Illuminating Key Microbial Players and Metabolic Processes Involved in the Remineralization of Particulate Organic Carbon in the Ocean's Twilight Zone by Metaproteomics

Abstract

The twilight zone (from the base of the euphotic zone to the depth of 1,000 m) is the major area of particulate organic carbon (POC) remineralization in the ocean, and heterotrophic microbes contribute to more than 70% of the estimated remineralization. However, little is known about the microbial community and metabolic activity directly associated with POC remineralization in this chronically understudied realm. Here, we characterized the microbial community proteomes of POC samples collected from the twilight zone of three contrasting sites in the Northwest Pacific Ocean using a metaproteomic approach. The particle-attached bacteria from Alteromonadales, Rhodobacterales, and Enterobacterales were the primary POC remineralizers. Hydrolytic enzymes, including proteases and hydrolases, that degrade proteinaceous components and polysaccharides, the main constituents of POC, were abundant and taxonomically associated with these bacterial groups. Furthermore, identification of diverse species-specific transporters and metabolic enzymes implied niche specialization for nutrient acquisition among these bacterial groups. Temperature was the main environmental factor driving the active bacterial groups and metabolic processes, and Enterobacterales replaced Alteromonadales as the predominant group under low temperature. This study provides insight into the key bacteria and metabolic processes involved in POC remineralization, and niche complementarity and species substitution among bacterial groups are critical for efficient POC remineralization in the twilight zone. IMPORTANCE The ocean's twilight zone is a critical zone where more than 70% of the sinking particulate organic carbon (POC) is remineralized. Therefore, the twilight zone determines the size of biological carbon storage in the ocean and regulates the global climate. Prokaryotes are major players that govern remineralization of POC in this region. However, knowledge of microbial community structure and metabolic activity is still lacking. This study unveiled microbial communities and metabolic activities of POC samples collected from the twilight zone of three contrasting environments in the Northwest Pacific Ocean using a metaproteomic approach. Alteromonadales, Rhodobacterales, and Enterobacterales were the major remineralizers of POC. They excreted diverse species-specific hydrolytic enzymes to split POC into solubilized POC or dissolved organic carbon. Temperature played a crucial role in regulating the community composition and metabolism. Furthermore, niche complementarity or species substitution among bacterial groups guaranteed the efficient remineralization of POC in the twilight zone.

Keywords: metaproteomics; microbe; ocean twilight zone; particulate organic carbon; remineralization.

FIG 1

(a) Taxonomic composition of prokaryote communities at depths of 200 m and 500 m from sites K2, B1, and B9 based on the metaproteomic analysis. Prokaryotic taxa with >1% relative abundance on average are displayed and named in the format, phylum_class_order. (b) The interaction network within the prokaryotic community based on Spearman’s correlation (P < 0.05). The red and green lines represent the positive and negative relationships, respectively. The circle size represents the abundance of the prokaryotic community. (c) Interactive chord diagram visualizing the relationship between prokaryotic communities and functional ontology (COG functions) based on metaproteomes collected at depths of 200 m and 500 m from sites of K2, B1, and B9. The number represents prokaryotic communities and the alphabet represents functional ontology (COG functions). The three dominant bacterial groups of Rhodobacterales, Alteromonadales, and Enterobacterales highlighted in red font are indicated by red arrows in panels a and c.

FIG 2

Vertical distribution and relative abundance of transporters, proteases, and hydrolases in Alteromonadales, Rhodobacterales, and Enterobacterales. The transporter, protease, and hydrolase proteins were grouped according to the predicted substrate specificity of the substrate-binding proteins. Samples are named in the format of site_depth;, for example, K2_200 indicates the sampling location at a depth of 200 m at the K2 site. AA, amino acid; ABC, ATP-binding cassette; AHL, N-acyl homoserine lactone; BCAAs, branched chain amino acids; DLH, dienelactone hydrolase; HAGH, hydroxyacylglutathione hydrolase; FAA, fatty acid amide; GTP, guanosine triohosphte; NagA, N-acetylglucosamine-6-phosphate deacetylase; NCA, N-carbamoyl-L-amino-acid; NCP, N-carbamoylputrescine; PRA-CH, phosphoribosyl-AMP cyclohdryrolase; SADH, succinylarginine dihydrolase; TBDTs, TonB-dependent transporters; TRAP, tripartite ATP-independent periplasmic; TTT, tripartite tricarboxylate.

FIG 3

Pairwise comparisons of environmental factors are shown with a color gradient denoting the Spearman’s correlation coefficients. The taxonomic (based on two independent methods: metaproteomics and 16S rRNA) and functional (based on COGs) community composition was correlated to each environmental factor using the partial (geographic distance-corrected) Mantel test. The edge width corresponds to the Mantel’s r statistic for the corresponding distance correlations, and the edge color denotes the statistical significance based on 9,999 permutations.

FIG 4

(a) The vertical distribution and relative abundance of heat and cold shock proteins present in metaproteomes collected at depths of 200 m and 500 m from the sites K2, B1, and B9. The black dots represent seawater temperatures at different sampling layers. (b) Taxonomic distribution of cold shock proteins. The relative abundances of these proteins from each taxon were averaged for all samples in the pie chart.

FIG 5

Schematic depiction of the key microbial players and essential enzymes involved in the remineralization of POC in the twilight zone. The relative abundances of the main processes/pathways are presented in yellow for each order type, which were calculated by comparing within their own group’s ratio. The relative abundance of different groups of membrane transporters is also presented, summing 100% for each group type. POC, particulate organic carbon; CM, carbohydrate metabolism; AAM, amino acids metabolism; NCM, nitrogen compound metabolism; MM, methane/methanol metabolism; OT, other transporter; TFO, transporter for organic substrate; TFI, transporter for inorganic substrate.