Effects of recA Mutations on Pilus Antigenic Variation and Phase Transitions in Neisseria gonorrhoeae

Genetics ◽  
1987 ◽  
Vol 117 (3) ◽  
pp. 391-398
Author(s):  
Michael Koomey ◽  
Emil C Gotschlich ◽  
Ken Robbins ◽  
Sven Bergström ◽  
John Swanson
Keyword(s):  
Homologous Recombination ◽  
Neisseria Gonorrhoeae ◽  
Genetic Basis ◽  
Antigenic Variation ◽  
Phase Variation ◽  
Intragenic Recombination ◽  
Gene Copies ◽  
Reduced Rate ◽  
Pilin Gene ◽  
Isogenic Strains

ABSTRACT Intragenic recombination between the single complete pilin gene (expression locus) and multiple, distinct, partial pilin gene copies (silent, storage loci) is thought to account for the generation of pilus antigenic diversity and piliation phase (on-off) changes exhibited by Neisseria gonorrhoeae. The mechanisms operating in the genomic rearrangements associated with these forms of pilus variation were investigated through the study of isogenic strains of gonococci bearing either wild-type or altered recA alleles. Examination of the rates of pilus phase variation and the genetic basis for changes in piliation status displayed by these strains show that recA mediated homologous recombination is required for these high frequency events and confirm that the nonpiliated state results from mutations in the expressed pilin gene. In a strain that is deficient in recA mediated homologous recombination, pilus phase variation occurs at a 100-1000-fold reduced rate and results predominantly from one class of spontaneous frameshift mutations within the pilin structural gene.

scholarly journals Neisseria gonorrhoeae MutS Affects Pilin Antigenic Variation through Mismatch Correction and Not bypilEGuanine Quartet Binding

2015 ◽  
Vol 197 (10) ◽  
pp. 1828-1838 ◽  
Author(s):  
Ella Rotman ◽  
H. Steven Seifert
Keyword(s):  
Homologous Recombination ◽  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Synthetic Lethality ◽  
Phase Variation ◽  
Surface Proteins ◽  
Loss Of Function ◽  
Content Type ◽  
Mismatch Correction ◽  
Guanine Quartet

ABSTRACTMany pathogens use homologous recombination to vary surface antigens to avoid immune surveillance.Neisseria gonorrhoeaeachieves this in part by changing the properties of its surface pili in a process called pilin antigenic variation (AV). Pilin AV occurs by high-frequency gene conversion reactions that transfer silentpilSsequences into the expressedpilElocus and requires the formation of an upstream guanine quartet (G4) DNA structure to initiate this process. The MutS and MutL proteins of the mismatch correction (MMC) system act to correct mismatches after replication and prevent homeologous (i.e., partially homologous) recombination, but MutS orthologs can also bind to G4 structures. A previous study showed that mutation of MutS resulted in a 3-fold increase in pilin AV, which could be due to the loss of MutS antirecombination properties or loss of G4 binding. We tested two site-directed separation-of-function MutS mutants that are both predicted to bind to G4s but are not able to perform MMC. Pilus phase variation assays and DNA sequence analysis ofpilEvariants produced in these mutants showed that all threemutSmutants and amutLmutant had similar increased frequencies of pilin AV. Moreover, themutSmutants all showed similar increased levels of pilin AV-dependent synthetic lethality. These results show that antirecombination by MMC is the reason for the effect that MutS has on pilin AV and is not due topilEG4 binding by MutS.IMPORTANCENeisseria gonorrhoeaecontinually changes its outer surface proteins to avoid recognition by the immune system.N. gonorrhoeaealters the antigenicity of the pilus by directed recombination between partially homologous pilin copies in a process that requires a guanine quartet (G4) structure. The MutS protein of the mismatch correction (MMC) system prevents recombination between partially homologous sequences and can also bind to G4s. We confirmed that loss of MMC increases the frequency of pilin antigenic variation and that two MutS mutants that are predicted to separate the two different functions of MutS inhibit pilin variation similarly to a complete-loss-of-function mutant, suggesting that interaction of MutS with the G4 structure is not a major factor in this process.

scholarly journals Analysis of the Piv Recombinase-Related Gene Family of Neisseria gonorrhoeae

2005 ◽  
Vol 187 (4) ◽  
pp. 1276-1286 ◽  
Author(s):  
Eric P. Skaar ◽  
Brian LeCuyer ◽  
Anne G. Lenich ◽  
Matthew P. Lazio ◽  
Donna Perkins-Balding ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Neisseria Gonorrhoeae ◽  
Gene Family ◽  
High Frequency ◽  
Antigenic Variation ◽  
Gene Copy ◽  
Related Gene ◽  
Multiple Loci ◽  
Gene Copies ◽  
Insertion Elements

ABSTRACT 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.

