Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G20

ABSTRACT The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alask...

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Autores principales:	Jennifer V. Kuehl, Morgan N. Price, Jayashree Ray, Kelly M. Wetmore, Zuelma Esquivel, Alexey E. Kazakov, Michelle Nguyen, Raquel Kuehn, Ronald W. Davis, Terry C. Hazen, Adam P. Arkin, Adam Deutschbauer
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
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spelling	oai:doaj.org-article:26a3f97aff804914a572f514337b17db2021-11-15T15:47:38ZFunctional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G2010.1128/mBio.01041-142150-7511https://doaj.org/article/26a3f97aff804914a572f514337b17db2014-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01041-14https://doaj.org/toc/2150-7511ABSTRACT The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5′ RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance. IMPORTANCE Sulfate-reducing bacteria contribute to global nutrient cycles and are a nuisance for the petroleum industry. Despite their environmental and industrial significance, the genomes of sulfate-reducing bacteria remain poorly characterized. Here, we describe a genetic approach to fill gaps in our knowledge of sulfate-reducing bacteria. We generated a large collection of archived, transposon mutants in Desulfovibrio alaskensis G20 and used the phenotypes of these mutant strains to infer the function of genes involved in gene regulation, methionine biosynthesis, and choline utilization. Our findings and mutant resources will enable systematic investigations into gene function, energy generation, stress response, and metabolism for this important group of bacteria.Jennifer V. KuehlMorgan N. PriceJayashree RayKelly M. WetmoreZuelma EsquivelAlexey E. KazakovMichelle NguyenRaquel KuehnRonald W. DavisTerry C. HazenAdam P. ArkinAdam DeutschbauerAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 3 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Jennifer V. Kuehl Morgan N. Price Jayashree Ray Kelly M. Wetmore Zuelma Esquivel Alexey E. Kazakov Michelle Nguyen Raquel Kuehn Ronald W. Davis Terry C. Hazen Adam P. Arkin Adam Deutschbauer Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G20
description	ABSTRACT The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5′ RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance. IMPORTANCE Sulfate-reducing bacteria contribute to global nutrient cycles and are a nuisance for the petroleum industry. Despite their environmental and industrial significance, the genomes of sulfate-reducing bacteria remain poorly characterized. Here, we describe a genetic approach to fill gaps in our knowledge of sulfate-reducing bacteria. We generated a large collection of archived, transposon mutants in Desulfovibrio alaskensis G20 and used the phenotypes of these mutant strains to infer the function of genes involved in gene regulation, methionine biosynthesis, and choline utilization. Our findings and mutant resources will enable systematic investigations into gene function, energy generation, stress response, and metabolism for this important group of bacteria.
format	article
author	Jennifer V. Kuehl Morgan N. Price Jayashree Ray Kelly M. Wetmore Zuelma Esquivel Alexey E. Kazakov Michelle Nguyen Raquel Kuehn Ronald W. Davis Terry C. Hazen Adam P. Arkin Adam Deutschbauer
author_facet	Jennifer V. Kuehl Morgan N. Price Jayashree Ray Kelly M. Wetmore Zuelma Esquivel Alexey E. Kazakov Michelle Nguyen Raquel Kuehn Ronald W. Davis Terry C. Hazen Adam P. Arkin Adam Deutschbauer
author_sort	Jennifer V. Kuehl
title	Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G20
title_short	Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G20
title_full	Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G20
title_fullStr	Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G20
title_full_unstemmed	Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G20
title_sort	functional genomics with a comprehensive library of transposon mutants for the sulfate-reducing bacterium <named-content content-type="genus-species">desulfovibrio alaskensis</named-content> g20
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/26a3f97aff804914a572f514337b17db
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Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium <named-content content-type="genus-species">Desulfovibrio alaskensis</named-content> G20

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