Identification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>

ABSTRACT Microbial growth arrest can be triggered by diverse factors, one of which is energy limitation due to scarcity of electron donors or acceptors. Genes that govern fitness during energy-limited growth arrest and the extent to which they overlap between different types of energy limitation are...

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Autores principales:	David W. Basta, Megan Bergkessel, Dianne K. Newman
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Pseudomonas aeruginosa Tn-seq fitness growth arrest slow growth energy limitation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/960eb6d124c54695a92945cf990cc064
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spelling	oai:doaj.org-article:960eb6d124c54695a92945cf990cc0642021-11-15T15:51:56ZIdentification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>10.1128/mBio.01170-172150-7511https://doaj.org/article/960eb6d124c54695a92945cf990cc0642017-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01170-17https://doaj.org/toc/2150-7511ABSTRACT Microbial growth arrest can be triggered by diverse factors, one of which is energy limitation due to scarcity of electron donors or acceptors. Genes that govern fitness during energy-limited growth arrest and the extent to which they overlap between different types of energy limitation are poorly defined. In this study, we exploited the fact that Pseudomonas aeruginosa can remain viable over several weeks when limited for organic carbon (pyruvate) as an electron donor or oxygen as an electron acceptor. ATP values were reduced under both types of limitation, yet more severely in the absence of oxygen. Using transposon-insertion sequencing (Tn-seq), we identified fitness determinants in these two energy-limited states. Multiple genes encoding general functions like transcriptional regulation and energy generation were required for fitness during carbon or oxygen limitation, yet many specific genes, and thus specific activities, differed in their relevance between these states. For instance, the global regulator RpoS was required during both types of energy limitation, while other global regulators such as DksA and LasR were required only during carbon or oxygen limitation, respectively. Similarly, certain ribosomal and tRNA modifications were specifically required during oxygen limitation. We validated fitness defects during energy limitation using independently generated mutants of genes detected in our screen. Mutants in distinct functional categories exhibited different fitness dynamics: regulatory genes generally manifested a phenotype early, whereas genes involved in cell wall metabolism were required later. Together, these results provide a new window into how P. aeruginosa survives growth arrest. IMPORTANCE Growth-arrested bacteria are ubiquitous in nature and disease yet understudied at the molecular level. For example, growth-arrested cells constitute a major subpopulation of mature biofilms, serving as an antibiotic-tolerant reservoir in chronic infections. Identification of the genes required for survival of growth arrest (encompassing entry, maintenance, and exit) is an important first step toward understanding the physiology of bacteria in this state. Using Tn-seq, we identified and validated genes required for fitness of Pseudomonas aeruginosa when energy limited for organic carbon or oxygen, which represent two common causes of growth arrest for P. aeruginosa in diverse habitats. This unbiased, genome-wide survey is the first to reveal essential activities for a pathogen experiencing different types of energy limitation, finding both shared and divergent activities that are relevant at different survival stages. Future efforts can now be directed toward understanding how the biomolecules responsible for these activities contribute to fitness under these conditions.David W. BastaMegan BergkesselDianne K. NewmanAmerican Society for MicrobiologyarticlePseudomonas aeruginosaTn-seqfitnessgrowth arrestslow growthenergy limitationMicrobiologyQR1-502ENmBio, Vol 8, Iss 6 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Pseudomonas aeruginosa Tn-seq fitness growth arrest slow growth energy limitation Microbiology QR1-502
spellingShingle	Pseudomonas aeruginosa Tn-seq fitness growth arrest slow growth energy limitation Microbiology QR1-502 David W. Basta Megan Bergkessel Dianne K. Newman Identification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>
description	ABSTRACT Microbial growth arrest can be triggered by diverse factors, one of which is energy limitation due to scarcity of electron donors or acceptors. Genes that govern fitness during energy-limited growth arrest and the extent to which they overlap between different types of energy limitation are poorly defined. In this study, we exploited the fact that Pseudomonas aeruginosa can remain viable over several weeks when limited for organic carbon (pyruvate) as an electron donor or oxygen as an electron acceptor. ATP values were reduced under both types of limitation, yet more severely in the absence of oxygen. Using transposon-insertion sequencing (Tn-seq), we identified fitness determinants in these two energy-limited states. Multiple genes encoding general functions like transcriptional regulation and energy generation were required for fitness during carbon or oxygen limitation, yet many specific genes, and thus specific activities, differed in their relevance between these states. For instance, the global regulator RpoS was required during both types of energy limitation, while other global regulators such as DksA and LasR were required only during carbon or oxygen limitation, respectively. Similarly, certain ribosomal and tRNA modifications were specifically required during oxygen limitation. We validated fitness defects during energy limitation using independently generated mutants of genes detected in our screen. Mutants in distinct functional categories exhibited different fitness dynamics: regulatory genes generally manifested a phenotype early, whereas genes involved in cell wall metabolism were required later. Together, these results provide a new window into how P. aeruginosa survives growth arrest. IMPORTANCE Growth-arrested bacteria are ubiquitous in nature and disease yet understudied at the molecular level. For example, growth-arrested cells constitute a major subpopulation of mature biofilms, serving as an antibiotic-tolerant reservoir in chronic infections. Identification of the genes required for survival of growth arrest (encompassing entry, maintenance, and exit) is an important first step toward understanding the physiology of bacteria in this state. Using Tn-seq, we identified and validated genes required for fitness of Pseudomonas aeruginosa when energy limited for organic carbon or oxygen, which represent two common causes of growth arrest for P. aeruginosa in diverse habitats. This unbiased, genome-wide survey is the first to reveal essential activities for a pathogen experiencing different types of energy limitation, finding both shared and divergent activities that are relevant at different survival stages. Future efforts can now be directed toward understanding how the biomolecules responsible for these activities contribute to fitness under these conditions.
format	article
author	David W. Basta Megan Bergkessel Dianne K. Newman
author_facet	David W. Basta Megan Bergkessel Dianne K. Newman
author_sort	David W. Basta
title	Identification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>
title_short	Identification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>
title_full	Identification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>
title_fullStr	Identification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>
title_full_unstemmed	Identification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>
title_sort	identification of fitness determinants during energy-limited growth arrest in <italic toggle="yes">pseudomonas aeruginosa</italic>
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/960eb6d124c54695a92945cf990cc064
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Identification of Fitness Determinants during Energy-Limited Growth Arrest in <italic toggle="yes">Pseudomonas aeruginosa</italic>

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