The Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor

ABSTRACT The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual...

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Autores principales:	Pablo I. Nikel, Francisco J. Romero-Campero, Joshua A. Zeidman, Ángel Goñi-Moreno, Víctor de Lorenzo
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/3fe1feeeeb1042ffb1ae6dc6b24feae8
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spelling	oai:doaj.org-article:3fe1feeeeb1042ffb1ae6dc6b24feae82021-11-15T15:41:33ZThe Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor10.1128/mBio.00340-152150-7511https://doaj.org/article/3fe1feeeeb1042ffb1ae6dc6b24feae82015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00340-15https://doaj.org/toc/2150-7511ABSTRACT The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the glp genes. Either deleting glpR (encoding the G3P-responsive transcriptional repressor that controls the expression of the glpFKRD gene cluster) or altering G3P formation (by overexpressing glpK, encoding glycerol kinase) abolished the bimodal glpD expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of glpR in trans restored the phenotypes lost in the ΔglpR mutant. The prolonged nongrowth regime of P. putida on glycerol could thus be traced to the regulatory device controlling the transcription of the glp genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states. IMPORTANCE Phenotypic variation is a widespread attribute of prokaryotes that leads, inter alia, to the emergence of persistent bacteria, i.e., live but nongrowing members within a genetically clonal population. Persistence allows a fraction of cells to avoid the killing caused by conditions or agents that destroy most growing bacteria (e.g., some antibiotics). Known molecular mechanisms underlying the phenomenon include genetic changes, epigenetic variations, and feedback-based multistability. We show that a prolonged nongrowing state of the bacterial population can be brought about by a distinct regulatory architecture of metabolic genes when cells face specific nutrients (e.g., glycerol). Pseudomonas putida may have adopted the resulting carbon source-dependent metabolic bet hedging as an advantageous trait for exploring new chemical and nutritional landscapes. Defeating such naturally occurring adaptive features of environmental bacteria is instrumental in improving the performance of these microorganisms as whole-cell catalysts in a bioreactor setup.Pablo I. NikelFrancisco J. Romero-CamperoJoshua A. ZeidmanÁngel Goñi-MorenoVíctor de LorenzoAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Pablo I. Nikel Francisco J. Romero-Campero Joshua A. Zeidman Ángel Goñi-Moreno Víctor de Lorenzo The Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor
description	ABSTRACT The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the glp genes. Either deleting glpR (encoding the G3P-responsive transcriptional repressor that controls the expression of the glpFKRD gene cluster) or altering G3P formation (by overexpressing glpK, encoding glycerol kinase) abolished the bimodal glpD expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of glpR in trans restored the phenotypes lost in the ΔglpR mutant. The prolonged nongrowth regime of P. putida on glycerol could thus be traced to the regulatory device controlling the transcription of the glp genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states. IMPORTANCE Phenotypic variation is a widespread attribute of prokaryotes that leads, inter alia, to the emergence of persistent bacteria, i.e., live but nongrowing members within a genetically clonal population. Persistence allows a fraction of cells to avoid the killing caused by conditions or agents that destroy most growing bacteria (e.g., some antibiotics). Known molecular mechanisms underlying the phenomenon include genetic changes, epigenetic variations, and feedback-based multistability. We show that a prolonged nongrowing state of the bacterial population can be brought about by a distinct regulatory architecture of metabolic genes when cells face specific nutrients (e.g., glycerol). Pseudomonas putida may have adopted the resulting carbon source-dependent metabolic bet hedging as an advantageous trait for exploring new chemical and nutritional landscapes. Defeating such naturally occurring adaptive features of environmental bacteria is instrumental in improving the performance of these microorganisms as whole-cell catalysts in a bioreactor setup.
format	article
author	Pablo I. Nikel Francisco J. Romero-Campero Joshua A. Zeidman Ángel Goñi-Moreno Víctor de Lorenzo
author_facet	Pablo I. Nikel Francisco J. Romero-Campero Joshua A. Zeidman Ángel Goñi-Moreno Víctor de Lorenzo
author_sort	Pablo I. Nikel
title	The Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor
title_short	The Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor
title_full	The Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor
title_fullStr	The Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor
title_full_unstemmed	The Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor
title_sort	glycerol-dependent metabolic persistence of <named-content content-type="genus-species">pseudomonas putida</named-content> kt2440 reflects the regulatory logic of the glpr repressor
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/3fe1feeeeb1042ffb1ae6dc6b24feae8
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The Glycerol-Dependent Metabolic Persistence of <named-content content-type="genus-species">Pseudomonas putida</named-content> KT2440 Reflects the Regulatory Logic of the GlpR Repressor

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