Stabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes

ABSTRACT Hypermutable simple sequence repeats (SSRs) are major drivers of phase variation in Campylobacter jejuni. The presence of multiple SSR-mediated phase-variable genes encoding enzymes that modify surface structures, including capsular polysaccharide (CPS) and lipooligosaccharide (LOS), genera...

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Autores principales:	Shouji Yamamoto, Sunao Iyoda, Makoto Ohnishi
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	Campylobacter genome editing natural transformation phase variation phenotypic engineering simple sequence repeats Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/c58d0d3b910b4f1b8356db5f23150915
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spelling	oai:doaj.org-article:c58d0d3b910b4f1b8356db5f231509152021-11-10T18:37:52ZStabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes10.1128/mBio.01401-212150-7511https://doaj.org/article/c58d0d3b910b4f1b8356db5f231509152021-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01401-21https://doaj.org/toc/2150-7511ABSTRACT Hypermutable simple sequence repeats (SSRs) are major drivers of phase variation in Campylobacter jejuni. The presence of multiple SSR-mediated phase-variable genes encoding enzymes that modify surface structures, including capsular polysaccharide (CPS) and lipooligosaccharide (LOS), generates extreme cell surface diversity within bacterial populations, thereby promoting adaptation to selective pressures in host environments. Therefore, genetically controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. Here, we show that natural “cotransformation” is an effective method for C. jejuni genome editing. Cotransformation is a trait of naturally competent bacteria that causes uptake/integration of multiple different DNA molecules, which has been recently adapted to multiplex genome editing by natural transformation (MuGENT), a method for introducing multiple mutations into the genomes of these bacteria. We found that cotransformation efficiently occurred in C. jejuni. To examine the feasibility of MuGENT in C. jejuni, we “locked” different polyG SSR tracts in strain NCTC11168 (which are located in the biosynthetic CPS/LOS gene clusters) into either the ON or OFF configurations. This approach, termed “MuGENT-SSR,” enabled the generation of all eight edits within 2 weeks and the identification of a phase-locked strain with a highly stable type of Penner serotyping, a CPS-based serotyping scheme. Furthermore, extensive genome editing of this strain by MuGENT-SSR identified a phase-variable gene that determines the Penner serotype of NCTC11168. Thus, MuGENT-SSR provides a platform for genetic and phenotypic engineering of genetically unstable C. jejuni, making it a reliable approach for elucidating the mechanisms underlying phase-variable expression of specific phenotypes. IMPORTANCE Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis in developed countries and occasionally progresses to the autoimmune disease Guillain-Barré syndrome. A relatively large number of hypermutable simple sequence repeat (SSR) tracts in the C. jejuni genome markedly decreases its phenotypic stability through reversible changes in the ON or OFF expression states of the genes in which they reside, a phenomenon called phase variation. Thus, controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. In this study, we developed a feasible and effective approach for genetically manipulate multiple SSR tracts in the C. jejuni genome using natural cotransformation, a trait of naturally transformable bacterial species that causes the uptake and integration of multiple different DNA molecules. This approach will greatly help to improve the genetic and phenotypic stability of C. jejuni to enable diverse applications in research and development.Shouji YamamotoSunao IyodaMakoto OhnishiAmerican Society for MicrobiologyarticleCampylobactergenome editingnatural transformationphase variationphenotypic engineeringsimple sequence repeatsMicrobiologyQR1-502ENmBio, Vol 12, Iss 4 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Campylobacter genome editing natural transformation phase variation phenotypic engineering simple sequence repeats Microbiology QR1-502
spellingShingle	Campylobacter genome editing natural transformation phase variation phenotypic engineering simple sequence repeats Microbiology QR1-502 Shouji Yamamoto Sunao Iyoda Makoto Ohnishi Stabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes
description	ABSTRACT Hypermutable simple sequence repeats (SSRs) are major drivers of phase variation in Campylobacter jejuni. The presence of multiple SSR-mediated phase-variable genes encoding enzymes that modify surface structures, including capsular polysaccharide (CPS) and lipooligosaccharide (LOS), generates extreme cell surface diversity within bacterial populations, thereby promoting adaptation to selective pressures in host environments. Therefore, genetically controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. Here, we show that natural “cotransformation” is an effective method for C. jejuni genome editing. Cotransformation is a trait of naturally competent bacteria that causes uptake/integration of multiple different DNA molecules, which has been recently adapted to multiplex genome editing by natural transformation (MuGENT), a method for introducing multiple mutations into the genomes of these bacteria. We found that cotransformation efficiently occurred in C. jejuni. To examine the feasibility of MuGENT in C. jejuni, we “locked” different polyG SSR tracts in strain NCTC11168 (which are located in the biosynthetic CPS/LOS gene clusters) into either the ON or OFF configurations. This approach, termed “MuGENT-SSR,” enabled the generation of all eight edits within 2 weeks and the identification of a phase-locked strain with a highly stable type of Penner serotyping, a CPS-based serotyping scheme. Furthermore, extensive genome editing of this strain by MuGENT-SSR identified a phase-variable gene that determines the Penner serotype of NCTC11168. Thus, MuGENT-SSR provides a platform for genetic and phenotypic engineering of genetically unstable C. jejuni, making it a reliable approach for elucidating the mechanisms underlying phase-variable expression of specific phenotypes. IMPORTANCE Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis in developed countries and occasionally progresses to the autoimmune disease Guillain-Barré syndrome. A relatively large number of hypermutable simple sequence repeat (SSR) tracts in the C. jejuni genome markedly decreases its phenotypic stability through reversible changes in the ON or OFF expression states of the genes in which they reside, a phenomenon called phase variation. Thus, controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. In this study, we developed a feasible and effective approach for genetically manipulate multiple SSR tracts in the C. jejuni genome using natural cotransformation, a trait of naturally transformable bacterial species that causes the uptake and integration of multiple different DNA molecules. This approach will greatly help to improve the genetic and phenotypic stability of C. jejuni to enable diverse applications in research and development.
format	article
author	Shouji Yamamoto Sunao Iyoda Makoto Ohnishi
author_facet	Shouji Yamamoto Sunao Iyoda Makoto Ohnishi
author_sort	Shouji Yamamoto
title	Stabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes
title_short	Stabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes
title_full	Stabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes
title_fullStr	Stabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes
title_full_unstemmed	Stabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes
title_sort	stabilizing genetically unstable simple sequence repeats in the <named-content content-type="genus-species">campylobacter jejuni</named-content> genome by multiplex genome editing: a reliable approach for delineating multiple phase-variable genes
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/c58d0d3b910b4f1b8356db5f23150915
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Stabilizing Genetically Unstable Simple Sequence Repeats in the <named-content content-type="genus-species">Campylobacter jejuni</named-content> Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes

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