Metagenomic profiling of a microbial assemblage associated with the California mussel: a node in networks of carbon and nitrogen cycling

Abstract

Mussels are conspicuous and often abundant members of rocky shores and may constitute an important site for the nitrogen cycle due to their feeding and excretion activities. We used shotgun metagenomics of the microbial community associated with the surface of mussels (Mytilus californianus) on Tatoosh Island in Washington state to test whether there is a nitrogen-based microbial assemblage associated with mussels. Analyses of both tidepool mussels and those on emergent benches revealed a diverse community of Bacteria and Archaea with approximately 31 million bp from 6 mussels in each habitat. Using MG-RAST, between 22.5-25.6% were identifiable using the SEED non-redundant database for proteins. Of those fragments that were identifiable through MG-RAST, the composition was dominated by Cyanobacteria and Alpha- and Gamma-proteobacteria. Microbial composition was highly similar between the tidepool and emergent bench mussels, suggesting similar functions across these different microhabitats. One percent of the proteins identified in each sample were related to nitrogen cycling. When normalized to protein discovery rate, the high diversity and abundance of enzymes related to the nitrogen cycle in mussel-associated microbes is as great or greater than that described for other marine metagenomes. In some instances, the nitrogen-utilizing profile of this assemblage was more concordant with soil metagenomes in the Midwestern U.S. than for open ocean system. Carbon fixation and Calvin cycle enzymes further represented 0.65 and 1.26% of all proteins and their abundance was comparable to a number of open ocean marine metagenomes. In sum, the diversity and abundance of nitrogen and carbon cycle related enzymes in the microbes occupying the shells of Mytilus californianus suggest these mussels provide a node for microbial populations and thus biogeochemical processes.

Figure 1. Taxonomic composition of surface-associated microbes of tidepool and emergent (bench) mussels.

a. The relative representation of microbial phylogenetic groups in both the tidepool and emergent (bench) mussel samples based on shotgun pyrosequencing. Proportional representation is based on 157,599 total contiguous sequences for the tidepool mussels and 141,293 for the bench mussels. In b., the taxonomic composition as related to enzymes for nitrogen metabolism in Fig. 3.

Figure 2. The relative proportional representation within the most commonly discovered bacterial orders on the surface of the mussel shells.

a. Cyanobacteria, b. α-Proteobacteria, c. β-Proteobacteria, and d. γ-Proteobacteria among the tidepool and emergent mussel shells samples. Y-axes differ due to differences in relative abundance (see Fig. 1a).

Figure 3. The number of proteins matched to nitrogen metabolism functions among multiple metagenome studies.

a. marine metagenomes and b. soil metagenomes. To facilitate comparison among studies, the number of matches is normalized to the ‘discovery rate’ for proteins in the dataset (number of protein matches per 100 fragments). For a. the marine metagenomes are as in Table 2, while b. uses Midwestern soil metagenomes (MG-RAST ids 4441091.3, 4442657.3, 4442656.3, 4442658.3, 4442659.3, 4441281.3, respectively).

Figure 4. The number of proteins involved in CO₂ fixation, including those of the Calvin-Benson cycle among a selection of marine metagenomes.

To facilitate comparison among studies, the number of matches is normalized to the ‘discovery rate’ for proteins in the dataset (number of protein matches per 100 fragments). The point symbols represent the percent of all proteins identified that are used in CO₂ fixation.