{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:43:01Z","timestamp":1785415381696,"version":"3.56.0"},"posted":{"date-parts":[[2026,7,1]]},"group-title":"Microbiology","reference-count":59,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:00:00Z","timestamp":1782864000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["1R35GM146904"],"award-info":[{"award-number":["1R35GM146904"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Bacteria frequently face challenges adapting to changing environmental conditions, such as shifting resource utilization, to survive and thrive. Methylotrophs capable of growth on reduced single-carbon compounds are prevalent in the phyllosphere (aerial plant surfaces), where they face continual and predictable shifts in the availability of different plant-produced carbon sources. We examined the ability of the methylotroph\n                  <jats:italic>Methylobacterium extorquens<\/jats:italic>\n                  PA1 to adapt to repeated shifts between two different carbon and energy sources: the one-carbon compound methanol and the multi-carbon organic acid succinate, both present in the phyllosphere. Evolved lineages of wild-type cells all increased their capacity for rapid transition between the carbon sources through high frequencies of loss-of-function mutations affecting a previously uncharacterized gene, named\n                  <jats:italic>cstR<\/jats:italic>\n                  for carbon source transition regulator, which encodes an orphan single-domain response receiver. Characterization showed that mutant strains were more competitive bidirectionally in the succinate-methanol transition. Though evolved populations of the \u0394\n                  <jats:italic>efgA<\/jats:italic>\n                  and \u0394\n                  <jats:italic>ttmR<\/jats:italic>\n                  strains, which are defective in the succinate-to-methanol transition, experienced similar phenotypic improvements in carbon-source transitions, we did not observe\n                  <jats:italic>cstR<\/jats:italic>\n                  mutations rise to prominence as extensively or frequently in these lineages.\n                <\/jats:p>\n                <jats:p>\n                  Transcriptomic work revealed loss-of-function to\n                  <jats:italic>cstR<\/jats:italic>\n                  impacted expression of genes involved in motility\/chemotaxis, energy metabolism, and stress response, among others, suggesting that it coordinates responses to metabolic cues that are prevalent in certain carbon source and growth phase transitions. Loss of\n                  <jats:italic>cstR<\/jats:italic>\n                  function did not compromise exogenous formaldehyde tolerance in the \u0394\n                  <jats:italic>efgA<\/jats:italic>\n                  and \u0394\n                  <jats:italic>ttmR<\/jats:italic>\n                  mutants, breaking a previously described tradeoff between these two phenotypes. However, this loss did lead to defects under exposure to certain stressors, including heat, desiccation, oxidative stress agents, and particularly pH stress. Altered levels of NAD+\/NADH across conditions, improved growth under acidic pH, and diminished ATP and increased mortality under heightened pH together support a model where CstR is responsible for coordinating cell signaling to manage the balance between growth and maintaining stress resilience.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    The ability of heterotrophic bacteria to navigate transitions between different carbon sources is critical to their growth and success. Facultative methylotrophs can grow upon multiple types of carbon sources, including reduced single-carbon compounds, and frequently exist in host-beneficial associations on the leaf surfaces of plants. The exposure to the methylotrophic substrate methanol is known to come in temporal bursts for bacteria residing in the phyllosphere. The utilization of methanol is accompanied by obligatory conversion and exposure to the toxic compound formaldehyde. We sought to investigate the capacity of\n                    <jats:italic>Methylobacterium extorquens<\/jats:italic>\n                    to adapt to repetitive transitions between multi-carbon succinate and single-carbon methanol, and in the process identified a novel signaling protein: a single-domain response regulator receiver that mediates cellular growth response to carbon source transitions, at the cost of resistance to certain stressors including oxidative stress and alkaline pH exposure.