Synthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-1

ABSTRACT Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transpos...

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Autores principales:	Iain C. Clark, Ryan A. Melnyk, Matthew D. Youngblut, Hans K. Carlson, Anthony T. Iavarone, John D. Coates
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/43da11ec97fe4e779b7ded9c153aeba0
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spelling	oai:doaj.org-article:43da11ec97fe4e779b7ded9c153aeba02021-11-15T15:49:02ZSynthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-110.1128/mBio.00282-152150-7511https://doaj.org/article/43da11ec97fe4e779b7ded9c153aeba02015-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00282-15https://doaj.org/toc/2150-7511ABSTRACT Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. IMPORTANCE The ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism.Iain C. ClarkRyan A. MelnykMatthew D. YoungblutHans K. CarlsonAnthony T. IavaroneJohn D. CoatesAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 3 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Iain C. Clark Ryan A. Melnyk Matthew D. Youngblut Hans K. Carlson Anthony T. Iavarone John D. Coates Synthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-1
description	ABSTRACT Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. IMPORTANCE The ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism.
format	article
author	Iain C. Clark Ryan A. Melnyk Matthew D. Youngblut Hans K. Carlson Anthony T. Iavarone John D. Coates
author_facet	Iain C. Clark Ryan A. Melnyk Matthew D. Youngblut Hans K. Carlson Anthony T. Iavarone John D. Coates
author_sort	Iain C. Clark
title	Synthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-1
title_short	Synthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-1
title_full	Synthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-1
title_fullStr	Synthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-1
title_full_unstemmed	Synthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-1
title_sort	synthetic and evolutionary construction of a chlorate-reducing <named-content content-type="genus-species">shewanella oneidensis</named-content> mr-1
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/43da11ec97fe4e779b7ded9c153aeba0
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Synthetic and Evolutionary Construction of a Chlorate-Reducing <named-content content-type="genus-species">Shewanella oneidensis</named-content> MR-1

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