis of these and other the elaboration of pili of altered antigenicity is not strains (M. KOOMEY,unpublished data). always associated with a discernible change in colonial The pilin mRNAs expressed by the pilus+ progeni- morphology, the frequency of productive pilin gene tor of strain VD302, three pilus- variants and a pilus+ conversion can most readily be assessed as the fre- revertant of each of these were sequenced by oligo- quency with which reverting pilus- organisms give nucleotide primer extension. The sequences obtained rise to pilus+ organisms. These data were determined from the nonpiliated variants were identical to one for two independent variants displaying the P-rp+ another but differed from that derived from the pi- phenotype derived from both strain VD300 and VD301 (Table 1). The values found using the recA+ liated predecessor and revertants by the deletion of a single nucleotide (Figure 3). Whereas the pilin tran- strain (VD300) were very similar to that previously reported for strain MS1 lmk (SWANSON et al. 1985) scripts from the pilus+ organisms contained eight cy- while those obtained from the recA insertion mutant tidines at nucleotide positions 559 through 566 (en- (VD30 1) were about tenfold lower than that seen with coding amino acids 75-77), the pilus- transcripts dis- the wild-type strains. These frequencies were much played a deletion of one of these cytidines. The loss higher than that found for the reverting pilus- var- of any one of these eight cytidines results in a frame- iants derived from the recA deletion mutant, strain shift mutation, premature translational termination VD302 (<5 x lO-'/cfu/generation). and expression of an immunologically undetectable, truncated polypeptide. Reinsertion of a cytidine DISCUSSION within this same stretch of nucleotides restored the integrity of the reading frame and therefore, the Past studies and the work detailed here have shown pilus+ revertants resulted from back mutations owing that nonpiliated variants of gonococci arise in vitro as to true reversions. a result of mutations within the single complete pilin Effects of altered recA alleles on the rates of structural gene. The reduced rates of phase variation productive pilin gene recombination: With the ex- and the phenotypes displayed by strain VD301 that ception of the recA deletion mutant noted, the spawn- expresses a reduced proficiency in recA associated ing of pilus+ revertants by pilus- organisms and the functions strongly suggests that recA gene product is direct generation of variants expressing antigenically involved in pilin gene conversion events. The approx- distinct pili by pilus+ organisms both correlate with imate tenfold reduction in the rates of pilus- to pilus+ 396 M. Koomey et al. transitions found for this strain parallels similar de- pair in the three pilus- variants we studied supports creases in two other recA associated functions, trans- the contention that base removal predominates in formability with chromosomal markers and UV re- spontaneous frameshift mutations of such sequences sisrance, exhibited by this strain (KOOMEYand FAL- in bacteria (ROTH 1974; STREISINCERand OWEN KOW 1987). Two classes of pilin gene lesions are found 1985). Another frameshift mutation in this same in recombination proficient derivatives of strain stretch of monotonous nucleotides was previously MSll: 1) nonreverting deletion mutations and 2) found in a pilus- variant of strain MS11,k but in that reverting frameshift, nonsense or missense mutations. case the forward mutation resulted from the insertion The loss of the unique 5’ end of the pilin gene that of a cytidine resulting in nine cytidines (BERGSTROM occurs in nonreverting pilus- variants can result from et al. 1986). The contention that this latter mutation either a conventional deletion of those sequences arose de novo from a recombinational event is sup- whose endpoints lie within the 3’ end of the expres- ported by the data found here. These sequences reside sion site and the tandemly arrayed partial pilin gene in what is known to be a variable segment of the gene copies that are found immediately upstream (SEGALet (BERGSTROMet al. 1986); so one would expect that al. 1985; SWANSONand KOOMEY1985; SWANSONet the introduction of the ArecA302 allele into variants al. 1986) or from the eviction of these sequences by a with differing sequences in that region might reveal gene conversion event (SWANSONet al. 1985). The other hotspots for pilin gene mutation. Although all failure to detect such nonreverting deletion mutants of the pilus- variants examined here and in our pre- in the strain carrying the ArecA302 allele (VD302) vious studies carry pilin mutations, a mutation in supports the contention that recAf function is required accessory genes involved in pilin expression, transport