Ring structure of the Escherichia coli DNA-binding protein RdgC associated with recombination and replication fork repair

The DNA-binding protein, RdgC, is associated with recombination and replication fork repair in Escherichia coli and with the virulence-associated, pilin antigenic variation mediated by RecA and other recombination proteins in Neisseria species. We solved the structure of the E. coli protein and refined it to 2.4A. RdgC crystallizes as a dimer with a head-to-head, tail-to-tail organization forming a ring with a 30 A diameter hole at the center. The protein fold is unique and reminiscent of a horseshoe with twin gates closing the open end. The central hole is lined with positively charged residues and provides a highly plausible DNA binding channel consistent with the nonspecific mode of binding detected in vitro and with the ability of RdgC to modulate RecA function in vivo.     

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.     

Mutation of the priA gene of Neisseria gonorrhoeae affects DNA transformation and DNA repair

In Escherichia coli, PriA is central to the restart of chromosomal replication when replication fork progression is disrupted and is also involved in homologous recombination and DNA repair. To investigate the role of PriA in recombination and repair in Neisseria gonorrhoeae, we identified, cloned, and insertionally inactivated the gonococcal priA homologue. The priA mutant showed a growth deficiency and decreased DNA repair capability and was completely for deficient in DNA transformation compared to the isogenic parental strain. The priA mutant was also more sensitive to the oxidative damaging agents H2O2 and cumene hydroperoxide compared to the parental strain. These phenotypes were complemented by supplying a functional copy of priA elsewhere in the chromosome. The N. gonorrhoeae priA mutant showed no alteration in the frequency of pilin antigenic variation. We conclude that PriA participates in DNA repair and DNA transformation processes but not in pilin antigenic variation.     

Insertion Mutations in pilE Differentially Alter Gonococcal Pilin Antigenic Variation

Pilus antigenic variation in Neisseria gonorrhoeae occurs by the high-frequency, unidirectional transfer of DNA sequences from one of several silent pilin loci (pilS) into the expressed pilin gene (pilE), resulting in a change in the primary pilin protein sequence. Previously, we investigated the effects of large or small heterologous insertions in conserved and variable portions of a pilS copy on antigenic variation. We observed differential effects on pilin recombination by the various insertions, and the severity of the defect correlated with the disruption or displacement of a conserved pilin DNA sequence called cys2. In this study, we show that disruption or displacement of the pilE cys2 sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in pilS, the analogous insertions in pilE impaired, but did not block, recombination of the flanking pilin sequences. These results, the change in the spectrum of donor silent copies used during variation, and our previous results with pilS mutations show that the donor pilS and recipient pilE play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.     

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.     

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.     

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.     

The RdgC protein employs a novel mechanism involving a finger domain to bind to circular DNA

The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.     

Purification and characterization of the RecA protein from Neisseria gonorrhoeae

The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.     

Genetic analysis of variant pilin genes from Neisseria gonorrhoeae P9 cloned in Escherichia coli: physical and immunological properties of encoded pilins

A series of genomic DNA fragments that encode gonococcal pilins from four well-characterized pilus variants of Neisseria gonorrhoeae strain P9 have been cloned in Escherichia coli K12. At least nine classes of cloned P9 pilin genes have been identified on the basis of restriction mapping of cloned pilin-encoding DNA and physical and immunological analysis of expressed pilin proteins. Each antigenic variant of strain P9 possesses many genomic segments of pilin gene information, although our results suggest that strain P9 contains only a single pilin-expressing (pilE) locus.     

Molecular models accounting for the gene conversion reactions mediating gonococcal pilin antigenic variation

The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high-frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high-frequency gene conversions that mediate pilin Av.     

Analysis of protein binding to the Sma/Cla DNA repeat in pathogenic Neisseriae.

Antigenic variation of the pilus is an essential component of Neisseria gonorrhoeae pathogenesis. Unidirectional recombination of silent pilin DNA into an expressed pilin gene allows for substantial sequence variation of this highly immunogenic surface structure. While the RecA protein is required for pilin gene recombination, the factors which maintain the silent reservoir of pilin sequences and/or allow unidirectional recombination from silent to expression loci remain undefined. We have previously shown that a conserved sequence at the 3'end of all pilin loci (the Sma/Cla repeat) is required to be present at the expression locus for efficient recombination from the silent loci. In this study, the binding of gonococcal proteins to this DNA sequence was investigated. Gel mobility shift assays and competition experiments using deletion derivatives of the repeat, show that multiple activities bind to different regions of the Sma/Cla repeat and define the boundaries of the binding sequences. Moreover, only the pathogenic Neisseria harbor proteins which specifically bind to this repeat, suggesting a correlation between the expression of these DNA binding proteins and the potential to cause disease.     

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.     

Inhibition of RecA protein function by the RdgC protein from Escherichia coli

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.     

Recombination, repair and replication in the pathogenic Neisseriae: the 3 R's of molecular genetics of two human-specific bacterial pathogens

Most of the detailed mechanisms that have been established for the molecular biological processes that mediate recombination, repair and replication of DNA have come from studies of the Escherichia coli paradigm. The human specific pathogens, Neisseria gonorrhoeae and N. meningitidis, are Gram-negative bacteria that have some molecular processes that are similar to E. coli and others that appear to be divergent. We propose that the pathogenic Neisseriae have evolved a specialized collection of molecular mechanisms to adapt to life limited to human hosts. In this MicroReview, we explore what is known about the basic processes of DNA repair, DNA recombination (genetic exchange and pilin variation) and DNA replication in these human specific pathogens.     

The Use of High-Throughput DNA Sequencing in the Investigation of Antigenic Variation: Application to Neisseria Species

Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3′ end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.