The Gram-negative bacterial pathogen Neisseria gonorrhoeae is naturally competent for transformation with species-related DNA. We show here that two phasevariable pilus-associated proteins, the major pilus subunit (pilin, or PilE) and PilC, a factor known to function in the assembly and adherence of gonococcal pili, are essential for transformation competence. The PilE and PilC proteins are necessary for the conversion of linearized plasmid DNA carrying the Neisseria-specific DNA uptake signal into a DNase-resistant form. The biogenesis of typical pilus fibers is neither essential nor sufficient for this process. DNA uptake deficiency of defined piliated pilCI,2 double mutants can be complemented by expression of a cloned pilC2 gene in trans. The PilC defect can also be restored by the addition ofpurified PilC protein, or better, pili containing PilC protein, to the mutant gonococci. Our data suggest that the two phasevariable Pil proteins act on the bacterial cell surface and cooperate in DNA recognition and/or outer membrane translocation.Neisseria gonorrhoeae, a strictly human-specific Gram-negative bacterial pathogen, belongs to a group of microorganisms that are naturally competent for DNA transformation (1). Natural transformation competence is considered to play a role in the horizontal exchange of genetic information between species, aside from transduction and conjugation (2, 3). DNA transformation appears to be particularly relevant in the case of N. gonorrhoeae, which, based on current knowledge, is not infected by any transducing phage (or phage in general) and is devoid of genetic elements capable of mobilizing chromosomal determinants. Thus, transformation is the mechanism that most probably accounts for the evolutionary signs of horizontal exchange in N. gonorrhoeae and related species (3-9).Transforming DNA is linearized during the uptake by N. gonorrhoeae (10). Rescue of linear plasmid DNA or chromosomal segments requires both a functional RecA protein and sufficient homology in the resident DNA pool; transformation is therefore limited to species-related DNA. Another restricting factor for species-related DNA is a specific nucleotide sequence required for the uptake of DNA (11,12) MATERIALS AND METHODSConstruction of Strains and Plasmids. All gonococcal strains used in this study are derived from N. gonorrhoeae MS11 (ref. 23; see Table 1). Plasmid pHEMK40 carrying the induciblepilEF3 gene (20) and pHTR93 encoding PilC (see Fig. 1) are derivatives of the conjugative N. gonorrhoeae plasmid ptetM25.2 constructed by gene replacement using the Hermes shuttle system (13,20). Gene replacements in the gonococcal genome or ptetM25.2 were carried out via transformation selected for in the presence of erythromycin (7 ,ug/ml for ermC) or chloramphenicol (Cm; 6, 12, or 20 pLg/ml for catLow, standard, or secondary catGC mutations, respectively). The conjugation of ptetM25.2 derivatives has been described (20). E. coli plasmid pES3 is plasmid pIP100 (4) with the ermC determinant inserted into the Bg...

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A novel genetic determinant (comA) has been identified and found to be required for the transformation of piliated Neisseria gonorrhoeae. Mutants in comA of strain MS11 grow normally and are DNA-uptake proficient but blocked in the translocation of DNA into the cytoplasm. Here we show by site-specific mutagenesis and genetic complementation that only one of two open reading frames identified in comA is essential for competence: it encodes a protein (ComA) with a predicted size of 74 kDa. The comA gene maps upstream of the iga locus and is transcribed in the opposite orientation, probably under the control of a putative sigma 54-type promoter. While DNA probes specific for the N. gonorrhoeae iga locus reveal only a little cross-reactivity with commensal Neisseria species, the neighbouring comA gene appears to be present in most of them. ComA fusion proteins were obtained by in vitro translation. The synthesized gene products migrated atypically in SDS gels indicating its strong hydrophobicity. Several transmembrane alpha-helices were predicted from the amino acid sequence of ComA which, in the context of an observed sequence similarity with other inner membrane proteins, suggests a location for the protein in the inner membrane. Using piliated and non-piliated comA mutants the consequences of transformation deficiency on pilin phase variation were assessed. We show that the comA defect affects some but not all types of DNA rearrangements associated with pilE variation. The results are in agreement with previous observations supporting the notion that multiple recombination pathways contribute to the variability of pilE.

A novel peptidoglycan-linked lipoprotein (ComL) has been identified which is required for efficient transformation of Neisseria gonorrhoeae by species-related DNA. Although most mutations in comL appear to be lethal, transposon shuttle mutagenesis was successful in generating a single viable comL mutant of N. gonorrhoeae strain MS11. This mutant, N457, exhibits a cratered and crinkled colony morphology and grows slower than wild-type MS11. However, as indicated by electron microscopy, this retardation is due to a small bacterial size rather than to a decreased generation time of the mutant bacteria. Complementation of N457 with an intact comL gene via the Hermes shuttle system fully reconstitutes bacterial size, colony morphology, and transformation competence of the wild-type strain. comL is a single-copy gene and maps downstream of the previously described comA gene. It is transcribed in the opposite direction, probably using the same transcriptional terminator. ComL has a predicted size of 29 kDa and is synthesized in Escherichia coli under the control of its native promoter, which is highly conserved with the E. coli promoter consensus sequence. The 5' end of the coding sequence reveals a lipoprotein secretion signal shown to be functional by gene fusion with alkaline phosphatase (phoA'). In E. coli, cloned ComL can be labelled with [3H]-palmitic acid, thus demonstrating its lipoproteinaceous nature. Palmitoylated ComL appears to be covalently bound to the murein sacculus of E. coli and N. gonorrhoeae since it resists boiling in 4% sodium dodecyl sulphate and is released only by lysozyme treatment. Homologous counterparts of the comL gene are found in Neisseria meningitidis as well as in several nonpathogenic Neisseria species.

We characterized a novel mutant phenotype (tetrapac, tpc) of Neisseria gonorrhoeae (Ngo) associated with a distinctive rough-colony morphology and bacterial growth in clusters of four. This phenotype, suggesting a defect in cell division, was isolated from a mutant library of Ngo MS11 generated with the phoA minitransposon TnMax4. The tpc mutant shows a 30% reduction in the overall murein hydrolase activity using Escherichia coli murein as substrate. Tetrapacs can be resolved by co-cultivation with wild-type Ngo, indicating that Tpc is a diffusible protein. Interestingly, Tpc is absolutely required for the natural transformation competence of piliated Ngo. Mutants in tpc grow normally, but show a approximately 10-fold reduction in their ability to invade human epithelial cells. The tpc sequence reveals an open reading frame of approximately 1 kb encoding a protein (Tpc) of 37 kDa. The primary gene product exhibits an N-terminal leader sequence typical of lipoproteins, but palmitoylation of Tpc could not be demonstrated. The ribosomal binding site of tpc is immediately downstream of the translational stop codon of the folC gene coding for an enzyme involved in folic acid biosynthesis and one-carbon metabolism. The tpc gene is probably co-transcribed from the folC promoter and a promoter located within the folC gene. The latter promoter sequence shares significant homology with E. coli gearbox consensus promoters. All three mutant phenotypes, i.e. the cell separation defect, the transformation deficiency and the defect in cell invasion can be restored by complementation of the mutant with an intact tpc gene. To some extent the tcp phenotype is reminiscent of iap in Listeria, lytA in Streptococcus pneumoniae and lyt in Bacillus subtilis, all of which are considered to represent murein hydrolase defects.