Interplay between autotrophic and heterotrophic prokaryotic metabolism in the bathypelagic realm revealed by metatranscriptomic analyses

Abstract

Background: Heterotrophic microbes inhabiting the dark ocean largely depend on the settling of organic matter from the sunlit ocean. However, this sinking of organic materials is insufficient to cover their demand for energy and alternative sources such as chemoautotrophy have been proposed. Reduced sulfur compounds, such as thiosulfate, are a potential energy source for both auto- and heterotrophic marine prokaryotes.

Methods: Seawater samples were collected from Labrador Sea Water (LSW, ~ 2000 m depth) in the North Atlantic and incubated in the dark at in situ temperature unamended, amended with 1 µM thiosulfate, or with 1 µM thiosulfate plus 10 µM glucose and 10 µM acetate (thiosulfate plus dissolved organic matter, DOM). Inorganic carbon fixation was measured in the different treatments and samples for metatranscriptomic analyses were collected after 1 h and 72 h of incubation.

Results: Amendment of LSW with thiosulfate and thiosulfate plus DOM enhanced prokaryotic inorganic carbon fixation. The energy generated via chemoautotrophy and heterotrophy in the amended prokaryotic communities was used for the biosynthesis of glycogen and phospholipids as storage molecules. The addition of thiosulfate stimulated unclassified bacteria, sulfur-oxidizing Deltaproteobacteria (SAR324 cluster bacteria), Epsilonproteobacteria (Sulfurimonas sp.), and Gammaproteobacteria (SUP05 cluster bacteria), whereas, the amendment with thiosulfate plus DOM stimulated typically copiotrophic Gammaproteobacteria (closely related to Vibrio sp. and Pseudoalteromonas sp.).

Conclusions: The gene expression pattern of thiosulfate utilizing microbes specifically of genes involved in energy production via sulfur oxidation and coupled to CO₂ fixation pathways coincided with the change in the transcriptional profile of the heterotrophic prokaryotic community (genes involved in promoting energy storage), suggesting a fine-tuned metabolic interplay between chemoautotrophic and heterotrophic microbes in the dark ocean. Video Abstract.

Keywords: Bathypelagic; Chemoautotrophy; Dissolved inorganic carbon (DIC) fixation; Dissolved organic matter (DOM); Gene expression; Heterotrophy; Labrador Sea Water; Metatranscriptome; Thiosulfate.

Fig. 1

Dissolved inorganic carbon (DIC) fixation in the bathypelagic prokaryotic community amended with ammonia (10 µM), sulfite (1 µM), thiosulfate (1 µM), thiosulfate + DOM (1 µM thiosulfate + 10 µM glucose + 10 µM acetate), DOM (10 µM glucose + 10 µM acetate), and the unamended control (control) after 72 h incubation. The bars represent the average ± standard error of three replicate bottles

Fig. 2

Heatmap representing normalized transcript abundance (transcript per million reads) of selected genes from bathypelagic prokaryotic communities in the different treatments and the original community (A). Light yellow to dark brown color range represents the increment in transcript abundance. Prokaryotic taxa affiliation of different gene transcripts (B). White bars indicate the absence of the corresponding gene. Natural sample stands for natural communities from Labrador Sea Water. Control T1 and Control T72 indicate prokaryotic communities filtered after 1 h and 72 h incubation, respectively. Thiosulfate T1 and Thiosulfate T72 indicate thiosulfate treated prokaryotic communities filtered after 1 h and 72 h incubation, respectively. Thiosulfate + DOM T1 and Thiosulfate + DOM T72 indicate thiosulfate + DOM (1 µM thiosulfate + 10 µM glucose + 10 µM acetate) treated prokaryotic communities filtered after 1 h and 72 h incubation, respectively

Fig. 3

Comparison of z-scores between five selected metabolic categories depicted by color codings, i.e., blue, red, green, maroon, and yellow. Box plot showing the distribution of relative transcript expression data and their interquartile ranges in each sample. Median value of all transcripts expression in each sample is depicted in the form of a horizontal line in every box plot. Significance is indicated by asterisks, i.e., control at T1 and T72 versus treatments

Fig. 4

Summary of key metabolic processes in selected bathypelagic prokaryotic communities amended with A thiosulfate and B thiosulfate + DOM. Red font indicates lower transcript abundance in the amended communities as compared to unamended communities. Metabolic pathways drawn in brown belong to the sulfur utilization pathways. Blue plotted pathways are part of the reductive TCA (tricarboxylic acid) cycle. Purple is used to depict biochemical reactions from the Calvin Benson cycle. Green is used to represent reactions involved in the anaplerotic carbon fixation. Gene abbreviations: ACK Acetate kinase, ACL ATP citrate lyase, ActP Acetate permease, Apr Adenylylsulfate reductase, CFA Cyclopropane fatty acid, CL Cardiolipin, CS Citrate synthase, CysK Cysteine synthase, DSR Dissimilatory-type sulfite reductase, FAS Fatty acid biosynthesis, FRD Fumarate reductase, GBE Glycogen branching enzyme, GSS Glutathione synthetase, IM Inner membrane, LamB Maltoporin, OGOR 2-Oxoglutarate:ferredoxin oxidoreductase, OM Outer membrane, OMP Outer membrane protein, oTCA cycle Oxidative tricarboxylic acid cycle, PA Phosphatidic acid, PE Phosphatidylethanolamine, PEPC Phosphoenolpyruvate carboxylase, PG Phosphatidylglycerol, PS Phosphatidylserine, PTA Phosphate acetyltransferase, PTS system, glucose-specific IIBC component, rTCA Reductive tricarboxylic acid cycle, SAT Sulfate adenylyltransferase, Sox Sulfur oxidation protein, SQR Succinate-ubiquinone oxidoreductase, SulP Sulfate transporter