scholarly journals A Double-Strand Break Does Not PromoteNeisseria gonorrhoeaePilin Antigenic Variation

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Lauren L. Prister ◽  
Jing Xu ◽  
H Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Dna Structure ◽  
Strand Break ◽  
Double Strand Break ◽  
Content Type ◽  
G4 Dna ◽  
Recombination System ◽  
Guanine Quadruplex ◽  
Pilin Gene

ABSTRACTThe major subunit of the type IV pilus (T4p) ofNeisseria gonorrhoeaeundergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of thepilEG4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entirepilEgene accumulated. Thus, thepilEG4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system.IMPORTANCENeisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.

scholarly journals Transposon Mutagenesis Identifies Sites Upstream of the Neisseria gonorrhoeae pilE Gene That Modulate Pilin Antigenic Variation

2007 ◽  
Vol 189 (9) ◽  
pp. 3462-3470 ◽  
Author(s):  
Kimberly A. Kline ◽  
Alison K. Criss ◽  
Anne Wallace ◽  
H. Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Gene Conversion ◽  
Dna Sequences ◽  
Antigenic Variation ◽  
Transposon Mutagenesis ◽  
Repeat Sequence ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Type Iv ◽  
Pilin Gene

ABSTRACT Gene conversion mediates the variation of virulence-associated surface structures on pathogenic microorganisms, which prevents host humoral immune responses from being effective. One of the best-studied gene conversion systems is antigenic variation (Av) of the pilin subunit of the Neisseria gonorrhoeae type IV pilus. To identify cis-acting DNA sequences that facilitate Av, the 700-bp region upstream of the pilin gene pilE was targeted for transposon mutagenesis. Four classes of transposon-associated mutations were isolated, distinguishable by their pilus-associated phenotypes: (i) insertions that did not alter Av or piliation, (ii) insertions that blocked Av, (iii) insertions that interfered with Av, and (iv) insertions that interfered with pilus expression and Av. Mutagenesis of the pilE promoter did not affect the frequency of Av, directly demonstrating that pilin Av is independent of pilE transcription. Two stretches of sequence upstream of pilE were devoid of transposon insertions, and some deletions in these regions were not recoverable, suggesting that they are essential for gonococcal viability. Insertions that blocked pilin Av were located downstream of the RS1 repeat sequence, and deletion of the region surrounding these insertions completely abrogated pilin Av, confirming that specific sequences 5′ to pilE are essential for the recombination events underlying pilin Av.

scholarly journals Insertion Mutations in pilEDifferentially Alter Gonococcal Pilin Antigenic Variation

1999 ◽  
Vol 181 (19) ◽  
pp. 6133-6141 ◽  
Author(s):  
Becky Howell-Adams ◽  
H. Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Protein Sequence ◽  
High Frequency ◽  
Antigenic Variation ◽  
Pilin Gene ◽  
Pilin Protein ◽  
Unidirectional Transfer ◽  
Insertion Mutations

ABSTRACT Pilus antigenic variation in Neisseria gonorrhoeaeoccurs 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 thepilE cys2 sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in pilS, the analogous insertions inpilE 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 pilSand recipient pilE play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.

scholarly journals Genetic basis of Neisseria gonorrhoeae lipooligosaccharide antigenic variation.

1995 ◽  
Vol 177 (24) ◽  
pp. 7275-7279 ◽  
Author(s):  
R J Danaher ◽  
J C Levin ◽  
D Arking ◽  
C L Burch ◽  
R Sandlin ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Genetic Basis ◽  
Antigenic Variation

Intragenic recombination leads to pilus antigenic variation in Neisseria gonorrhoeae

Nature ◽  
1985 ◽  
Vol 315 (6015) ◽  
pp. 156-158 ◽  
Author(s):  
Per Hagblom ◽  
Ellyn Segal ◽  
Elizabeth Billyard ◽  
Magdalene So
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Intragenic Recombination

scholarly journals Antigenic Variation in Neisseria gonorrhoeae Occurs Independently of RecQ-Mediated Unwinding of the pilE G Quadruplex

2019 ◽  
Vol 202 (3) ◽  
Author(s):  
Andrew F. Voter ◽  
Melanie M. Callaghan ◽  
Ramreddy Tippana ◽  
Sua Myong ◽  
Joseph P. Dillard ◽  
...  
Keyword(s):  
Immune System ◽  
Homologous Recombination ◽  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Dna Unwinding ◽  
Duplex Dna ◽  
Recq Helicase ◽  
Content Type ◽  
G4 Dna ◽  
Guanine Quadruplex

