Dataset Information

H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

ABSTRACT: Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/?-10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.

SUBMITTER: Masters TL

PROVIDER: S-EPMC6190205 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

Masters Thao L TL Wachter Shaun S Wachter Jenny J Hill Stuart A SA

Microbiology (Reading, England) 20151016 1

Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/ -10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative ...[more]

PMID: 26475082

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Project description: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.

Dataset Information

H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

Publications

H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

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