TETRAPAC (TPC), A NOVEL GENOTYPE OF NEISSERIA-GONORRHOEAE AFFECTING EPITHELIAL-CELL INVASION, NATURAL TRANSFORMATION COMPETENCE AND CELL-SEPARATION

We characterized a novel mutant phenotype (tetrapac, tpc) of Neisseria gonorrhoeae(Ngo) associated with a distinctive rough-colony morpholog y 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 mini-transposon TnMax4, The tpc mutant sh ows a 30% reduction in the overall murein hydrolase activity using Esc herichia coli murein as substrate. Tetrapacs can be resolved by co-cul tivation with wild-type Ngo, indicating that Tpc is a diffusible prote in, Interestingly, Tpc is absolutely required for the natural transfor mation competence of piliated Ngo. Mutants in tpc grow normally, but s how a similar to 10-fold reduction in their ability to invade human ep ithelial cells. The tpc sequence reveals an open reading frame of simi lar to 1kb encoding a protein (Tpc) of 37 kDa, The primary gene produc t 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 biosynth esis and one-carbon metabolism. The tpc gene is probably co-transcribe d 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 cel l separation defect, the transformation deficiency and the defect in c ell invasion can be restored by complementation of the mutant with an intact tpc gene. To some extent the tcp phenotype is reminiscent of la p in Listeria, lytA in Streptococcus pneumoniae and lyt in Bacillus su btilis, all of which are considered to represent murein hydrolase defe cts.