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A phosphoketolase bypasses reductive stress in P. aeruginosa under anaerobic survival

ABSTRACT:

Across diverse contexts, bacteria experience loss of electron acceptors due to fluctuating environmental conditions, requiring strategies to avoid metabolic arrest due to reductive stress. Yet, microbial metabolism has been primarily studied with cells growing aerobically under nutrient replete conditions. To study how cells metabolically preserve redox homeostasis and viability during growth-arrest, we explored how the opportunistic pathogen Pseudomonas aeruginosa remodels its metabolism during anaerobic survival and oxidant limitation. During anaerobic survival on glucose, P. aeruginosa relies on flux through the pentose phosphate pathway (PPP) and a previously undescribed phosphoketolase (here-in termed xfp). This re-routing bypasses P. aeruginosa’s canonical glucose-catabolizing Entner-Doudoroff pathway (EDP) avoiding reducing equivalent generation. Moreover, anaerobic survival on diverse carbon sources triggers PPP metabolite accumulation and ribonucleotide salvage, with this phosphoketolase being essential to mediating ribonucleotide homeostasis and avoiding lethal metabolic dysregulation. This study expands our understanding of P. aeruginosa’s anaerobic survival strategies and suggests that phosphoketolases play a previously unappreciated role in avoiding reductive stress and mediating ribonucleotide homeostasis. Our work serves as a reminder that large gaps remain in our understanding of growth arrest physiology even in well-studied model organisms, highlighting the potential for basic discovery in the realm of non-growth metabolism.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse phase

PROVIDER: MTBLS13293 | MetaboLights | 2026-06-03

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS13293_LC-MS_negative_reverse-phase_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS13293_LC-MS_negative_reverse-phase_metabolite_profiling_v2_maf.tsv	Tabular
	s_MTBLS13293.txt	Txt

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Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators. We performed three comparisons to identify genes induced under anaerobic denitrifying conditions in the P. aeruginosa wild type strain and genes which are under control of the Anr or Dnr regulators under these anaerobic conditions. Since the anr and dnr mutant strains do not grow under anaerobic denitrifying conditions, we applied anaerobic shift experiments. Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 ÂµM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 oC and 300 rpm. The aerobic culture was grown to an OD578 of 0.3. For the aerobic culture, cells were harvested at this point. For the anaerobic shift experiments 130 ml of the respective aerobic culture were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions. Within these 2h incubation period no growth of the wild type, the anr mutant or the dnr mutant strain was observed. First comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown under aerobic conditions up to an OD578 of 0.3 with the transcriptome profile of the PAO1 strain, which was first grown under aerobic conditions up to an OD578 of 0.3 and than shifted to anaerobic conditions by transfer to a sealed serum flask and further incubated for two hours under anaerobic conditions. Second comparison: Identification of genes regulated differently in the anr mutant strain PAO6261. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa anr mutant strain PAO6261. Both strains were harvested after 2h incubation under anaerobic conditions. Third comparison: Identification of genes regulated differently in the dnr mutant strain RM536. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa dnr mutant strain RM536. Both strains were harvested after 2h incubation under anaerobic conditions.

Project description:Sohn2010 - Genome-scale metabolic network of Pseudomonas putida (PpuMBEL1071) This model is described in the article: In silico genome-scale metabolic analysis of Pseudomonas putida KT2440 for polyhydroxyalkanoate synthesis, degradation of aromatics and anaerobic survival. Sohn SB, Kim TY, Park JM, Lee SY. Biotechnol J 2010 Jul; 5(7): 739-750 Abstract: Genome-scale metabolic models have been appearing with increasing frequency and have been employed in a wide range of biotechnological applications as well as in biological studies. With the metabolic model as a platform, engineering strategies have become more systematic and focused, unlike the random shotgun approach used in the past. Here we present the genome-scale metabolic model of the versatile Gram-negative bacterium Pseudomonas putida, which has gained widespread interest for various biotechnological applications. With the construction of the genome-scale metabolic model of P. putida KT2440, PpuMBEL1071, we investigated various characteristics of P. putida, such as its capacity for synthesizing polyhydroxyalkanoates (PHA) and degrading aromatics. Although P. putida has been characterized as a strict aerobic bacterium, the physiological characteristics required to achieve anaerobic survival were investigated. Through analysis of PpuMBEL1071, extended survival of P. putida under anaerobic stress was achieved by introducing the ackA gene from Pseudomonas aeruginosa and Escherichia coli. This model is hosted on BioModels Database and identified by: MODEL1507180043. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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A phosphoketolase bypasses reductive stress in P. aeruginosa under anaerobic survival

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