Force production by type IV pilus retraction is critical for infectivity of Neisseria gonorrhoeae and DNA transfer. We investigated the roles of pilus number and the retraction motor, PilT, in force generation in vivo at the single-molecule level and found that individual retraction events are generated by a single pilus fiber, and only one PilT complex powers retraction. Retraction velocity is constant at low forces but decreases at forces greater than 40 pN, giving a remarkably high average stall force of 110 ± 30 pN. Further insights into the molecular mechanism of force generation are gained from the effect of ATP-depletion, which reduces the rate of retraction but not the stall force. Energetic considerations suggest that more than one ATP is involved in the removal of a single pilin subunit from a pilus. The results are most consistent with a model in which the ATPase PilT forms an oligomer that disassembles the pilus by a cooperative conformational change.

Pathogens can utilize DNA recombination to promote antigenic variation of surface structures to avoid immune detection. We identified a cis-acting DNA sequence near the antigenically variable pilin locus of the human pathogen, Neisseria gonorrhoeae. This 16 base pair G-rich sequence was required for pilin antigenic variation and formed a guanine quartet (G4) structure in vitro. Individual mutations that disrupted the structure also blocked pilin antigenic variation and prevented nicks required for recombination from occurring within the G4 region. A compound that binds and stabilizes G4 structures also inhibited pilin antigenic variation and prevented nicks from occurring on the G-rich strand. This site constitutes a recombination initiation sequence/structure that directs gene conversion to a specific chromosomal locus.DNA recombination is a process shared by all DNA carrying organisms that is used for a variety of cellular processes including DNA repair, genetic exchange, and meiotic chromosome segregation (1). Additionally, recombination mediates many high frequency gene diversification systems including yeast mating type switches, immunoglobin diversity and pathogenesis-associated antigenic variation (Av) (2-4). Most recombination reactions occur at low frequency, but several diversity generating systems can enact programmed recombination reactions between specific loci at relatively high frequencies (2,3,5,6).Neisseria gonorrhoeae is the sole causative agent of gonorrhea and has evolved three highfrequency, diversity-generation systems to avoid immune surveillance (7). This antigenic variability of gonococcal populations is one reason that natural immunity to re-infection has never been demonstrated and has prevented development of an effective vaccine. One of these Av systems is mediated by high-frequency gene conversion events between one of many silent pilin loci and the single expressed pilin locus, pilE. This produces variant pilin proteins (2) that form antigenically divergent pili, which are the hair-like appendages expressed by many bacteria.This system uses normal homologous recombination factors to mediate specialized gene conversion reactions (8-12), but no DNA element required for pilin Av has been described. Previous work showed that some transposon insertions in the intergenic region upstream of pilE block pilin Av without altering pilin expression (11,13). To define the DNA element being disrupted by these transposons, the pilE upstream region was randomly mutagenized, and the mutations were introduced into the gonococcal chromosome by DNA transformation and linkage to a transposon insertion that does not affect pilin Av (Fig. S1A) (13). Two independent screens were performed. In both screens, mutants unable to undergo pilin Av were selected by screening for a stable, piliated colony morphology (Avd phenotype, Fig. S1B NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript second screen both Av deficient (Avd) and Av transformants were analyzed (Table S1) (14). DNA...

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation‐like secretion system. Consistent with this hypothesis, a wild‐type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.

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SummaryNeisseria gonorrhoeae (Gc) pili undergo antigenic variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin antigenic variation occurs by RecA-dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high-frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin antigenic and phase variation and DNA repair but are not involved in natural DNA transformation. This suggests that a RecF-like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA transformation and DNA repair but not in pilin antigenic variation.

CRISPR interference confers adaptive, sequence-based immunity against viruses and plasmids and is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from spacer-repeat units. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here, our studies of crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp. reveal a unique crRNA maturation pathway in which crRNAs are transcribed from promoters that are embedded within each repeat, yielding crRNA 5’ ends formed by transcription and not by processing. Although crRNA 3’ end formation involves RNase III and trans-encoded tracrRNA, as in other Type II CRISPR systems, this processing is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/cas system characterized to date. Endogenous CRISPR spacers limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in the human pathogen N. meningitidis.

SummarySymptomatic gonococcal infection, caused by the pathogen Neisseria gonorrhoeae (Gc), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess several mechanisms of oxidative killing, intact Gc can be found associated with PMNs, suggesting that gonococcal defences against oxidative stress are crucial for its ability to evade killing by PMNs. We used microarrays to identify genes that were differentially expressed after transient exposure of Gc to hydrogen peroxide (H 2 O 2 ). Of the 75 genes found to be upregulated after H 2 O 2 treatment, over one-quarter, including two of the most highly upregulated genes (NGO1686 and NGO554), were predicted to encode proteins with unknown functions. Further characterization of a subset of these upregulated genes demonstrated that NGO1686, a putative zinc metalloprotease, protects against oxidative damage caused by both H 2 O 2 and cumene hydroperoxide, and that NGO554, a Gc-specific protein, acts to protect against damage caused by high levels of H 2 O 2 . Our current study also ascribes a role in H 2 O 2 damage protection to recN , a gene previously characterized for its role in DNA repair. A PMN survival assay demonstrated that the recN and NGO1686 mutants were more susceptible to killing than the parent strain FA1090. These results define for the first time the robust transcriptional response to H 2 O 2 by this strict human pathogen and underscore the importance of this system for survival to host defences.

SummarySymptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40-70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase-and RecA-deficient Gc, which are more sensitive to ROS in vitro, had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.