Community ecology of hot spring cyanobacterial mats: predominant populations and their functional potential

Abstract

Phototrophic microbial mat communities from 60°C and 65°C regions in the effluent channels of Mushroom and Octopus Springs (Yellowstone National Park, WY, USA) were investigated by shotgun metagenomic sequencing. Analyses of assembled metagenomic sequences resolved six dominant chlorophototrophic populations and permitted the discovery and characterization of undescribed but predominant community members and their physiological potential. Linkage of phylogenetic marker genes and functional genes showed novel chlorophototrophic bacteria belonging to uncharacterized lineages within the order Chlorobiales and within the Kingdom Chloroflexi. The latter is the first chlorophototrophic member of Kingdom Chloroflexi that lies outside the monophyletic group of chlorophototrophs of the Order Chloroflexales. Direct comparison of unassembled metagenomic sequences to genomes of representative isolates showed extensive genetic diversity, genomic rearrangements and novel physiological potential in native populations as compared with genomic references. Synechococcus spp. metagenomic sequences showed a high degree of synteny with the reference genomes of Synechococcus spp. strains A and B', but synteny declined with decreasing sequence relatedness to these references. There was evidence of horizontal gene transfer among native populations, but the frequency of these events was inversely proportional to phylogenetic relatedness.

Figure 1

A network map of the core scaffold clusters observed in the Celera assemblies. Scaffolds with similar oligonucleotide frequency profiles that group together in the same cluster are connected by lines colored to indicate the percentage of times they cluster together (in ⩾90% of 100 trials). The isolate genomes included in this analysis are indicated by large white circles, whereas metagenomic scaffolds that contain characterized phylogenetic marker genes are indicated by medium-sized circles colored according to taxonomic grouping. The area of each ellipse is proportional to the amount of metagenomic sequence data contained within each respective scaffold cluster.

Figure 2

Histograms of disjointly recruited (green), jointly recruited syntenous (red) and jointly recruited, non-syntenous (blue) metagenomic sequences that can be associated confidently with a reference genome presented as a function of their % NT ID relative to the reference genomes that recruited them in the BLASTN analysis. % NT ID, percent nucleotide identity.

Figure 3

The PufL and PufM phylogeny and genomic context. The neighbor-joining phylogenetic tree of the PufL and PufM sequences from a novel Chloroflexi metagenomic scaffold from Cluster-6 and from sequenced genomes; asterisks at nodes indicate bootstrap support >50% (1000 replications). A more detailed tree is shown in Supplementary Figure 12. The genomic context of the genes encoding the type-2 reaction center and the light-harvesting polypeptides in the metagenomic scaffolds and chromosomes of Chloroflexus and Roseiflexus isolates is also shown. The jagged lines indicate positions on scaffolds that are interrupted by a lack of overlapping sequence data between contigs.

Figure 4

Position of alignments and the corresponding % NT ID to the Synechococcus sp. A genome of syntenous (red) and non-syntenous (blue) sequences jointly recruited by the Synechococcus sp. A genome from the Mushroom Spring ∼65 °C metagenome. Each end sequence is connected by a line to its clone mate. Sequences suspected to originate from Synechococcus sp. A′-like populations ranging from 83to 92% NT ID are indicated on the right side of the graph. % NT ID, percent nucleotide identity.

Figure 5

Synteny conservation between the Synechococcus sp. strain-A genome and metagenomic sequences and other genomes. The open circles represent alignments of metagenomic sequences relative to the Synechococcus sp. strain-A genome. Metagenome sequences were categorized as Synechococcus A, A′ or B′ based on % NT ID ranges to the Synechococcus spp. strain-A and B′ recruitment bins. The closed circles represent alignments of genome sequences from cultivated cyanobacteria (T. elongatus, Gloeobacter violaceus, Synechococcus sp. strain WH8102, Nostoc sp. strain PCC7120) and the out-group organism Roseiflexus sp. RS-1 relative to the Synechococcus sp. strain-A genome. These genome fragments were generated in silico to represent the same proportion of insert sizes as observed in the distribution of metagenome sequences that were recruited by the Synechococcus sp. strain-A genome. % NT ID, percent nucleotide identity.