Genome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters

ABSTRACT Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific comp...

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Autores principales:	Tyler C. Helmann, Caitlin L. Ongsarte, Jennifer Lam, Adam M. Deutschbauer, Steven E. Lindow
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	endophytes epiphytes fitness Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/c70cf94171a44eb19c8932064e760435
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spelling	oai:doaj.org-article:c70cf94171a44eb19c8932064e7604352021-11-15T15:59:41ZGenome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters10.1128/mBio.02614-192150-7511https://doaj.org/article/c70cf94171a44eb19c8932064e7604352019-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02614-19https://doaj.org/toc/2150-7511ABSTRACT Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants. IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.Tyler C. HelmannCaitlin L. OngsarteJennifer LamAdam M. DeutschbauerSteven E. LindowAmerican Society for MicrobiologyarticleendophytesepiphytesfitnessMicrobiologyQR1-502ENmBio, Vol 10, Iss 5 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	endophytes epiphytes fitness Microbiology QR1-502
spellingShingle	endophytes epiphytes fitness Microbiology QR1-502 Tyler C. Helmann Caitlin L. Ongsarte Jennifer Lam Adam M. Deutschbauer Steven E. Lindow Genome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters
description	ABSTRACT Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants. IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.
format	article
author	Tyler C. Helmann Caitlin L. Ongsarte Jennifer Lam Adam M. Deutschbauer Steven E. Lindow
author_facet	Tyler C. Helmann Caitlin L. Ongsarte Jennifer Lam Adam M. Deutschbauer Steven E. Lindow
author_sort	Tyler C. Helmann
title	Genome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters
title_short	Genome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters
title_full	Genome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters
title_fullStr	Genome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters
title_full_unstemmed	Genome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters
title_sort	genome-wide transposon screen of a <italic toggle="yes">pseudomonas syringae mexb</italic> mutant reveals the substrates of efflux transporters
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/c70cf94171a44eb19c8932064e760435
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Genome-Wide Transposon Screen of a <italic toggle="yes">Pseudomonas syringae mexB</italic> Mutant Reveals the Substrates of Efflux Transporters

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