(Eco)system proteomics

Metalloenzyme Mechanisms

(Eco)system Proteomics

Biogeochemical Coupling

The entirety of proteins present in pure cultures of single microbes (proteome) or natural mixed microbial communities (metaproteome) directly reflects the functional status of the organisms. For example, highly responsive protein biomarkers indicate nutrient stress levels. As such, proteomics is a powerful tool to understand cellular physiology, metabolic pathways, and adaptive responses to environmental conditions of marine plankton, thus enabling new insights into function, interactions, and ecosystem-level processes.

Protein biomarkers of nutrient stress (nitrogen, iron, phosphorus, cobalt/zinc), showing cohesive responses in different ocean environments (stations 1-4).

Highlights:

Atypical phosphatases drive dissolved organic phosphorus utilization by phosphorus-stressed phytoplankton in the California Current EcosystemIn the ocean, dissolved organic phosphorus (DOP) supports the health and productivity of marine phytoplankton, a phenomenon most often investigated under inorganic phosphate (Pi) scarcity. However, microbial DOP acquisition occurring in Pi replete ocean environments remains poorly understood. Here, we conducted a combination of nutrient addition experiments, alkaline phosphatase (AP) rate measurements, and metatranscriptomics analyses along an onshore-to-offshore gradient in the California Current Ecosystem (CCE), a relatively Pi-rich upwelling region. We found that AP activity (APA) and eukaryotic transcripts for DOP utilization were present throughout the CCE. In bottle incubations, APA was upregulated in response to iron (Fe) and nitrogen (N) additions. Major contributors to these trends included atypical alkaline phosphatases (APaty) of diatoms in the coastal upwelling area, and unclassified non-cytoplasmic P-diesterases (PDEnc) of multiple eukaryotic taxa in the offshore regime. APA and gene expression dynamics were not coupled to phytoplankton growth, suggesting that these organisms experience underlying P stress, a state of cellular metabolism caused by Pi scarcity, even in regions primarily growth-limited by other elements. While APaty and PDEnc were highly abundant among the microbial community phosphatase pool, these genes were missing from a widely used annotation database, highlighting the importance of manual curation for the detection of these atypical and unclassified proteins. Altogether, these results emphasize the functional diversity of phosphatases sustaining microbial community health in diverse and productive marine habitats. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, https://ror.org/021nxhr62, OCE-1637632, OCE-2224726, OCE-1851230, OCE-1756884, OCE- 2326965, OCE-1948042 Simons Foundation, 970820, 678537 National Oceanic and Atmospheric Administration, NA19NOS4780181 Scripps Institution of Oceanography, https://ror.org/04v7hvq31, Earth Section Small Grant

Proteomics reveal temperature-coupled cobalamin homeostasis and pathogenicity in Pseudomonas aeruginosaThe opportunistic pathogen Pseudomonas aeruginosa 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 P. aeruginosa , 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 B12 (cobalamin) during host infection remains unclear. Here, we investigate the environmental isolate P. aeruginosa PA254 using high-resolution global proteomics and cellular cobalamin measurements over a temperature gradient spanning environmental, host-associated, and heat-stress conditions (22–42 °C). PA254 occupies a continuum between an ambient-temperature virulent state characterized by versatile secreted factors, exopolysaccharide-rich biofilms, and planktonic swimmers and surface swarmers; and a host-associated virulent state characterized by potent secretion effectors, 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 strong overexpression of alternative iron acquisition strategies relying on heme and siderophores. Proteins of the cobalamin biosynthetic pathway declined significantly above ambient temperatures, despite constant intracellular B12 concentrations across all conditions. This decoupling of biosynthesis from cellular pools implies prioritization and recycling within B12-dependent processes, while the lack of B12 production at human body temperatures creates avenues for therapeutics interfering with B12 supply. Altogether, this work highlights a gradual rather than stepwise reprogramming of the P. aeruginosa proteome in response to environmental cues, and highlights proteomics as a tool to investigate system level mechanisms of challenging pathogens. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R01GM135709 Simons Foundation, Microbial Oceanography Project Award

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