The PilB protein of Neisseria gonorrhoeae has been reported to be involved in the regulation of pilin gene transcription, but it also possesses significant homology to the peptide methionine sulfoxide reductase family of enzymes, specifically MsrA and MsrB from Escherichia coli. MsrA and MsrB in E. coli are able to reduce methionine sulfoxide residues in proteins to methionines. In addition, the gonococcal PilB protein encodes for both MsrA and MsrB activity associated with the repair of oxidative damage to proteins. In this work, we demonstrate that the PilB protein of Neisseria gonorrhoeae is not involved in pilus expression. Additionally, we show that wild-type N. gonorrhoeae produces two forms of this polypeptide, one of which contains a signal sequence and is secreted from the bacterial cytoplasm to the outer membrane; the other lacks a signal sequence and is cytoplasmic. Furthermore, we show that the secreted form of the PilB protein is involved in survival in the presence of oxidative damage.

The penC resistance gene was previously characterized in a FA19 penA mtrR penB gonococcal strain (PR100) as a spontaneous mutation that increased resistance to penicillin and tetracycline. We show here that antibiotic resistance mediated by penC is the result of a Glu-666 to Lys missense mutation in the pilQ gene that interferes with the formation of the SDS-resistant high-molecular-mass PilQ secretin complex, disrupts piliation, and decreases transformation frequency by 50-fold. Deletion of pilQ in PR100 confers the same level of antibiotic resistance as the penC mutation, but increased resistance was observed only in strains containing the mtrR and penB resistance determinants. Sitesaturation mutagenesis of Glu-666 revealed that only acidic or amidated amino acids at this position preserved PilQ function. Consistent with early studies suggesting the importance of cysteine residues on stability of the PilQ multimer, mutation of either of the two cysteine residues in FA19 PilQ led to a similar phenotype as penC: increased antibiotic resistance, loss of piliation, intermediate levels of transformation competence, and absence of SDS-resistant PilQ oligomers. These data show that a functional secretin complex can enhance the entry of antibiotics into the cell and suggest that the PilQ oligomer forms a pore in the outer membrane through which antibiotics diffuse into the periplasm.

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.

The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-␤-D-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitchingmotility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.

A spontaneous point mutation in pilQ (pilQ1) resulted in phenotypic suppression of a hemoglobin (Hb) receptor mutant (hpuAB mutant), allowing gonococci to grow on Hb as the sole source of iron. PilQ, formerly designated OMP-MC, is a member of the secretin family of proteins located in the outer membrane and is required for pilus biogenesis. The pilQ1 mutant also showed decreased piliation and transformation efficiency. Insertional inactivation of pilQ1 resulted in the loss of the Hb utilization phenotype and decreased entry of free heme. Despite the ability of the pilQ1 mutant to use Hb for iron acquisition and porphyrin, there was no demonstrable binding of Hb to the cell surface. The pilQ1 mutant was more sensitive to the toxic effect of free heme in growth medium and hypersensitive to the detergent Triton X-100 and multiple antibiotics. Double mutation in pilQ1 and tonB had no effect on these phenotypes, but a double pilQ1 pilT mutant showed a reduction in Hb-dependent growth and decreased sensitivity to heme and various antimicrobial agents. Insertional inactivation of wild-type pilQ also resulted in reduced entry of heme, Triton X-100, and some antibiotics. These results show that PilQ forms a channel that allows entry of heme and certain antimicrobial compounds and that a gain-of function point mutation in pilQ results in TonB-independent, PilT-dependent increase of entry.

Pilus-mediated adherence to mucosal epithelial cells is a critical step for Neisseria gonorrhoeae to establish an infection in the human host. CD46, the defined receptor for the gonococcal pilus, is a complement-regulatory protein that is expressed on all human nucleated cells. It was observed that a piliated, Opa N variant of gonococcal strain FA1090 adhered with different efficiencies to the human epithelial cell lines tested (Chang, ME180, HEC-1B and PC-3). Surprisingly, these differences in adherence levels did not correlate with levels of CD46 expressed by these cell lines. In fact, there was an inverse relationship between total surface-exposed CD46 and gonococcal adherence. Four major isoforms of CD46 are produced due to alternative RNA splicing of a surfaceexposed region and the cytoplasmic tail. The relative isoform surface expression of each cell line was determined, and each was found to express different ratios of the four CD46 isoforms. No correlation could be derived between CD46 isoform surface expression and pilus-mediated gonococcal adherence, indicating that CD46 does not act as a classic receptor for gonococcal pili.

SummaryPiv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and sitespecific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.

The physical properties of most bacterial genomes are largely unexplored. We have previously demonstrated that the strict human pathogen Neisseria gonorrhoeae is polyploid, carrying an average of three chromosome copies per cell and only maintaining one pair of replication forks per chromosome (D. M. Tobiason and H. S. Seifert, PLos Biol. 4:1069-1078, 2006). We are following up this initial report to test several predictions of the polyploidy model of gonococcal chromosome organization. We demonstrate that the N. gonorrhoeae chromosomes exist solely as monomers and not covalently linked dimers, and in agreement with the monomer status, we show that distinct nucleoid regions can be detected by electron microscopy. Two different approaches to isolate heterozygous N. gonorrhoeae resulted in the formation of merodiploids, showing that even with more than one chromosome copy, these bacteria are genetically haploid. We show that the closely related bacterium Neisseria meningitidis is also polyploid, while the commensal organism Neisseria lactamica maintains chromosomes in single copy. We conclude that the pathogenic Neisseria strains are homozygous diploids.