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. 2023 Jun 15;11(1):134.
doi: 10.1186/s40168-023-01571-5.

Gene expression dynamics of natural assemblages of heterotrophic flagellates during bacterivory

Aleix Obiol  1 , David López-Escardó  2 , Eric D Salomaki  3 , Monika M Wiśniewska  3   4 , Irene Forn  2 , Elisabet Sà  2 , Dolors Vaqué  2 , Martin Kolísko  3   4 , Ramon Massana  5
Affiliations

Gene expression dynamics of natural assemblages of heterotrophic flagellates during bacterivory

Aleix Obiol et al. Microbiome. .

Abstract

Background: Marine heterotrophic flagellates (HF) are dominant bacterivores in the ocean, where they represent the trophic link between bacteria and higher trophic levels and participate in the recycling of inorganic nutrients for regenerated primary production. Studying their activity and function in the ecosystem is challenging since most of the HFs in the ocean are still uncultured. In the present work, we investigated gene expression of natural HF communities during bacterivory in four unamended seawater incubations.

Results: The most abundant species growing in our incubations belonged to the taxonomic groups MAST-4, MAST-7, Chrysophyceae, and Telonemia. Gene expression dynamics were similar between incubations and could be divided into three states based on microbial counts, each state displaying distinct expression patterns. The analysis of samples where HF growth was highest revealed some highly expressed genes that could be related to bacterivory. Using available genomic and transcriptomic references, we identified 25 species growing in our incubations and used those to compare the expression levels of these specific genes. Video Abstract CONCLUSIONS: Our results indicate that several peptidases, together with some glycoside hydrolases and glycosyltransferases, are more expressed in phagotrophic than in phototrophic species, and thus could be used to infer the process of bacterivory in natural assemblages.

Keywords: Bacterivory; Functional genes; Glycosidases; Heterotrophic flagellates; Metatranscriptomics; Peptidases; Phagocytosis; Unamended incubations.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Cell counts and taxonomic dynamics during the four incubations. A Cell counts of heterotrophic bacteria, Synechococcus, heterotrophic flagellates (HF), and phototrophic flagellates (PF) conducted by epifluorescence microscopy. The background of the plots is colored by the different incubation states of the HF community (“lag”, “growth”, and “decline”). White dots in HF curves represent time points from which we obtained metatranscriptomic data. B Relative read abundance of the main taxonomic eukaryotic groups during the incubations as seen by 18S-V4 mTags. Groups are divided into 2 plots by their overall dynamics: increasing (upper panels) or decreasing (bottom panels) their relative abundance
Fig. 2
Fig. 2
General functional dynamics of the four incubations. A Expression values per sample of the 25 most expressed KOs (KEGG orthologs) in the incubations represented by boxplots colored by incubation state. B Non-metric multidimensional scaling (NMDS) plot using Bray–Curtis dissimilarities between the expression of KOs in the different samples. Samples are grouped by their incubation state. C Heatmap displaying the expression of main categories as represented by KEGG BRITE classifications in all samples (i.e., each column represents a sample). Values shown are computed by scaling TPM values to a 0–100 scale per category and incubation (i.e., TPM values belonging to a category and incubation are divided by their maximum value). Boxplots display the actual TPM values per sample of each category
Fig. 3
Fig. 3
Most expressed functions and genes in the growth state of the incubations. A Expression values (TPM) of the 359 most expressed genes in “growth” samples pooled into custom categories, delineated to report each KO to a single category (see Table S1 for further details). B Fold change (FC) between “lag” and “growth” incubation states and TPM expression values of genes (KOs) annotated as peptidases, translocases (proton pumps), and CAZy enzymes. Dots representing FC larger than the average FC of housekeeping genes are colored in dark blue. KOs displaying overlap in functional annotations (i.e., different KOs associated to the same transcript) needed to be grouped into broader sets (asterisk, see Fig. S5 for further details). “Cysteine peptidases” include cathepsins B, F, H, K, L, O, and X, as well as KDEL-tailed endopeptidase and xylem cysteine peptidase; “aspartyl peptidase” includes cathepsins D and E, phytepsin and saccharopepsin; “serine peptidase” includes cathepsin A, serine carboxypeptidase-like clades I and II and vitellogenic carboxypeptidase-like protein. For CAZy enzymes, “GH7” groups cellulose 1,4-beta-cellobiosidase and cellulase
Fig. 4
Fig. 4
Expression dynamics of a selection of species with genomic data found in the metatranscriptomes. See the full list of the 25 detected species in Table 2 and the display of their expression dynamics in Fig. S7. Values represent pseudocounts per million, obtained after correcting the abundance profiles by gene length and sequencing depth (see the “Methods” section for details)
Fig. 5
Fig. 5
Expression of selected genes in species with different trophic modes. Points represent the relative expression of the gene in a single species and sample. Values were computed by dividing the expression of the selected gene by the total expression for each species and sample. Values are separated by the trophic mode of the species they come from (Table 2). Genes within dashed rectangles have a higher relative expression in phagotrophs than in phototrophs and could be good candidates to infer bacterivory in natural assemblages (see Table S2 for more details)
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