Moiecuiar Microbiology (1995) 16(3), 451-464 identification and characterization of pilG, a highly conserved piius-assembiy gene in pathogenic Neisseria Tone Tonjum,^* Nancy E. Freitag,^ Ellen Namork^ and Michael Koomey^ 'Kaptein W. Witlieimsen og Frues Bakteriotogiske Instituit Rikshospitalet (National Hospital), University of Osto, N-0Q27 Osto t Norway ^Department of Microbtology and Immunology. University of Midiigan Medicat Sctioot, Ann Arbor, Michigan 48109-0620, USA. ^^Etectron Microscopy Unit, Nafiotiat Institute of Public Healfh, 0462 Oslo. Norway. Summary Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human patho- gens Neisseria gonorrhoeae and Neisseria meningiti- dis. The assembly of these colonization factors is a complex process. This report describes a new pilus- assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre-pilin leader peptidase. The nucleotide sequence of this region revealed a single comptete open reading frame whose derived polypeptide displayed significant identities to the piius-assembty protein PilC of Pseudomonas aeruginosa and other polytopic integral cytopiasmic membrane constituents involved in protein export and competence. A unique polypeptide of M^ 38 kDa corresponding to the gene product was identified. A highly related gene and fianking sequences were cloned from a group E polysaccharide-producing strain of N. tneningit/dis (Me). The results indicate that the pilG genes and genetic organization at these loci in Gc and Me are extremely conserved. Hybridiza- tion studies strongly suggest that p//G-related genes exist in commensal Neisseria species and other spe- cies known to express type IV pili. Defined genetic lesions were created by using insertionai and transpo- son mutagenesis and moved into the Gc and Me chro- mosomes by allellc replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed Received 25 October, 1994; revised 11 January, 1995; accepted 13 Januaiy, 1995- '"Present address for correspondence; Department of Microbiology. Ulleval Hospital, University of Oslo, N-0407 Oslo, Norway- Tel. 22 11 88 1S\ Fat; 22 74 15 96, dramatically reduced competence for transformation. These findings could not be ascribed to pilin-gene alterations or to polarity exerted on pilD expression. The resulls indicated that PilG exerts its own indepen- dent role in neisserial pilus biogenesis. Introduction Infections caused by Neisseria gonorrhoeae (Gc) and Neisseria rneningitidis (Me), pathogenic tnembers of the genus Neisseria. are associated WiVn signiiicant morbidity and mortality for their exclusively human hosts. Despite the unique disease manifestations associated with these species, it appears that their basic strategies for success- ful colonization of man are highly conseived. Many studies have focused on understanding the structures and func- tions of surface-localized and extracellular neisserial com- ponents. The expression ot pili, which are filamentous-like structures emanating from the bacterial surface, appears to be of paramount importance to ihe pathogenic process (Heckels, 1989), Gc and Me pilus filaments are ordered arrays of polymerized protein subunits termed pilin. The short leader sequences and proximal 30 amino acids of their pre-pilins (Meyer et ai, 1984; Potts et ai, 1988) show a high degree of homology witb pre-pilins of other Gram-negative buman pathogens including Pseudomo- tias aeruginosa (Pasloske ef ai, 1985). Vibrio choierae (Shaw and Taytor, 1990) and certain strains oi entero- pathogenic Escheridiia coit (Giron ef ai. 1991). Collec- tively, members of ihis fatnily of pilus colonization factors have been termed type IV pili. Evidence for critical roles of Gc and Me pili can be found in the Invariable recoveries of piliated organisms frorn primary cultures (Jyssum and Lie, 1965; Kellogg et at., 1968; Swanson et ai., 1987) and lhe capacities of these stnjclures to undergo anti- genic variation (Tinsley and Heckels. 1986: Swanson ef at., 1987). Gc pilus variation results from homologous recombination between a single complete pilin gene or expression locus and multiple partial pilin gene copies or silent alleles (Haas and Meyer, 1986; Swanson et at.. 1986; Koomey efat., 1987) and an analogous mechanism is thought to operate in Me pilus variation (Abo and Cannon, 1988; Perry etai, 1988; Blake etai, 1989). Neisserial piii appear to promote infectivity by mediating attachment to mucosal epithelia (Swanson, 1973; Stephens 1995 Blackwell Science Ltd