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.30.735679","type":"posted-content","created":{"date-parts":[[2026,7,2]],"date-time":"2026-07-02T04:35:17Z","timestamp":1782966917000},"source":"Crossref","is-referenced-by-count":0,"title":["Adaptation of\n                  <i>Methylobacterium extorquens<\/i>\n                  to alternating carbon sources identifies the regulator CstR as an intersectional hub of cellular carbon metabolic dynamics and stress response"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6369-4180","authenticated-orcid":false,"given":"Eric L.","family":"Bruger","sequence":"first","affiliation":[{"name":"Department of Plant and Microbial Biology, University of Minnesota, Twin Cities, Saint Paul, MN, United States of America"},{"name":"Microbial and Plant Genomics Institute, University of Minnesota, Twin Cities, Minnesota, United States of America"},{"name":"Biotechnology Institute, University of Minnesota, Twin Cities, Minnesota, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabella","family":"Ikobe","sequence":"additional","affiliation":[{"name":"Department of Plant and Microbial Biology, University of Minnesota, Twin Cities, Saint Paul, MN, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7694-0715","authenticated-orcid":false,"given":"Chandler N.","family":"Hellenbrand","sequence":"additional","affiliation":[{"name":"Department of Plant and Microbial Biology, University of Minnesota, Twin Cities, Saint Paul, MN, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Uly","family":"Zigmund","sequence":"additional","affiliation":[{"name":"Department of Plant and Microbial Biology, University of Minnesota, Twin Cities, Saint Paul, MN, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9012-2260","authenticated-orcid":false,"given":"Jannell V.","family":"Bazurto","sequence":"additional","affiliation":[{"name":"Department of Plant and Microbial Biology, University of Minnesota, Twin Cities, Saint Paul, MN, United States of America"},{"name":"Microbial and Plant Genomics Institute, University of Minnesota, Twin Cities, Minnesota, United States of America"},{"name":"Biotechnology Institute, University of Minnesota, Twin Cities, Minnesota, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026070408300694000_2026.06.30.735679v1.1","doi-asserted-by":"publisher","DOI":"10.1104\/pp.108.4.1359"},{"key":"2026070408300694000_2026.06.30.735679v1.2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0150820"},{"key":"2026070408300694000_2026.06.30.735679v1.3","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0107887"},{"key":"2026070408300694000_2026.06.30.735679v1.4","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0048271"},{"key":"2026070408300694000_2026.06.30.735679v1.5","doi-asserted-by":"publisher","DOI":"10.1093\/jxb\/erm038"},{"key":"2026070408300694000_2026.06.30.735679v1.6","first-page":"2593","article-title":"Environmental controls over methanol emission from leaves","volume":"4","year":"2007","journal-title":"Biogeosci Discuss"},{"key":"2026070408300694000_2026.06.30.735679v1.7","doi-asserted-by":"crossref","first-page":"395","DOI":"10.5194\/bg-4-395-2007","article-title":"Methanol exchange between grassland and the atmosphere","volume":"4","year":"2007","journal-title":"Biogeosciences"},{"key":"2026070408300694000_2026.06.30.735679v1.8","doi-asserted-by":"crossref","first-page":"7555","DOI":"10.5194\/acp-17-7555-2017","article-title":"Effect of mid-term drought on Quercus pubescens BVOCs\u2019 emission seasonality and their dependency on light and\/or temperature","volume":"17","year":"2017","journal-title":"Atmos Chem Phys"},{"key":"2026070408300694000_2026.06.30.735679v1.9","doi-asserted-by":"crossref","unstructured":"Niinemets \u00dc , Reichstein M . 2003. 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Retrieved 2026."},{"key":"2026070408300694000_2026.06.30.735679v1.26","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-0554-6_12"},{"key":"2026070408300694000_2026.06.30.735679v1.27","doi-asserted-by":"publisher","DOI":"10.1186\/1756-0500-1-1"},{"key":"2026070408300694000_2026.06.30.735679v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1183-784"},{"key":"2026070408300694000_2026.06.30.735679v1.29","doi-asserted-by":"crossref","first-page":"e3001208","DOI":"10.1371\/journal.pbio.3001208","article-title":"EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde","volume":"19","year":"2021","journal-title":"PLoS Biol"},{"key":"2026070408300694000_2026.06.30.735679v1.30","doi-asserted-by":"crossref","first-page":"evab082","DOI":"10.1093\/gbe\/evab082","article-title":"Genetic context significantly influences the maintenance and evolution of degenerate pathways","volume":"13","year":"2021","journal-title":"Genome Biol Evol"},{"key":"2026070408300694000_2026.06.30.735679v1.31","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-62703-646-7_6"},{"key":"2026070408300694000_2026.06.30.735679v1.32","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-024-07487-W"},{"key":"2026070408300694000_2026.06.30.735679v1.33","doi-asserted-by":"crossref","first-page":"eadv5109","DOI":"10.1126\/sciadv.adv5109","article-title":"Gaia: An AI-enabled genomic context-aware platform for protein sequence annotation","volume":"11","year":"2025","journal-title":"Sci Adv"},{"key":"2026070408300694000_2026.06.30.735679v1.34","doi-asserted-by":"crossref","unstructured":"Baller J , Kono T , Herman A , Zhang Y . 2019. 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