Metabolism Interactions Promote the Overall Functioning of the Episymbiotic Chemosynthetic Community of Shinkaia crosnieri of Cold Seeps

Abstract

Remarkably diverse bacteria have been observed as biofilm aggregates on the surface of deep-sea invertebrates that support the growth of hosts through chemosynthetic carbon fixation. Growing evidence also indicates that community-wide interactions, and especially cooperation among symbionts, contribute to overall community productivity. Here, metagenome-guided metatranscriptomic and metabolic analyses were conducted to investigate the taxonomic composition, functions, and potential interactions of symbionts dwelling on the seta of Shinkaia crosnieri lobsters in a methane cold seep. Methylococcales and Thiotrichales dominated the community, followed by the Campylobacteriales, Nitrosococcales, Flavobacteriales, and Chitinophagales Metabolic interactions may be common among the episymbionts since many separate taxon genomes encoded complementary genes within metabolic pathways. Specifically, Thiotrichales could contribute to detoxification of hydroxylamine that is a metabolic by-product of Methylococcales. Further, Nitrosococcales may rely on methanol leaked from Methylococcales cells that efficiently oxidize methane. Elemental sulfur may also serve as a community good that enhances sulfur utilization that benefits the overall community, as evidenced by confocal Raman microscopy. Stable intermediates may connect symbiont metabolic activities in cyclical oxic-hypoxic fluctuating environments, which then enhance overall community functioning. This hypothesis was partially confirmed via in situ experiments. These results highlight the importance of microbe-microbe interactions in symbiosis and deep-sea adaptation. IMPORTANCE Symbioses between chemosynthetic bacteria and marine invertebrates are common in deep-sea chemosynthetic ecosystems and are considered critical foundations for deep-sea colonization. Episymbiotic microorganisms tend to form condensed biofilms that may facilitate metabolite sharing among biofilm populations. However, the prevalence of metabolic interactions among deep-sea episymbionts and their contributions to deep-sea adaptations are not well understood due to sampling and cultivation difficulties associated with deep-sea environments. Here, we investigated metabolic interactions among the episymbionts of Shinkaia crosnieri, a dominant chemosynthetic ecosystem lobster species in the Northwest Pacific Ocean. Meta-omics characterizations were conducted alongside in situ experiments to validate interaction hypotheses. Furthermore, imaging analysis was conducted, including electron microscopy, fluorescent in situ hybridization (FISH), and confocal Raman microscopy (CRM), to provide direct evidence of metabolic interactions. The results support the Black Queen Hypothesis, wherein leaked public goods are shared among cohabitating microorganisms to enhance the overall adaptability of the community via cooperation.

Keywords: adaptation; chemosynthesis; cold seep; episymbiont; interaction.

FIG 1

Shinkaia crosnieri seta symbiont sampling information. (A) The geographic location of the cold seep site studied here (i.e., the F site) and image of the S. crosnieri community adjacent to a methane seepage taken by the ROV Faxian. (B) Ventral and dorsal view of S. crosnieri, with seta on the appendages highlighted. (C) Scanning electron microscopy (SEM) image showing the distribution of key episymbiont taxa on the seta based on FISH analyses. Coloring of taxa is as follows: blue, Methylococcales; red, Thiotrichales; yellow, Campylobacteriales; green: Methylococcales or Thiotrichales. (D) Work flow for investigations of this study.

FIG 2

Comparison of order-level 16S rRNA gene phylotype relative abundances among S. crosnieri episymbionts and environmental microbial populations within lobster habitats. SEEP_1 and 2, environmental seep samples; SE_1 to 5, seta samples. Quantification was conducted with reconstructed SSU sequences from metagenomic sequencing data sets using phyloFlash.

FIG 3

Phylogenomic assignment, relative abundance, and metabolic potential of the dominant MAGs of the setae along with reference genomes. Phylogeny of 28 high-quality MAGs recovered from the S. crosnieri setae. The colors of the tree represent order-level taxonomic groups for MAGs. Heat map colors represent completeness of KEGG modules.

FIG 4

Conceptual schemes showing proposed interactions among representative episymbiont orders inhabiting the S. crosnieri setae. (A) Relative expression of critical genes normalized to RPKM values. Gray indicates a gene absent in the transcriptomic data set. (B) Major metabolic pathways predicted to be utilized by the dominant orders in addition to inferred shared goods among symbionts. Individual pathways are indicated with the same color. Detailed descriptions of pathways are provided in Table S2. SRS, sulfur relay system; GSH, glutathione; AA, amino acid.

FIG 5

Transmission electron micrographs of S. crosnieri setae episymbionts. (A) Overview of the setae and associated bacterial cells. (B to D) Observed morphotypes. White arrow, methanotrophs with intracellular stacked membranes that are typical of type I methanotrophs. Black arrow, Thiotrichales cells harboring intracellular sulfur globules.

FIG 6

Metabolic strategies adopted by S. crosnieri episymbionts in oxic-hypoxic fluctuating environments. (A) Schematic of the predicted overall community strategy. During the hypoxic phase, the symbionts oxidize methane and sulfide to methanol and sulfur globules by coupling to nitrate respiration. The stable sulfur and methane intermediates can then be stored as inclusions or released to the environment and taken up by other symbionts. During the oxic phase, episymbionts can use oxygen to oxidize sulfur and methanol to conserve additional energy to support cellular growth. (B) Temporal measurements of DO and methane concentrations inside the S. crosnieri community. (C) Nutrient and dissolved oxygen concentrations of environmental seawaters. (D) Gene expression comparison among episymbionts under oxic and hypoxic conditions within in situ experiments. (E) Confocal Raman microscopy imaging showing elemental sulfur in the episymbiont communities.