{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:19:02Z","timestamp":1792790342568,"version":"4.5.0"},"posted":{"date-parts":[[2026,1,27]]},"group-title":"Microbiology","reference-count":67,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T00:00:00Z","timestamp":1769472000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["R01GM135709"],"award-info":[{"award-number":["R01GM135709"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Simons Foundation","award":["Microbial Oceanography Project Award"],"award-info":[{"award-number":["Microbial Oceanography Project Award"]}],"id":[{"id":"https:\/\/ror.org\/01cmst727","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,10,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The opportunistic pathogen\n                  <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                  is highly adaptable to different environmental conditions due to its versatile sensing and metabolic capabilities. Both external temperature and metal availability have a strong influence on the virulence and pathogenicity of\n                  <jats:italic>P. aeruginosa<\/jats:italic>\n                  , but the coupling between these two factors is not well understood. While iron is recognized as major player in nutritional immunity, the role of cobalt and the cobalt-containing vitamin B (cobalamin) during host infection remains unclear. Here, we investigate the environmental isolate\n                  <jats:italic>P. aeruginosa<\/jats:italic>\n                  PA254 using high-resolution global proteomics and cellular cobalamin measurements over a temperature gradient spanning environmental, host-associated, and heat-stress conditions (22\u201342 \u00b0C). PA254 occupies a continuum between an ambient-temperature state characterized by secreted virulence factors, exopolysaccharide-rich biofilms, and planktonic swimmers and surface swarmers; and a host-associated state characterized by a distinct set of virulence factors, alginate-dominated biofilms, and a strong proportion of surface twitching motility. Pathway analyses indicate a shift toward carbon sparing, energy conservation, redox control, and metabolic maintenance during a host-adapted lifestyle, along with the increased production of alternative iron acquisition strategies relying on heme and siderophores. Proteins of the cobalamin biosynthetic pathway declined significantly above ambient temperatures, despite constant intracellular B\n                  <jats:sub>12<\/jats:sub>\n                  concentrations and sustained abundance of B\n                  <jats:sub>12<\/jats:sub>\n                  -dependent enzymes across all conditions. This decoupling of biosynthesis from cellular pools implies temperature-dependent changes in prioritization, biosynthetic efficiency, or recycling within B\n                  <jats:sub>12<\/jats:sub>\n                  -dependent processes, while the lack of B\n                  <jats:sub>12<\/jats:sub>\n                  production at human body temperatures creates avenues for therapeutics interfering with B\n                  <jats:sub>12<\/jats:sub>\n                  supply. Altogether, this work highlights a gradual rather than stepwise reprogramming of the\n                  <jats:italic>P. aeruginosa<\/jats:italic>\n                  proteome in response to environmental cues, and highlights proteomics as a comprehensive tool to investigate system level mechanisms of challenging pathogens.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author Summary<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                    is a bacterial pathogen that causes severe infections in human patients. This organism restructures its cellular machinery significantly between the low temperatures in the environment and the high body temperatures of a human host. Several factors influence its virulence and pathogenicity, such as the supply of essential metals (iron, zinc, cobalt) and vitamins as bacterial nutrients. However, how the availability of cobalt, together with varying temperature, regulates\n                    <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                    remains poorly understood. Here, we applied measurements of proteomics and cobalamin, a cobalt-containing essential vitamin, to understand the changes of this pathogen in response to changing environmental conditions. Our study reveals that this bacterium reacts to varying temperatures by continuously modulating biofilm formation, bacterial movement, cellular metabolism, and virulence expression. We also found that\n                    <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                    \u2019s capacity to produce its own cobalamin declines at host-associated temperatures, while the vitamin\u2019s concentration is kept constant within the cell at all temperatures. These outcomes are important because starving this pathogen of cobalamin could present a novel therapeutic strategy for combating\n                    <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                    infections.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.01.27.701802","type":"posted-content","created":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T03:45:52Z","timestamp":1769571952000},"source":"Crossref","is-referenced-by-count":0,"title":["Proteomics reveal temperature-coupled cobalamin homeostasis and pathogenicity in\n                  <i>Pseudomonas aeruginosa<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0833-3531","authenticated-orcid":false,"given":"Viktoria","family":"Steck","sequence":"first","affiliation":[{"name":"Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America"},{"name":"Department of Biogeochemistry, GEOMAR Helmholtz Centre for Ocean Research, Kiel, Schleswig-Holstein, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5301-8365","authenticated-orcid":false,"given":"Matthew R.","family":"McIlvin","sequence":"additional","affiliation":[{"name":"Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-4841-4690","authenticated-orcid":false,"given":"Annie","family":"Stefanides","sequence":"additional","affiliation":[{"name":"Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4085-7730","authenticated-orcid":false,"given":"Eric A.","family":"Webb","sequence":"additional","affiliation":[{"name":"Biological Sciences, Marine and Environmental Biology Section, University of Southern California, Los Angeles, California, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6040-9295","authenticated-orcid":false,"given":"Mak A.","family":"Saito","sequence":"additional","affiliation":[{"name":"Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026101504051611000_2026.01.27.701802v2.1","unstructured":"Organization WH. 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Impact of temperature-dependent phage expression on Pseudomonas aeruginosa biofilm formation","volume":"7","year":"2021","journal-title":"npj Biofilms Microbiomes"},{"key":"2026101504051611000_2026.01.27.701802v2.7","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-2920.2011.02495.x"},{"key":"2026101504051611000_2026.01.27.701802v2.8","doi-asserted-by":"crossref","unstructured":"Chang C-Y . Surface Sensing for Biofilm Formation in Pseudomonas aeruginosa . Front Microbiol. 2018;8.","DOI":"10.3389\/fmicb.2017.02671"},{"key":"2026101504051611000_2026.01.27.701802v2.9","doi-asserted-by":"publisher","DOI":"10.1007\/s00216-006-0774-x"},{"key":"2026101504051611000_2026.01.27.701802v2.10","doi-asserted-by":"crossref","unstructured":"Saito MA , McIlvin MR . The iron metalloproteome of Pseudomonas aeruginosa under oxic and anoxic conditions. 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Heme protects Pseudomonas aeruginos and Staphylococcus aureus from calprotectin-induced iron starvation. J Biol Chem. 2021;296.","DOI":"10.1074\/jbc.RA120.015975"},{"key":"2026101504051611000_2026.01.27.701802v2.23","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.359265"},{"issue":"7","key":"2026101504051611000_2026.01.27.701802v2.24","doi-asserted-by":"crossref","first-page":"722","DOI":"10.1016\/j.bbagrm.2018.01.018","article-title":"Pseudomonas aeruginosa zinc homeostasis: Key issues for an opportunistic pathogen","volume":"1862","year":"2019","journal-title":"Biochim Biophys Acta, Gene Regul Mech"},{"key":"2026101504051611000_2026.01.27.701802v2.25","doi-asserted-by":"crossref","unstructured":"Meyer ACS , McIlvin MR , Lopez P , Searle BC , Saito MA . Proteomic profiling of zinc homeostasis mechanisms in Pseudomonas aeruginosa through data-dependent and data-independent acquisition mass spectrometry. 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Aerobic Vitamin B12 Biosynthesis Is Essential for Pseudomonas aeruginosa Class II Ribonucleotide Reductase Activity During Planktonic and Biofilm Growth. Front Microbiol. 2018;9.","DOI":"10.3389\/fmicb.2018.00986"},{"key":"2026101504051611000_2026.01.27.701802v2.32","doi-asserted-by":"crossref","unstructured":"Michetti E , Secli V , Astolfi ML , Demingo C , Pacello F , Ammendola S , Battistoni A . Cobalt mitigates zinc-starvation effects in Pseudomonas aeruginosa . 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Pseudomonas aeruginosa Exhibits Deficient Biofilm Formation in the Absence of Class II and III Ribonucleotide Reductases Due to Hindered Anaerobic Growth. 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