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 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 Guanine Quadruplex DNA Regulates Gamma Radiation Response of Genome Functions in the Radioresistant Bacterium Deinococcus radiodurans

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Shruti Mishra ◽  
Reema Chaudhary ◽  
Sudhir Singh ◽  
Swathi Kota ◽  
Hari S. Misra
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Deinococcus Radiodurans ◽  
Dna Structure ◽  
Protein Translation ◽  
Content Type ◽  
Cellular Processes ◽  
G4 Dna ◽  
Guanine Quadruplex ◽  
Damage Response

ABSTRACT Guanine quadruplex (G4) DNA/RNA are secondary structures that regulate the various cellular processes in both eukaryotes and bacteria. Deinococcus radiodurans, a Gram-positive bacterium known for its extraordinary radioresistance, shows a genomewide occurrence of putative G4 DNA-forming motifs in its GC-rich genome. N-Methyl mesoporphyrin (NMM), a G4 DNA structure-stabilizing drug, did not affect bacterial growth under normal conditions but inhibited the postirradiation recovery of gamma-irradiated cells. Transcriptome sequencing analysis of cells treated with both radiation and NMM showed repression of gamma radiation-responsive gene expression, which was observed in the absence of NMM. Notably, this effect of NMM on the expression of housekeeping genes involved in other cellular processes was not observed. Stabilization of G4 DNA structures mapped at the upstream of recA and in the encoding region of DR_2199 had negatively affected promoter activity in vivo, DNA synthesis in vitro and protein translation in Escherichia coli host. These results suggested that G4 DNA plays an important role in DNA damage response and in the regulation of expression of the DNA repair proteins required for radioresistance in D. radiodurans. IMPORTANCE Deinococcus radiodurans can recover from extensive DNA damage caused by many genotoxic agents. It lacks LexA/RecA-mediated canonical SOS response. Therefore, the molecular mechanisms underlying the regulation of DNA damage response would be worth investigating in this bacterium. D. radiodurans genome is GC-rich and contains numerous islands of putative guanine quadruplex (G4) DNA structure-forming motifs. Here, we showed that in vivo stabilization of G4 DNA structures can impair DNA damage response processes in D. radiodurans. Essential cellular processes such as transcription, DNA synthesis, and protein translation, which are also an integral part of the double-strand DNA break repair pathway, are affected by the arrest of G4 DNA structure dynamics. Thus, the role of DNA secondary structures in DNA damage response and radioresistance is demonstrated.

scholarly journals PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Egon A. Ozer ◽  
Lauren L. Prister ◽  
Shaohui Yin ◽  
Billy H. Ward ◽  
Stanimir Ivanov ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Amplicon Sequencing ◽  
Common Mechanism ◽  
Macrophage Infection ◽  
Gene Encoding ◽  
Transmitted Infection ◽  
Variable Regions ◽  
Content Type ◽  
Strain Fa1090

ABSTRACT Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection. IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.

scholarly journals Altering the Neisseria gonorrhoeae pilE Guanine Quadruplex Loop Bases Affects Pilin Antigenic Variation

Biochemistry ◽  
2020 ◽  
Vol 59 (10) ◽  
pp. 1104-1112
Author(s):  
Lauren L. Prister ◽  
Shaohui Yin ◽  
Laty A. Cahoon ◽  
H Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Guanine Quadruplex

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 Biased Gene Conversion in Rhizobium etli Is Caused by Preferential Double-Strand Breaks on One of the Recombining Homologs

2015 ◽  
Vol 198 (3) ◽  
pp. 591-599 ◽  
Author(s):  
Fares Osam Yáñez-Cuna ◽  
Mildred Castellanos ◽  
David Romero
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Biased Gene Conversion ◽  
Resident Plasmid

ABSTRACTGene conversion, the nonreciprocal transfer of information during homologous recombination, is the main process that maintains identity between members of multigene families. Gene conversion in the nitrogenase (nifH) multigene family ofRhizobium etliwas analyzed by using a two-plasmid system, where each plasmid carried a copy ofnifH. One of thenifHcopies was modified, creating restriction fragment length polymorphisms (RFLPs) spaced along the gene. Once the modified plasmid was introduced intoR. etli, selection was done for cointegration with a resident plasmid lacking the RFLPs. Most of the cointegrate molecules harbor gene conversion events, biased toward a gain of RFLPs. This bias may be explained under the double-strand break repair model by proposing that thenifHgene lacking the RFLPs suffers a DNA double-strand break, so the incoming plasmid functions as a template for repairing the homolog on the resident plasmid. To support this proposal, we cloned an SceI site into thenifHhomolog that had the RFLPs used for scoring gene conversion.In vivoexpression of the meganuclease I-SceI allowed the generation of a double-strand break on this homolog. Upon introduction of this modified plasmid into anR. etlistrain lacking I-SceI, biased gene conversion still favored the retention of markers on the incoming plasmid. In contrast, when the recipient strain ectopically expressed I-SceI, a dramatic reversal in gene conversion bias was seen, favoring the preservation of resident sequences. These results show that biased gene conversion is caused by preferential double-strand breaks on one of the recombining homologs.IMPORTANCEIn this work, we analyzed gene conversion by using a system that entails horizontal gene transfer followed by homologous recombination in the recipient cell. Most gene conversion events are biased toward the acquisition of the incoming sequences, ranging in size from 120 bp to 800 bp. This bias is due to preferential cutting of resident DNA and can be reversed upon introduction of a double-strand break on the incoming sequence. Since conditions used in this work are similar to those in horizontal gene transfer, it provides evidence that, upon transfer, the resident DNA preferentially acquires gene variants.

scholarly journals Marker Recycling in Candida albicans through CRISPR-Cas9-Induced Marker Excision

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Manning Y. Huang ◽  
Aaron P. Mitchell
Keyword(s):  
Candida Albicans ◽  
Genetic Manipulation ◽  
Strand Break ◽  
Double Strand Break ◽  
Selection Marker ◽  
Content Type ◽  
Marker Recycling ◽  
Marker Excision ◽  
Selection For ◽  
Selection Markers

ABSTRACT It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology. We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ability of the CRISPR-Cas9 system to make a targeted double-strand break in DNA and the expectation that a double-strand break within a selection marker may promote recombination between directly repeated sequences that flank the marker. We call the approach CRISPR-Cas9-induced marker excision (CRIME). We tested the utility of this approach with the fungal pathogen Candida albicans, which is typically diploid. We used two selection markers, modified to include flanking direct repeats. In a proof-of-principle study, we created successive homozygous deletions in three genes through use of the two markers and had one of the markers available in the final strain for further selection and recycling. This strategy will accelerate the creation of multiple-mutant strains in C. albicans. CRISPR-Cas9 systems have been applied to many organisms, so the genetic design principles described here may be broadly applicable. IMPORTANCE It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology.

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 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.

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