Methylobacterium extorquens PA1 utilizes multiple strategies to maintain formaldehyde homeostasis during methylotrophic growth
Zachary T. Hying, Anya M. Rushmer, Chin Yi Loh, Eric L. Bruger, Jannell V. Bazurto
Abstract
Metabolic homeostasis is a central organizing principle of physiology whereby dynamic processes work to maintain a balanced internal state. Highly reactive essential metabolites are ideally maintained at equilibrium to prevent cellular damage. In the facultative methylotrophic bacterium Methylobacterium extorquens PA1, the utilization of one-carbon growth substrates, including methanol, generates formaldehyde as an obligate intermediate.
Introduction
Metabolic homeostasis is the tendency for metabolites to be maintained at relatively constant concentrations that support the growth and viability of the cell and is a central organizing principle of physiology [1]. Changes in environmental or regulatory conditions can perturb metabolic homeostasis and trigger cellular responses that return the cell to homeostatic conditions.
Materials and method
Bacterial strains, media, and chemicals
Bacterial strains used in this study (S1 Table) are derived from Methylobacterium extorquens PA [41] with cellulose synthase genes deleted (ΔbcsABZ) to prevent aggregation and optimize growth measurements in liquid culture [42]. Bacterial strains were cultivated using Methylobacterium piparazine-N,N’-bis(2-ethanesulfonic acid) (MP) medium [42] with 3.5 mM succinate; 15 mM MeOH, 20 mM acetate, 20 mM oxalate, 20 mM formate, or 20 mM methylamine as a sole source of carbon and energy. When grown on solid MP medium (15 g/L Bacto agar), 15 mM succinate was provided as the sole carbon. Chemicals and reagents were purchased from Sigma Aldrich.
Results
EfgA and TtmR independently mitigate FA imbalance during the transition to methylotrophy
During the transition to methylotrophy, strains lacking either efgA or ttmR have comparable extended lag phase defects and suffer FA imbalance, as determined by increased FA release into the supernatant [27]. To determine whether EfgA and TtmR act independently or are redundant parts of a common underlying process, wild-type (WT), each single mutant (ΔefgA and ΔttmR), and the double mutant (ΔefgA ΔttmR) were transitioned to methylotrophy via a carbon source switch experiment (S1 Fig). Here, stationary phase cells grown on succinate were inoculated into fresh medium supplemented with MeOH. We assayed growth and extracellular FA at the end of lag phase for each strain. Hereon, cells experiencing a new transition to methylotrophy are described as ‘FA naïve’ and cells already acclimated to growth on MeOH as a carbon source are described as ‘FA acclimated’.
Discussion
Herein, we demonstrated that EfgA and TtmR play independent, but complementary, roles in maintaining FA homeostasis during transitions to FA-generating carbon sources. The novel FA sensor EfgA plays the primary role in mediating homeostasis by coupling FA sensing to translation inhibition during transient FA imbalance to prevent cell death. The regulatory protein TtmR plays a secondary role in maintaining C1 homeostasis and utilization by mediating release of obligate C1 intermediates, including FA, to the extracellular space.
Acknowledgments
We would like to thank members of the Bazurto lab for reviewing/editing drafts, and members of Kathryn R. Fixen’s lab for providing E. coli S17-1 and feedback on the data herein during our joint lab meetings.
Citation: Hying ZT, Rushmer AM, Loh CY, Bruger EL, Bazurto JV (2025) Methylobacterium extorquens PA1 utilizes multiple strategies to maintain formaldehyde homeostasis during methylotrophic growth. PLoS Genet 21(6): e1011736. https://doi.org/10.1371/journal.pgen.1011736
Editor: Jue D. Wang, University of Wisconsin-Madison, UNITED STATES OF AMERICA
Received: January 20, 2025; Accepted: May 19, 2025; Published: June 9, 2025
Copyright: © 2025 Hying et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data generated from this work can be found in a supplemental “all data” excel file where the data underlying each figure and the "SX Table" files where the supplemental tables are provided.
Funding: This work was supported by funding from National Institute of Health (NIH), National Institute of General Medical Sciences (NIGMS, https://www.nigms.nih.gov/) to JVB under award number 1R35GM146904-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
