Molecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome

ABSTRACT Vibrionaceae family members are interesting models for studying DNA replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique yet highly conserved elements, the origin DNA sequence oriCII and the initiator pr...

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Autores principales:	Matthew A. Gerding, Michael C. Chao, Brigid M. Davis, Matthew K. Waldor
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1601cdfe0dab43b79ad1bb502e34329d
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spelling	oai:doaj.org-article:1601cdfe0dab43b79ad1bb502e34329d2021-11-15T15:41:26ZMolecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome10.1128/mBio.00973-152150-7511https://doaj.org/article/1601cdfe0dab43b79ad1bb502e34329d2015-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00973-15https://doaj.org/toc/2150-7511ABSTRACT Vibrionaceae family members are interesting models for studying DNA replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique yet highly conserved elements, the origin DNA sequence oriCII and the initiator protein RctB. The minimum functional region of oriCII, oriCII-min, contains multiple elements that are bound by RctB in vitro, but little is known about the specific requirements for individual elements during oriCII initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of mutant forms of oriCII-min and assessed binding to various mutant forms by RctB. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired oriCII-min-based replication. RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of origin components other than RctB binding sites (e.g., AT-rich sequence, DnaA target site) also abolished replicative capacity. Comprehensive mutagenesis and deep-sequencing-based screening (OriSeq) allowed the identification of a previously uncharacterized methylated domain in oriCII that is required for origin function. Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII. IMPORTANCE The genome of the enteric pathogen Vibrio cholerae consists of two chromosomes. While the chromosome I replication origin and its cognate replication initiator protein resemble those of Escherichia coli, the factors responsible for chromosome II replication initiation display no similarity to any other known initiation systems. Here, to enhance our understanding of how this DNA sequence, oriCII, and its initiator protein, RctB, function, we used both targeted mutagenesis and a new random-mutagenesis approach (OriSeq) to finely map the oriCII structural features and sequences required for RctB-mediated DNA replication. Collectively, our findings reveal the extraordinary evolutionary honing of the architecture and motifs that constitute oriCII and reveal a new role for methylation in oriCII-based replication. Finally, our findings suggest that the OriSeq approach is likely to be widely applicable for defining critical bases in cis-acting sequences.Matthew A. GerdingMichael C. ChaoBrigid M. DavisMatthew K. WaldorAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 4 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Matthew A. Gerding Michael C. Chao Brigid M. Davis Matthew K. Waldor Molecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome
description	ABSTRACT Vibrionaceae family members are interesting models for studying DNA replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique yet highly conserved elements, the origin DNA sequence oriCII and the initiator protein RctB. The minimum functional region of oriCII, oriCII-min, contains multiple elements that are bound by RctB in vitro, but little is known about the specific requirements for individual elements during oriCII initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of mutant forms of oriCII-min and assessed binding to various mutant forms by RctB. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired oriCII-min-based replication. RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of origin components other than RctB binding sites (e.g., AT-rich sequence, DnaA target site) also abolished replicative capacity. Comprehensive mutagenesis and deep-sequencing-based screening (OriSeq) allowed the identification of a previously uncharacterized methylated domain in oriCII that is required for origin function. Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII. IMPORTANCE The genome of the enteric pathogen Vibrio cholerae consists of two chromosomes. While the chromosome I replication origin and its cognate replication initiator protein resemble those of Escherichia coli, the factors responsible for chromosome II replication initiation display no similarity to any other known initiation systems. Here, to enhance our understanding of how this DNA sequence, oriCII, and its initiator protein, RctB, function, we used both targeted mutagenesis and a new random-mutagenesis approach (OriSeq) to finely map the oriCII structural features and sequences required for RctB-mediated DNA replication. Collectively, our findings reveal the extraordinary evolutionary honing of the architecture and motifs that constitute oriCII and reveal a new role for methylation in oriCII-based replication. Finally, our findings suggest that the OriSeq approach is likely to be widely applicable for defining critical bases in cis-acting sequences.
format	article
author	Matthew A. Gerding Michael C. Chao Brigid M. Davis Matthew K. Waldor
author_facet	Matthew A. Gerding Michael C. Chao Brigid M. Davis Matthew K. Waldor
author_sort	Matthew A. Gerding
title	Molecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome
title_short	Molecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome
title_full	Molecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome
title_fullStr	Molecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome
title_full_unstemmed	Molecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome
title_sort	molecular dissection of the essential features of the origin of replication of the second <named-content content-type="genus-species">vibrio cholerae</named-content> chromosome
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/1601cdfe0dab43b79ad1bb502e34329d
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Molecular Dissection of the Essential Features of the Origin of Replication of the Second <named-content content-type="genus-species">Vibrio cholerae</named-content> Chromosome

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