Metagenomics reads the DNA of a whole community at once. Applied to microbialites and mats over the past decade, it has shown who lives in them, what they are capable of, and which questions are still open. Each summary below links to the original paper.
Freshwater microbialites
Most early molecular work on microbialites was marine. Studies of two Canadian freshwater systems, a cold lake and a flooded mine pit, filled a large gap.
Pavilion Lake microbialites host a distinct community
Microbialites from Pavilion Lake, British Columbia, differed from the neighbouring sediment and water in community profile, metabolic potential and pathway distribution, which suggests a true microbialite community rather than a passive collection of lake microbes. They were enriched in cyanobacteria and acidobacteria, likely contributors to mineral formation. Genes associated with viral infection, including CRISPR-Cas, phage shock and phage excision genes, were abundant, a sign that viruses exert selection on the community.
The microbialites change shape dramatically with depth, yet their metagenomes did not vary significantly with depth or morphology. Something other than gene content, such as gene expression or the physical environment, likely shapes their form.
White RA III, Chan AM, Gavelis GS, Leander BS, Brady AL, Slater GF, Lim DSS, Suttle CA (2016). Metagenomic analysis suggests modern freshwater microbialites harbor a distinct core microbial community. Frontiers in Microbiology 6:1531. doi:10.3389/fmicb.2015.01531
Mine-pit microbialites look like polar mats
At Clinton Creek, Yukon, rapidly growing microbialites in an abandoned, flooded asbestos pit were dominated by proteobacteria: Alphaproteobacteria in the microbialites, Gammaproteobacteria in the sediment. Filamentous cyanobacteria and sulfate-reducing Deltaproteobacteria were present too, consistent with a shared, global microbialite microbiome. Compared with sediment, the microbialites carried more genes for photosynthesis, pigment biosynthesis and carbon fixation.
Their overall functional potential was strikingly similar to non-lithifying mats from the Canadian High Arctic and Antarctica, and distinct from Yellowstone hot-spring mats. Because the pit is so young, it offers a view of microbialite formation on a human timescale.
White RA III, Power IM, Dipple GM, Southam G, Suttle CA (2015). Metagenomic analysis reveals that modern microbialites and polar microbial mats have similar taxonomic and functional potential. Frontiers in Microbiology 6:966. doi:10.3389/fmicb.2015.00966
Genomes of isolates
Culturing complements metagenomics. A culturing effort on Pavilion Lake microbialites yielded more than a hundred bacterial isolates, two of which now have complete genomes.
Agrococcus pavilionensis, a new species
Strain RW1, isolated from a thrombolite collected 20 m down in Pavilion Lake, provided the first complete genome for its genus: a 2.6 million base-pair chromosome plus two plasmids. The genome shows considerable genetic promiscuity, with a prophage-like element and plasmid genes for integration, transposition and heavy-metal stress. A carotenoid gene cluster likely explains the yellow pigment typical of the genus, and a predicted pathway that releases ammonia from amino acids could raise the carbonate saturation state and favour precipitation.
White RA III, Gavelis G, Soles SA, Gosselin E, Slater GF, Lim DSS, Leander B, Suttle CA (2018). Frontiers in Microbiology 9:2180. doi:10.3389/fmicb.2018.02180. Draft genome: White RA III, Grassa CJ, Suttle CA (2013), