Growth-stage-related shifts in diatom endometabolome composition set the stage for bacterial heterotrophy

Abstract

Phytoplankton-derived metabolites fuel a large fraction of heterotrophic bacterial production in the global ocean, yet methodological challenges have limited our understanding of the organic molecules transferred between these microbial groups. In an experimental bloom study consisting of three heterotrophic marine bacteria growing together with the diatom Thalassiosira pseudonana, we concurrently measured diatom endometabolites (i.e., potential exometabolite supply) by nuclear magnetic resonance (NMR) spectroscopy and bacterial gene expression (i.e., potential exometabolite uptake) by metatranscriptomic sequencing. Twenty-two diatom endometabolites were annotated, with nine increasing in internal concentration in the late stage of the bloom, eight decreasing, and five showing no variation through the bloom progression. Some metabolite changes could be linked to shifts in diatom gene expression, as well as to shifts in bacterial community composition and their expression of substrate uptake and catabolism genes. Yet an overall low match indicated that endometabolome concentration was not a good predictor of exometabolite availability, and that complex physiological and ecological interactions underlie metabolite exchange. Six diatom endometabolites accumulated to higher concentrations in the bacterial co-cultures compared to axenic cultures, suggesting a bacterial influence on rates of synthesis or release of glutamate, arginine, leucine, 2,3-dihydroxypropane-1-sulfonate, glucose, and glycerol-3-phosphate. Better understanding of phytoplankton metabolite production, release, and transfer to assembled bacterial communities is key to untangling this nearly invisible yet pivotal step in ocean carbon cycling.

Fig. 1. Time course of microbial abundances.

A Cell abundance based on flow cytometric analysis for co-cultures (5 time points) and axenic cultures (day 15 only) (n = 3). The intensive sampling dates for the early and late bloom comparisons are marked with gray boxes. B Mean relative abundance of bacterial species is based on CFUs (n = 3). The day 0 samples were collected 8 h after inoculation.

Fig. 2. Relative endometabolite abundance in diatom cells.

Abundance is expressed as mean Z-score of per cell concentration in early bloom co-cultures (day 3), late bloom co-cultures (day 15), and axenic late bloom cultures (day 15 AX). Metabolites present in significantly different per cell concentrations are linked by brackets (T-test, p ≤ 0.05, n = 3); no statistical comparisons were made between day 3 and day 15 AX. Row A Endometabolites with significantly higher concentration in day 15 co-cultures compared to day 3 co-cultures; Row B Endometabolites with significantly higher concentration in day 3 co-cultures compared to day 15 co-cultures; Row C Endometabolites not significantly different between day 3 and day 15. Bold font highlights the metabolites accumulating to higher concentrations in 15 d co-cultures compared to 15 d axenic cultures. Plots are colored according to metabolite class. Error bars represent standard deviations. See Table S3 for metabolite intensity per cell.

Fig. 3. Integration of diatom endometabolite and gene expression data for early-stage (left panel) and late-stage (right panel) bloom phases.

Black lines indicate relative gene expression that is significantly higher in one growth stage compared to the other, gray lines indicate expression that is not significantly higher. Green font indicates significantly higher metabolite concentration in early-stage cells, and blue font indicates higher concentration in late-stage cells (see Fig. 2). A Carbon and nitrogen assimilation. B Glycolysis/Gluconeogenesis. C TCA cycle. D Urea cycle. Metabolite abbreviations are as follows: Ala alanine, Ace acetate, Ac-CoA acetyl-CoA, Arg arginine, β-1,3-glu β-1,3-glucan, Citr citrulline, Cyst cysteate, Arg-S Arginine-succinate, Fum fumarate, GlcNac N-acetyl-D-glucosamine, Glu glutamate, Gln glutamine, Glyo glyoxylate, Mal malate, G3P glyceraldehyde-3-phosphate, Gro3P glycerol-3-phosphate, 2-OG oxoglutarate, OAA oxaloacetate, PEP phosphoenolpyruvate, Orn ornithine, Pyr pyruvate, PGA phosphoglycerate, Put putrescine, Spe spermidine.