Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS

ABSTRACT Although much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenabl...

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Autores principales:	Ryan A. Melnyk, Iain C. Clark, Annette Liao, John D. Coates
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
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spelling	oai:doaj.org-article:1c833658baae4eddbb354bb49de760422021-11-15T15:45:10ZTransposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS10.1128/mBio.00769-132150-7511https://doaj.org/article/1c833658baae4eddbb354bb49de760422014-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00769-13https://doaj.org/toc/2150-7511ABSTRACT Although much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism. IMPORTANCE Although perchlorate is generated naturally in the environment, groundwater contamination is largely a result of industrial activity. Bacteria capable of respiring perchlorate and remediating contaminated water have been isolated, but relatively little is known about the biochemistry and genetics of this process. Here we used two complementary approaches to identify genes involved in perchlorate reduction. Most of these genes are located on a genomic island, which is potentially capable of moving between organisms. Some of the genes identified are known to be directly involved in the metabolism of perchlorate, but other new genes likely regulate the metabolism in response to environmental signals. This work has uncovered new questions about the regulation, energetics, and evolution of perchlorate reduction but also presents the tools to address them.Ryan A. MelnykIain C. ClarkAnnette LiaoJohn D. CoatesAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 1 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Ryan A. Melnyk Iain C. Clark Annette Liao John D. Coates Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS
description	ABSTRACT Although much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism. IMPORTANCE Although perchlorate is generated naturally in the environment, groundwater contamination is largely a result of industrial activity. Bacteria capable of respiring perchlorate and remediating contaminated water have been isolated, but relatively little is known about the biochemistry and genetics of this process. Here we used two complementary approaches to identify genes involved in perchlorate reduction. Most of these genes are located on a genomic island, which is potentially capable of moving between organisms. Some of the genes identified are known to be directly involved in the metabolism of perchlorate, but other new genes likely regulate the metabolism in response to environmental signals. This work has uncovered new questions about the regulation, energetics, and evolution of perchlorate reduction but also presents the tools to address them.
format	article
author	Ryan A. Melnyk Iain C. Clark Annette Liao John D. Coates
author_facet	Ryan A. Melnyk Iain C. Clark Annette Liao John D. Coates
author_sort	Ryan A. Melnyk
title	Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS
title_short	Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS
title_full	Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS
title_fullStr	Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS
title_full_unstemmed	Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS
title_sort	transposon and deletion mutagenesis of genes involved in perchlorate reduction in <italic toggle="yes">azospira suillum</italic> ps
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/1c833658baae4eddbb354bb49de76042
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Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in <italic toggle="yes">Azospira suillum</italic> PS

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