Unravelling core microbial metabolisms in the hypersaline microbial mats of Shark Bay using high-throughput metagenomics

Abstract

Modern microbial mats are potential analogues of some of Earth's earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic next-generation sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.

Figure 1

Sampling site and sample descriptions. (a) The location of sample site in Hamelin Pool. (b) Shark Bay smooth mat sample. (c) Shark Bay pustular mat sample. (d) Shark Bay columnar stromatolite sample.

Figure 2

Community composition across microbial mats, thrombolites and stromatolites based on protein-coding genes (RefSeq Database). (a) Composition and abundance of bacteria, archaea and eukaryote sequences in Shark Bay microbial mats and stromatolites, as well as Highbourne Cay stromatolites and thrombolites. (b) PCA plots were constructed from similarity matrices utilising protein coding sequence recruitment. Proportion variance was explained by each component printed next to the axes labels.

Figure 3

Community composition across microbial mats, thrombolites and stromatolites based on 16 s rDNA. (a) Bacterial composition through 16 S rDNA recruitment from Shark Bay stromatolites metagenomes as well as Highbourne Cay thrombolites and stromatolites. (b) PCA plots were constructed from similarity matrices utilising 16 S rDNA recruitment. Proportion variance was explained by each component printed next to the axes labels. Refseq databases were used for these analyses.

Figure 4

MetaCyc pathway analysis using Metapathways (ePGDBs). (a) Venn diagram of MetaCyc pathways across Shark Bay mats and stromatolites (pustular, smooth, columnar) (b) Shared abundance of nitrogen, primary ethanol and carbon-related pathway among Shark Bay mats and stromatolites.

Figure 5

Principal coordinate analyses comparison of the metabolic potential of Shark Bay and Highbourne Cay metagenomes. (a) PCA plots show the results from the STAMP ANOVA analyses. ANOVA used multiple groups and a post hoc test (Tukey-Kramer at 0.95), an effect size (Eta-squared), and a q-value (<0.05) of 11 active only features for SEED level I. A multiple-test correction using Benjamini-Hochberg FDR was employed for microbial community structure using class-level classification. (b) The chart depicts post-havoc confidence interval plots (>95%) based on ANOVA parameters including SEED level I for Highbourne Cay vs Shark Bay carried out in STAMP. Similarities and differences between Highbourne Cay and Shark Bay microbial mats are shown.