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.