Metagenomic sequencing of marine periphyton: taxonomic and functional insights into biofilm communities

Abstract

Periphyton communities are complex phototrophic, multispecies biofilms that develop on surfaces in aquatic environments. These communities harbor a large diversity of organisms comprising viruses, bacteria, algae, fungi, protozoans, and metazoans. However, thus far the total biodiversity of periphyton has not been described. In this study, we use metagenomics to characterize periphyton communities from the marine environment of the Swedish west coast. Although we found approximately ten times more eukaryotic rRNA marker gene sequences compared to prokaryotic, the whole metagenome-based similarity searches showed that bacteria constitute the most abundant phyla in these biofilms. We show that marine periphyton encompass a range of heterotrophic and phototrophic organisms. Heterotrophic bacteria, including the majority of proteobacterial clades and Bacteroidetes, and eukaryotic macro-invertebrates were found to dominate periphyton. The phototrophic groups comprise Cyanobacteria and the alpha-proteobacterial genus Roseobacter, followed by different micro- and macro-algae. We also assess the metabolic pathways that predispose these communities to an attached lifestyle. Functional indicators of the biofilm form of life in periphyton involve genes coding for enzymes that catalyze the production and degradation of extracellular polymeric substances, mainly in the form of complex sugars such as starch and glycogen-like meshes together with chitin. Genes for 278 different transporter proteins were detected in the metagenome, constituting the most abundant protein complexes. Finally, genes encoding enzymes that participate in anaerobic pathways, such as denitrification and methanogenesis, were detected suggesting the presence of anaerobic or low-oxygen micro-zones within the biofilms.

Keywords: biodiversity; biofouling; marine biofilms; microbial ecology; next generation sequencing; pathway analysis; shotgun metagenomics; shotgun sequencing.

FIGURE 1

Phylogenetic relationships among the taxa recovered in the periphyton samples. LCA tree based on the BLAST matches of extracted 16S and 18S SSU rRNA gene sequences from periphyton metagenome in the SILVA database v 111. The size of the circles correspond to the abundance of the 16S and 18S sequences, and the circle size of the Arthropoda clade corresponds to 177 sequences.

FIGURE 2

Ranked abundances of phyla identified in the periphyton metagenome. Relative abundances of phyla found in periphyton according to whole metagenome similarity searches to public databases. Red boxes labeled with “DNA” are the relative abundances of phyla according to the blastn matches in the NCBI nt database. Black boxes labeled with “Protein” are the relative abundances of phyla according to the blastx matches in the NCBI nr database.

FIGURE 3

Abundances and linkages of KEGG functions in periphyton. CIRCOS plot based on the functional analysis of periphyton metagenome data by using the KB database as reference. The outer and inner circular heat maps shows the mean relative abundances for the broadest hierarchy level (Level 1) and the second hierarchy level in the KB database, respectively. The top and bottom color codes represent the relative abundances of the outer and inner circular heat maps, respectively. The bars in the innermost circle show the mean relative abundances of genes encoding proteins at the pathway level. The curved lines in the center represent the enzyme-pathway linkage information as indexed in KEGG. Genes encoding enzymes and proteins that are shared among different pathways are illustrated by these curved lines. Gray highlights on top of pathway names are the manually curated biofilm-related subcategories as defined in Table 2. Full names of the abbreviated KB categories shown in this plot can be found in Supplementary Table S13.