The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae

The pilin antigenic variation (Av) system of Neisseria gonorrhoeae (Gc) mediates unidirectional DNA recombination from silent gene copies into the pilin expression locus. A DNA sequencing assay was developed to accurately measure pilin Av in a population of Gc strain FA1090 arising from a defined pilin progenitor under non-selective culture conditions. This assay employs a piliated parental Gc variant with a recA allele whose promoter is replaced by lac-regulatory elements, allowing for controlled induction of pilin Av. From this assay, the frequency of pilin Av was measured as 0.13 recombination events per cell, with a corresponding rate of pilin Av of 4x10(-3) events per cell per generation. Most pilin variants retained the parental piliation phenotype, providing the first comprehensive analysis of piliated variants arising from a piliated progenitor. Sequence analysis of pilin variants revealed that a subset of possible recombination events predominated, which differed between piliated and non-piliated progeny. Pilin Av exhibits the highest reported frequency of any pathogenic gene conversion system and can account for the extensive pilin variation detected during human infection.     

Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair

Neisseria 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.     

The recX gene potentiates homologous recombination in Neisseria gonorrhoeae

In the pathogen Neisseria gonorrhoeae (Gc), the RecA protein is necessary for DNA repair, DNA transformation and pilus antigenic variation. Many bacteria contain a gene, recX, which has been suggested to downregulate recA through an unknown mechanism. To investigate the possible role of recX in Gc, we cloned and insertionally inactivated the recX gene. The recX loss-of-function mutant showed decreases in pilus phase variation, DNA transformation and DNA repair ability compared with wild type. We were able to complement all these deficiencies by supplying a functional copy of recX elsewhere in the chromosome. The recX mutant still showed increases in pilus phase variation under conditions of iron starvation, and the recX mutant showed levels of RecA protein equivalent to wild type. Although the precise role of recX in recombination remains unclear, RecX aids all RecA-related processes in Gc, and this is the first demonstration of a role for recX in homologous recombination in any organism.     

Nucleotide sequence of the structural gene for class I pilin from Neisseria meningitidis: homologies with the pilE locus of Neisseria gonorrhoeae

The nucleotide sequence has been determined for the expressed pilin (pilE) locus of Neisseria meningitidis strain C311 which produces class I pili that are antigenically and structurally similar to those of gonococci. The deduced amino acid sequence of the N. meningitidis pilE translation product contains a 7 amino acid N-terminal pre-pilin leader sequence which is identical to that found in gonococcal pilin and which is characteristic of N-methylphenylalanine pili in general. The succeeding N-terminal 53 amino acids are identical to those found in the equivalent position in antigenically variant gonococcal pilins and confirm direct peptide sequencing of the amino-terminus of at least one type of meningococcal pilin. Other regions that are conserved in variant pilin polypeptides from Neisseria gonorrhoeae are conserved at the amino acid level in the class I meningococcal pilin but the coding DNA contains numerous base substitutions when compared with the equivalent gonococcal pil sequence. Sequences extending downstream for about 140 bp on the 3' side of the coding region for both pilin genes are only about 85% homologous.     

Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affect Type IV pilus assembly, dynamics and associated functions

Type IV pili (Tfp) are multifunctional surface appendages expressed by many Gram negative species of medical, environmental and industrial importance. The N-terminally localized, so called alpha-helical spine is the most conserved structural feature of pilin subunits in these organelles. Prevailing models of pilus assembly and structure invariably implicate its importance to membrane trafficking, organelle structure and related functions. Nonetheless, relatively few studies have examined the effects of missense substitutions within this domain. Using Neisseria gonorrhoeae as a model system, we constructed mutants with single and multiple amino acid substitutions localized to this region of the pilin subunit PilE and characterized them with regard to pilin stability, organelle expression and associated phenotypes. The consequences of simultaneous expression of the mutant and wild-type PilE forms were also examined. The findings document for the first time in a defined genetic background the phenomenon of pilin intermolecular complementation in which assembly defective pilin can be rescued into purifiable Tfp by coexpression of wild-type PilE. The results further demonstrate that pilin subunit composition can impact on organelle dynamics mediated by the PilT retraction protein via a process that appears to monitor the efficacy of subunit-subunit interactions. In addition to confirming and extending the evidence for PilE multimerization as an essential component for competence for natural genetic transformation, this work paves the way for detailed studies of Tfp subunit-subunit interactions including self-recognition within the membrane and packing within the pilus polymer.     

Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae

Pilus fibers are long protein filaments on many pathogenic bacteria that participate in attachment to host cells. Although the self-assembling protein pilin is the major structural component of the Neisseria gonorrhoeae pilus fiber, several other proteins co-purified with pilin through the repeated solubilization-reassociation steps of the biochemical purification. Pilin solubilized in the nondenaturing detergent n-octyl-beta-D-glucopyranoside remained an aggregate of about 100 kDa at pH 9.5, but was reduced to a 40-kDa dimer at pH 10.5, suggesting that assembly involves electrostatic interactions of lysine, tyrosine, or other side chains with high pKa values. Pilin dimers and aggregates of higher molecular mass were partially stable even in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Removal of pilus-associated proteins and stabilization of pilin multimers permitted the reproducible crystallization of pilin. Three-dimensional needle- and plate-shaped crystals of purified N. gonorrhoeae pilin (strain MS11 variant C30) grew from 36 to 40% polyethylene glycol 400, pH 8.0-9.0, in space group C222, with cell dimensions a = 126.4, b = 121.2, c = 26.7 A and Vm = 2.84 A3/dalton for one molecule per asymmetric unit. The best crystals diffracted to 2.4 A resolution using synchrotron radiation, were stable to x-ray damage, and appear suitable for determination of the atomic structure. This approach of stabilizing and crystallizing an intermediate assembly state may be useful for other fiber-forming proteins, which have previously not been successfully crystallized in forms that diffract to atomic resolution.     

The repertoire of silent pilus genes in Neisseria gonorrhoeae: evidence for gene conversion

To investigate the significance of silent gene loci for pilus antigenic variation in N. gonorrhoeae, we determined the nucleotide sequence of the major silent locus, pilS1. The pilS1 locus contains six tandem pilus gene copies linked by a 39 bp repeat sequence also present in the expression loci. All silent copies lack the common N-terminal coding sequence of pilin, containing instead variant sequence information that constitutes a semivariable (SV) and a hypervariable (HV) domain. The SV and HV domains are interspersed with short, strictly conserved (C) regions flanking small cassettes of variable sequence information. It appears that such minicassettes from silent copies can be duplicated and transferred to other silent or expression genes by means of gene conversion.     

A genetic screen identifies genes and sites involved in pilin antigenic variation in Neisseria gonorrhoeae

It has previously been shown that the frequency of pilin antigenic variation in Neisseria gonorrhoeae (the gonococcus, Gc) is regulated by iron availability. To identify factors involved in pilin variation in an iron-dependent or an iron-independent manner, we conducted a genetic screen of transposon-mutated gonococci using a pilus-dependent colony morphology phenotype to detect antigenic variation deficient mutants. Forty-six total mutants representing insertions in 30 different genes were shown to have reduced colony morphology changes resulting from impaired pilin variation. Five mutants exhibited an iron-dependent decrease in pilin variation, while the remaining 41 displayed an iron-independent decrease in pilin variation. Based on the levels of antigenic variation impairment, we defined the genes as being essential for, important for, or involved in antigenic variation. DNA repair and DNA transformation frequencies of each mutant were measured to determine whether other recombination-based processes were also affected in the mutants. Each mutant was placed into one of six classes based on their pilin variation, DNA repair and DNA transformation phenotypes. Among the many genes identified, recR is shown to be an additional member of the gonococcal RecF-like recombination pathway. In addition, recG and ruvA represent the first evidence that the processing of Holliday junctions is required for pilin antigenic variation. Moreover, two independent insertions in a non-coding region upstream of the pilE gene suggest that cis-acting sequences important for pilin variation are found in that region. Finally, insertions that effect expression of the thrB and thrC genes suggest that molecules in the threonine biosynthetic pathway are important for pilin variation. Many of the other genes identified in this genetic screen do not have an obvious role in pilin variation, DNA repair, or DNA transformation.     

Inversion of Moraxella lacunata type 4 pilin gene sequences by a Neisseria gonorrhoeae site-specific recombinase.

