Life in the dark: metagenomic evidence that a microbial slime community is driven by inorganic nitrogen metabolism

Abstract

Beneath Australia's large, dry Nullarbor Plain lies an extensive underwater cave system, where dense microbial communities known as 'slime curtains' are found. These communities exist in isolation from photosynthetically derived carbon and are presumed to be chemoautotrophic. Earlier work found high levels of nitrite and nitrate in the cave waters and a high relative abundance of Nitrospirae in bacterial 16S rRNA clone libraries. This suggested that these communities may be supported by nitrite oxidation, however, details of the inorganic nitrogen cycling in these communities remained unclear. Here we report analysis of 16S rRNA amplicon and metagenomic sequence data from the Weebubbie cave slime curtain community. The microbial community is comprised of a diverse assortment of bacterial and archaeal genera, including an abundant population of Thaumarchaeota. Sufficient thaumarchaeotal sequence was recovered to enable a partial genome sequence to be assembled, which showed considerable synteny with the corresponding regions in the genome of the autotrophic ammonia oxidiser Nitrosopumilus maritimus SCM1. This partial genome sequence, contained regions with high sequence identity to the ammonia mono-oxygenase operon and carbon fixing 3-hydroxypropionate/4-hydroxybutyrate cycle genes of N. maritimus SCM1. Additionally, the community, as a whole, included genes encoding key enzymes for inorganic nitrogen transformations, including nitrification and denitrification. We propose that the Weebubbie slime curtain community represents a distinctive microbial ecosystem, in which primary productivity is due to the combined activity of archaeal ammonia-oxidisers and bacterial nitrite oxidisers.

Figure 1

Photographs of Weebubbie cave environment and microbial slime communities. (a) Aerial view of Weebubbie cave doline. (b) Submerged chamber of Weebubbie cave. (c) Image of slime ‘tentacles' attached to cave walls and roof.

Figure 2

The inferred taxonomic composition of the Weebubbie cave community. Taxonomic designations were assigned at phylum level based on the (a) complete metagenomic data set, and (b) 16S rRNA amplicon data set; species level for the phylum Thaumarchaeota based on the (c) complete metagenomic data set, and (d) 16S rRNA amplicon data set; and species level for the phylum Proteobacteria based on (e) the complete metagenomic data set, and (f) the 16S rRNA amplicon data set. The taxonomic analyses based on the metagenome data used MG-RAST, and sequences were compared with the M5 non-redundant protein database with a maximum e-value cutoff of 1e⁻¹⁵. Unclassified sequences accounted for 7.31% of metagenome reads, and were not included in this figure. The taxonomic analyses based on the 16S rRNA data used MG-RAST, and sequences were compared with the M5 rRNA non-redundant rRNA database with a maximum e-value cutoff of 1e⁻¹⁵. Unclassified sequences accounted for 26.35% of 16S rRNA amplicon reads. Full colour breakdown of the organisms represented in the pie charts in (a) and (b) can be found in Supplementary Figure 1.

Figure 3

MAUVE alignment of the Weebubbie contigs and N. maritimus SCM1 genome. Coloured, boxed sections show regions of synteny, with the connecting lines between the two alignments linking matching regions in the N. maritimus SCM1 genome and Weebubbie contigs. The height of the coloured lines within these regions indicates how similar the gene sequences of the matching genes are, with identical gene sequences having a bar that extends to the top border of the region. Gaps indicate regions of sequence present in one genome, but absent in the other. Panel (a) shows the complete alignment over the entire N. maritimus SCM1 genome while (b) shows a subsection, where a region of aligned Weebubbie contigs contains sequence not observed in N. maritimus (in this panel extended red vertical lines indicate contig boundaries, with one large contig spanning most of the length of this region).

Figure 4

Phylogenetic tree showing the relationship of recovered archaeal Weebubbie cave 16S rRNA gene to that of other archaeal representatives. The maximum likelihood tree was constructed from an alignment of 695 nucleotide positions in the 16S rRNA gene. The tree was inferred with the GTR genetic distance model, using Thermotoga maritima MSB-8 as an outgroup. Bootstraps were calculated based on a total of 1000 replications, with bootstrap values displayed at the nodes.

Figure 5

Functional comparison of the Weebubbie cave metagenome with other selected aquatic metagenomes. A complete cluster analysis with Bray-Curtis distances was performed using normalised abundances of SEED Subsystem categories, comparing the Weebubbie cave metagenome (♦) with freshwater (●) and marine (▪) metagenomes (scale bar indicates log transformed, normalised abundance values). SEED categories from left to right: (1) Fatty Acids, Lipids, and Isoprenoids; (2) Phages, Prophages, Transposable Elements, Plasmids; (3) Cofactors, Vitamins, Prosthetic Groups, Pigments; (4) Virulence, Disease and Defence; (5) Carbohydrates; (6) Protein Metabolism; (7) Clustering-based Subsystems; (8) Respiration; (9) Stress Response; (10) DNA Metabolism; (11) Amino Acids and Derivatives; (12) Miscellaneous; (13) RNA Metabolism; (14) Nitrogen Metabolism; (15) Cell Wall and Capsule; (16) Metabolism of Aromatic Compounds; (17) Membrane Transport; (18) Nucleosides and Nucleotides; (19) Cell Division and Cell Cycle; (20) Sulphur Metabolism; (21) Motility and Chemotaxis; (22) Regulation and Cell Signalling; (23) Phosphorus Metabolism; (24) Potassium Metabolism; (25) Secondary Metabolism; (26) Dormancy and Sporulation; (27) Photosynthesis; (28) Iron Acquisition and Metabolism.

Figure 6

Bacterial and archaeal abundances for key nitrogen metabolism enzymes in the Weebubbie cave metagenome. The total abundance of gene fragments for each enzyme is shown in parentheses next to the enzyme name. The grey and white shading with the boxes indicates the relative proportions of bacterial and archaeal reads matching each enzyme. Solid arrows represent reactions performed by a known enzyme that was found in the Weebubbie cave metagenome. Dotted lines represent known nitrogen transformations for which no evidence was obtained from the Weebubbie cave metagenome.