for, or at least significantly facilitates, some part of or assembly would also display a pilus- phenotype. the deletion process (ALBERTINIet al. 1982). Previous The introduction of the recA deletion mutation into investigations of pilin frameshift and missense muta- the MSll variant that has two complete pilin genes tions found in reverting pilus- variants of strain would appear to be a straight forward way to facilitate MSl l,k (responsible for the P-rp- and P-rp+ pheno- the search for such mutants and the identification of types, respectively) have demonstrated that these for- those genes and gene products. In that case, pilus- ward mutations encompass clustered multiple nucleo- variants with second site mutations that preclude the tide substitutions as do the back mutations present in biogenesis of pili, but not pilin synthesis, or that co- pilus+ revertants (BERGSTROMet al. 1986; SWANSON ordinately repress pilin gene expression should pre- et al. 1986). Prior to those observations, such proc- dominate. esses of reversible phenotypic variation involving mu- The precise role played by the recA gene product tation mediated by gene conversion had not been in catalyzing the DNA rearrangements of pilin gene documented in prokaryotes. The finding of one pilus- conversion is obscured by the pleiotropic nature of variant in strain VD301, of the eighteen examined, recA mutations (CLARK1973) and although it is nec- that contains a single base change shows that such essary for the process, it alone is probably not suffi- mutations do occur in recombination proficient strains cient. It should be noted in this regard that the mu- although their presence is obscured by the more prev- tations in both strains carrying altered recA alleles alent variants bearing multiple substitutions. It also involve uncharacterized flanking DNA sequences that shows that the P-rp+ phenotype can result from pilin may contain other genes that effect pilin gene recom- gene nonsense mutations as well as from assembly bination, as previously acknowledged (KOOMEYand missense mutations. FALKOW1987) and experiments to address this possi- Data obtained using the VD302 strain that bears bility in a definitive manner will require the construc- the ArecA302 allele and is deficient in recA mediated tion of mutants bearing lesions wholely contained functions provide substantial evidence that homolo- within the recA structural gene. Since gonococci are gous recombination functions are required for pilin known to be highly competent for transformation gene conversion associated with phase variation and using chromosomal DNA, they clearly have a well the generation of antigenically diverse pilins. Phase developed system for the mismatch repair of DNA variation occurs at greatly reduced rates in this strain heteroduplexes. It seems likely then that pilin gene by reversible spontaneous frameshift mutation within conversion occurs by similar mismatch repair of het- a stretch of redundant C/G basepairs. This region eroduplexes formed at the pilin expression locus be- represents a classical example for such alterations tween resident sequences and those that originate (STREISINGERand OWEN1985) and virtually identical from the multiple partial pilin genes. The recA gene mutational hotspots have been documented in pro- product would probably be involved in both the initial karyotes (OESCHGARand HARTMAN1970; CALOSand pairing and heteroduplex formation as well as in the MILLER1981) and eukaryotes (WILSONet al. 1986). repair that follows. Of more pertinence to the study The deletion (as opposed to the insertion) of a C/G of microbial pathogenesis, this work implies that the Pilus Variation in N. gonorrhoeae 397 recA gene plays an indirect role in the virulence of N. This work was supported in part by U.S. Public Health Service gonorrhoeae. Since it has previously been shown to be grants A110615 and AI19469 and a grant from the World Health Organization. S.B. was partially supported by grant project 6937 involved in amplification of toxin genes of Vibrio chol- from the Swedish Medical Research Council. erae (GOLDBERGand MEKALANOS1986) and in the instability of capsule production in Haemophilus influ- enzae type b (HOISETH,MOXON and SILVER-1986) it LITERATURE CITED appears that homologous recombination mediated in ABRAHAM,J. M., C. S. FREITAG,J. R. CLEMENTSand B. I. EISEN- part by the recA gene product may be a fundamental STEIN,1985 An invertible element of DNA controls phase mechanism by which pathogenic bacteria -modulate variation of type 1 fimbriae of Escherichia coli. Proc. Natl. Acad. the expression of virulence associated genes that exist Sci. USA 62: 5724-5727. as duplicated elements within the chromosome. ALBERTINI,A. M., M. HOFER,M. P. CALOSand J. H. 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