A plasmid library of Neisseria gonorrhoeae sequences was screened for the ability to mediate recombinations on a sequence containing the Moraxella lacunata type 4 pilin gene invertible region in Escherichia coli. A plasmid containing the N. gonorrhoeae sequence encoding the putative recombinase (gcr) was identified and sequenced. Plasmids containing gcr were able to mediate site-specific recombinations despite a weak amino acid homology to Piv, the native M. lacunata pilin gene invertase. The gcr gene is present only in pathogenic strains of Neisseria tested; however, in our assays gene knockouts of gcr did not alter the variation of surface features that play a role in the pathogenesis of N. gonorrhoeae.     

Role of the Rep helicase gene in homologous recombination in Neisseria gonorrhoeae

In Escherichia coli, the Rep helicase has been implicated in replication fork progression, replication restart, homologous recombination, and DNA repair. We show that a Neisseria gonorrhoeae rep mutant is deficient in the homologous-recombination-mediated processes of DNA transformation and pilus-based colony variation but not in DNA repair.     

The fbpABC locus of Neisseria gonorrhoeae functions in the periplasm-to-cytosol transport of iron.

We have determined that the DNA sequence downstream of the well-characterized gonococcal fbp gene contains two open reading frames: one designated fbpB, which encodes a protein proposed to function as a cytoplasmic permease, and one designated fbpC, which encodes a protein proposed to function as a nucleotide-binding protein. The fpbABC operon composes an iron transport system that is homologous to the sfu and hit operons previously reported for Serratia marcescens and Haemophilus influenzae, respectively, and displays elements characteristic of ATP binding cassette transporters. The fpbABC operon differs from these loci in that it is lethal when overexpressed in Escherichia coli.     

RecQ DNA helicase HRDC domains are critical determinants in Neisseria gonorrhoeae pilin antigenic variation and DNA repair

Neisseria gonorrhoeae (Gc), an obligate human bacterial pathogen, utilizes pilin antigenic variation to evade host immune defences. Antigenic variation is driven by recombination between expressed ( pilE ) and silent ( pilS ) copies of the pilin gene, which encodes the major structural component of the type IV pilus. We have investigated the role of the GcRecQ DNA helicase (GcRecQ) in this process. Whereas the vast majority of bacterial RecQ proteins encode a single 'Helicase and RNase D C-terminal' (HRDC) domain, GcRecQ encodes three tandem HRDC domains at its C-terminus. Gc mutants encoding versions of GcRecQ with either two or all three C-terminal HRDC domains removed are deficient in pilin variation and sensitized to UV light-induced DNA damage. Biochemical analysis of a GcRecQ protein variant lacking two HRDC domains, GcRecQΔHRDC2,3, shows it has decreased affinity for single-stranded and partial-duplex DNA and reduced unwinding activity on a synthetic Holliday junction substrate relative to full-length GcRecQ in the presence of Gc single-stranded DNA-binding protein (GcSSB). Our results demonstrate that the multiple HRDC domain architecture in GcRecQ is critical for structure-specific DNA binding and unwinding, and suggest that these features are central to GcRecQ's roles in Gc antigenic variation and DNA repair.     

DNA circle formation in Neisseria gonorrhoeae: a possible intermediate in diverse genomic recombination processes

An important attribute that contributes to the virulence of Neisseria gonorrhoeae is its phenotypic variability, which is based on recombination within complex gene families in the genome. In this study we report on the in vivo amplification of large segments of the genome and the existence of circular DNA intermediates in the cell, which might help to explain the evolution of these gene families and provide possible clues as to how genetic variability is maintained. Using an inserted chromosomal marker (cat) in the N. gonorrhoeae MS11 genome that confers low-level resistance to chloramphenicol (Cm), we isolated variants that express resistance to high levels of Cm. Genetic analysis revealed that virtually all variants harboured single or multiple tandem amplifications of the respective genome segments carrying the cat insert. This process occurred independently of both the location of the cat insertion site and of the presence of a functional recA gene. Analysis of the genetically well characterised pilC region revealed a head-to-tail orientation of the amplified segments, with the junctions being located within direct repeats. Identical junctions were detected in extra-chromosomal circular DNA molecules isolated from non-selected wild-type and recA strains, suggesting that both types of structure arise by related processes. The existence of DNA circles was shown by their banding behaviour in caesium chloride/ethidium bromide density centrifugation and their resistance to digestion by exonuclease. The possible roles of such circles in processes such as pilin gene recombination, chromosomal gene amplification and genetic transformation are discussed.     