ABSTRACT The obligate human pathogen Neisseria gonorrhoeae alters its cell surface antigens to evade the immune system in a process known as antigenic variation (AV). During pilin AV, portions of the expressed pilin gene (pilE) are replaced with segments of silent pilin genes (pilS) through homologous recombination. The pilE-pilS exchange is initiated by formation of a parallel guanine quadruplex (G4) structure near the pilE gene, which recruits the homologous recombination machinery. The RecQ helicase, which has been proposed to aid AV by unwinding the pilE G4 structure, is an important component of this machinery. However, RecQ also promotes homologous recombination through G4-independent duplex DNA unwinding, leaving the relative importance of its G4 unwinding activity unclear. Previous investigations revealed a guanine-specific pocket (GSP) on the surface of RecQ that is required for G4, but not duplex, DNA unwinding. To determine whether RecQ-mediated G4 resolution is required for AV, N. gonorrhoeae strains that encode a RecQ GSP variant that cannot unwind G4 DNA were created. In contrast to the hypothesis that G4 unwinding by RecQ is important for AV, the RecQ GSP variant N. gonorrhoeae strains had normal AV levels. Analysis of a purified RecQ GSP variant confirmed that it retained duplex DNA unwinding activity but had lost its ability to unwind antiparallel G4 DNA. Interestingly, neither the GSP-deficient RecQ variant nor the wild-type RecQ could unwind the parallel pilE G4 nor the prototypical c-myc G4. Based on these results, we conclude that N. gonorrhoeae AV occurs independently of RecQ-mediated pilE G4 resolution. IMPORTANCE The pathogenic bacteria Neisseria gonorrhoeae avoids clearance by the immune system through antigenic variation (AV), the process by which immunogenic surface features of the bacteria are exchanged for novel variants. RecQ helicase is critical in AV and its role has been proposed to stem from its ability to unwind a DNA secondary structure known as a guanine quadruplex (G4) that is central to AV. In this work, we demonstrate that the role of RecQ in AV is independent of its ability to resolve G4s and that RecQ is incapable of unwinding the G4 in question. We propose a new model of RecQ’s role in AV where the G4 might recruit or orient RecQ to facilitate homologous recombination.

scholarly journals Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

2016 ◽  
Vol 198 (18) ◽  
pp. 2470-2482 ◽  
Author(s):  
Ella Rotman ◽  
David M. Webber ◽  
H. Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Biological Diversity ◽  
Antigenic Variation ◽  
Immune Surveillance ◽  
454 Sequencing ◽  
Phase Variation ◽  
Transmitted Infection ◽  
Major Barrier ◽  
Content Type ◽  
Type Iv

ABSTRACTMany pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance.Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). TheN. gonorrhoeaechromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transferpilSsequences into thepilElocus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity.IMPORTANCEMeasuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation.

scholarly journals Mismatch Correction Modulates Mutation Frequency and Pilus Phase and Antigenic Variation in Neisseria gonorrhoeae

2009 ◽  
Vol 192 (1) ◽  
pp. 316-325 ◽  
Author(s):  
Alison K. Criss ◽  
Kevin M. Bonney ◽  
Rhoda A. Chang ◽  
Paul M. Duffin ◽  
Brian E. LeCuyer ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Phase Variation ◽  
Single Nucleotide ◽  
Mismatch Correction ◽  
Nucleotide Mismatch ◽  
Specific Pathogen ◽  
Dna Mismatches ◽  
Nucleotide Insertions ◽  
Human Specific

ABSTRACT The mismatch correction (MMC) system repairs DNA mismatches and single nucleotide insertions or deletions postreplication. To test the functions of MMC in the obligate human pathogen Neisseria gonorrhoeae, homologues of the core MMC genes mutS and mutL were inactivated in strain FA1090. No mutH homologue was found in the FA1090 genome, suggesting that gonococcal MMC is not methyl directed. MMC mutants were compared to a mutant in uvrD, the helicase that functions with MMC in Escherichia coli. Inactivation of MMC or uvrD increased spontaneous resistance to rifampin and nalidixic acid, and MMC/uvrD double mutants exhibited higher mutation frequencies than any single mutant. Loss of MMC marginally enhanced the transformation efficiency of DNA carrying a single nucleotide mismatch but not that of DNA with a 1-kb insertion. Unlike the exquisite UV sensitivity of the uvrD mutant, inactivating MMC did not affect survival after UV irradiation. MMC and uvrD mutants exhibited increased PilC-dependent pilus phase variation. mutS-deficient gonococci underwent an increased frequency of pilin antigenic variation, whereas uvrD had no effect. Recombination tracts in the mutS pilin variants were longer than in parental gonococci but utilized the same donor pilS loci. These results show that gonococcal MMC repairs mismatches and small insertion/deletions in DNA and also affects the recombination events underlying pilin antigenic variation. The differential effects of MMC and uvrD in gonococci unexpectedly reveal that MMC can function independently of uvrD in this human-specific pathogen.

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