An Enrichment of CRISPR and Other Defense-Related Features in Marine Sponge-Associated Microbial Metagenomes

Abstract

Many marine sponges are populated by dense and taxonomically diverse microbial consortia. We employed a metagenomics approach to unravel the differences in the functional gene repertoire among three Mediterranean sponge species, Petrosia ficiformis, Sarcotragus foetidus, Aplysina aerophoba and seawater. Different signatures were observed between sponge and seawater metagenomes with regard to microbial community composition, GC content, and estimated bacterial genome size. Our analysis showed further a pronounced repertoire for defense systems in sponge metagenomes. Specifically, clustered regularly interspaced short palindromic repeats, restriction modification, DNA phosphorothioation and phage growth limitation systems were enriched in sponge metagenomes. These data suggest that defense is an important functional trait for an existence within sponges that requires mechanisms to defend against foreign DNA from microorganisms and viruses. This study contributes to an understanding of the evolutionary arms race between viruses/phages and bacterial genomes and it sheds light on the bacterial defenses that have evolved in the context of the sponge holobiont.

Keywords: CRISPR; defense; metagenomes; restriction modification; seawater; sponge microbiome.

FIGURE 1

Barplot showing the relative genomic diversity and associated hierarchical clustering of the metagenomic samples using the Bray–Curtis dissimilarity and complete linkage. The relative abundance is scaled on the x-axis. Taxonomic assignments are based on MG-RAST annotated phylum and class level (indicated by (c)). For the groups contributing ≥5% of relative abundance (Proteobacteria, Bacteroidetes, Firmicutes) class level assignments are given. The group ‘other’ comprises eukaryotes, viruses, archaea, fungi, bacteria at ≤1% abundance and unclassified sequences.

FIGURE 2

(A) Plot of metagenomics samples showing the relative distribution of the GC content of filtered reads. (B) Calculated average genome sizes for bacteria of each metagenomic sample.

FIGURE 3

Heatmap of COG functional categories for the four analyzed metagenomes. The color scale ranges from 0 (black) to 100 (white) and indicates copies per megabase metagenome (cpm). Functional dissimilarities (Bray–Curtis) are indicated by the dendrogram on top. The term “enriched feature” relates to COG classes which are on average at least 1.5-fold higher in seawater (circle) or in sponges (cross) over all sponge samples. COG classes are ordered from high to low copy numbers.

FIGURE 4

Heatmap of defense mechanisms in (A) COG functional categories and (B) additional searches for the phage growth limitation and DNA phosphorothiotation in the COG and Pfam databases. The color scale ranges from 0 (black) to 2 (white) and indicates copies per megabase metagenome. Bray–Curtis dissimilarity is indicated by the dendrogram on top. Enriched feature relates to COG classes which are on average >1.5-fold higher in seawater (circle) or in sponges (cross) over all sponge samples. Similar COG annotations are labeled on the left side of the heatmap and ordered from high to low copy numbers.

FIGURE 5

Barplot showing the abundance of CRISPR arrays and cas genes in the four metagenomes. The x-axis shows their abundance in copy number per megabase.

FIGURE 6

Plots showing the origin (left side of circles) and targets (right side of circles) of spacer sequences for the three sponge datasets. The two outermost rings indicate the percentage of target found for each spacer and vice versa. The inner ring indicates the number of spacers connected to the origin and target, respectively.

FIGURE 7

Presence of types I–III restriction modification systems in the sponge and seawater metagenomes along with additional taxonomic assignments on the phylum and class (indicated through the c in brackets) level. The size of each bubble indicates the gene copy number per megabase. Bray–Curtis dissimilarity for each RMS type is indicated by the dendrograms.