Allelic polymorphism and site-specific recombination in the opc locus of Neisseria meningitidis

The opc gene is widespread in epidemic and endemic Neisseria meningitidis , but most strains of certain epidemic clones (ET-37 complex, Cluster A4) and a few random endemic isolates lack an opc gene. Four percent of the 1148 bp that contain opc plus the surrounding intergenic region was polymorphic (18 alleles), and many of the alleles contained a 230 bp insertion at a fixed location in the intergenic region. The presence or absence of the insertion reflects site-specific recombination. The alleles are stably inherited within clonal groupings for up to at least 50 years, with rare cases of horizontal genetic exchange. Most statistical methods indicated significant intragenic recombination events within this dataset.     

Intragenic variation by site-specific recombination in the cryptic plasmid of Neisseria gonorrhoeae.

Cryptic plasmid DNA of Neisseria gonorrhoeae was found integrated into the gonococcal chromosome in both plasmid-bearing strains and plasmid-free strains. At several chromosomal locations only segments of the plasmid were found. However, in at least two strains an intact copy of the plasmid seemed to be present with the joints between the plasmid and the chromosomal DNA being located within the cppB gene of the cryptic plasmid. The cppB gene was shown to undergo a sequence-specific intragenic deletion. The deletion removed 54 base pairs, representing 18 amino acids, and did not affect the reading frame. It is proposed that the cryptic plasmid integrates into the chromosome and other gonococcal plasmids within this site-specific deletion region. Models for the site-specific recombination are presented.     

The opaH locus of Neisseria gonorrhoeae MS11A is involved in epithelial cell invasion

In order to produce a successful infection, Neisseria gonorrhoeae (GC) must attach to and invade mucosal epithelial cells. To identify GC gene products involved in this early interaction with host cells we constructed a gene bank derived from a clinical isolate of GC, and isolated a clone which had the capacity to adhere to the human endometrial adenocarcinoma tissue-culture line HEC-1-B. The cloned sequence was identified as a member of the opa gene family whose protein products have been associated with virulence. The GC chromosome contains numerous variant opa genes which, in MS11, are designated opaA-K. Previous work showed that expression of opaC confers a highly invasive phenotype upon strain MS11. When our cloned opa gene was mutated and returned to the GC MS11A chromosome by transformation and homologous recombination, we isolated one transformation that was significantly reduced in its invasive capacity. The locus mutated in this transformant was identified as opaH. Our resuits indicate that invasive-ness of GC for human epithelail cells can be determined by more than one opa gene in strain MS11 A.     

Homologous and non-homologous recombination differentially affect DNA damage repair in mice

Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the ability of cells to repair the inflicted DNA damage. Here we demonstrate that homologous recombination-deficient mRAD54(-/-) mice are hypersensitive to ionizing radiation at the embryonic but, unexpectedly, not at the adult stage. However, at the adult stage mRAD54 deficiency dramatically aggravates the ionizing radiation sensitivity of severe combined immune deficiency (scid) mice that are impaired in DNA double-strand break repair through DNA end-joining. In contrast, regardless of developmental stage, mRAD54(-/-) mice are hypersensitive to the interstrand DNA crosslinking compound mitomycin C. These results demonstrate that the two major DNA double-strand break repair pathways in mammals have overlapping as well as specialized roles, and that the relative contribution of these pathways towards repair of ionizing radiation-induced DNA damage changes during development of the animal.     

The epidemiology of Neisseria gonorrhoeae in Europe

This review addresses the occurrence, the trends, and the risk groups of Neisseria gonorrhoeae in Europe over the past decade. A decline has been observed in most of Europe since the 1980s, except for an increase in the Baltic countries (early 1990s) and an increase among men who have sex with men (between 1989 and 1991), and heterosexuals in some countries (between 1995 and 1997). Despite the overall fall in the incidence of gonorrhoea, plasmid-mediated resistance to penicillin and tetracycline increased in Europe. More recently, resistance to fluoroquinolones has been documented, mainly imported from Southeast Asia. Until now, no resistance to third-generation cephalosporins has been observed.     

Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics

Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilA(PAK) pilin subunit in N. gonorrhoeae. We show here that, although PilA(PAK) pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilA(PAK) pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilA(PAK) pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilA(PAK) pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilA(PAK) pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.