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 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 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.

A β-lactamase-producing plasmid from Neisseria gonorrhoeae carrying a unique 6 bp deletion in blaTEM-1 encoding a truncated 24 kDa TEM-1 penicillinase that hydrolyses ampicillin slowly

2019 ◽  
Vol 74 (10) ◽  
pp. 2904-2912 ◽  
Author(s):  
Reema Singh ◽  
Sumudu R Perera ◽  
George S Katselis ◽  
Paulos Chumala ◽  
Irene Martin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Neisseria Gonorrhoeae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Start Codon ◽  
Surveillance Program ◽  
Binding Properties ◽  
Antimicrobial Surveillance ◽  
Agar Dilution ◽  
Hydrolysis Of

AbstractBackgroundSeven structurally related β-lactamase-producing plasmids have been characterized in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates. We characterized a variant (i.e. pJRD20, Canada type) of the Africa-type (pJD5) plasmid isolated from N. gonorrhoeae strain 8903.ObjectivesTo compare the DNA sequence of pJRD20 with that of pJD5 and pJD4 (Asia-type) and their TEM-1 β-lactamases.MethodsN. gonorrhoeae 8903 was identified as part of the Gonococcal Antimicrobial Surveillance Program in Canada. β-Lactamase production was assessed using nitrocefin. MICs were determined by agar dilution and Etest methods (CLSI). The DNA sequences of pJRD20, pJD5 and pJD4 were assembled and annotated. The structure of TEM-1 and its penicillin-binding properties were determined by in silico molecular modelling and docking. TEM-1 proteins were characterized by western blot, mass spectrometry and ampicillin hydrolysis assays.ResultsN. gonorrhoeae 8903 exhibited intermediate susceptibility to penicillin with slow β-lactamase activity (i.e. 35 min to hydrolyse nitrocefin). Except for a novel 6 bp deletion starting at the G of the ATG start codon of blaTEM-1, the DNA sequence of pJRD20 was identical to that of pJD5. The TEM-1 β-lactamase produced by pJRD20 is 24 kDa and hydrolyses ampicillin only after several hours.ConclusionsThis unusual PPNG isolate might have been characterized as a non-PPNG owing to its low MIC of penicillin and its very slow hydrolysis of nitrocefin. Given the unusual nature of its TEM-1 β-lactamase, laboratories might consider extending the duration of nitrocefin hydrolysis assays.

scholarly journals Frequency of Pilin Antigenic Variation inNeisseria gonorrhoeae

1998 ◽  
Vol 180 (7) ◽  
pp. 1955-1958 ◽  
Author(s):  
Carla D. Serkin ◽  
H. Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Stationary Phase ◽  
Dna Sequences ◽  
Reverse Transcription ◽  
Antigenic Variation ◽  
Colony Morphology ◽  
Pcr Assay ◽  
Twofold Increase ◽  
Reverse Transcription Pcr

ABSTRACT Variation of the pilus of Neisseria gonorrhoeae occurs by the recombination of silent pilin DNA sequences into the pilin expression locus. We have developed a quantitative, competitive reverse transcription-PCR assay which measures the frequency of pilin antigenic variation independently of changes in gonococcal colony morphology and have determined this frequency within a gonococcal population. We have also studied the frequency of antigenic variation during growth and have concluded that growth does not dramatically influence the frequency of pilin antigenic variation, although a reproducible, twofold increase is observed upon the transition into late log/stationary phase.

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 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.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

scholarly journals DNA thermodynamics shape chromosome organization and topology

2013 ◽  
Vol 41 (2) ◽  
pp. 548-553 ◽  
Author(s):  
Andrew A. Travers ◽  
Georgi Muskhelishvili
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Chromosome Organization ◽  
Topological Properties ◽  
Genetic Organization ◽  
And Topology ◽  
Dna Translocases

How much information is encoded in the DNA sequence of an organism? We argue that the informational, mechanical and topological properties of DNA are interdependent and act together to specify the primary characteristics of genetic organization and chromatin structures. Superhelicity generated in vivo, in part by the action of DNA translocases, can be transmitted to topologically sensitive regions encoded by less stable DNA sequences.

scholarly journals An application of slow feature analysis to the genetic sequences of coronaviruses and influenza viruses

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Anastasios A. Tsonis ◽  
Geli Wang ◽  
Lvyi Zhang ◽  
Wenxu Lu ◽  
Aristotle Kayafas ◽  
...  
Keyword(s):  
Mathematical Method ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Transmission Rate ◽  
Complex Structure ◽  
Mortality Rates ◽  
Influenza Viruses ◽  
Feature Analysis ◽  
Slow Feature Analysis ◽  
Genetic Sequences

Abstract Background Mathematical approaches have been for decades used to probe the structure of DNA sequences. This has led to the development of Bioinformatics. In this exploratory work, a novel mathematical method is applied to probe the DNA structure of two related viral families: those of coronaviruses and those of influenza viruses. The coronaviruses are SARS-CoV-2, SARS-CoV-1, and MERS. The influenza viruses include H1N1-1918, H1N1-2009, H2N2-1957, and H3N2-1968. Methods The mathematical method used is the slow feature analysis (SFA), a rather new but promising method to delineate complex structure in DNA sequences. Results The analysis indicates that the DNA sequences exhibit an elaborate and convoluted structure akin to complex networks. We define a measure of complexity and show that each DNA sequence exhibits a certain degree of complexity within itself, while at the same time there exists complex inter-relationships between the sequences within a family and between the two families. From these relationships, we find evidence, especially for the coronavirus family, that increasing complexity in a sequence is associated with higher transmission rate but with lower mortality. Conclusions The complexity measure defined here may hold a promise and could become a useful tool in the prediction of transmission and mortality rates in future new viral strains.

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.

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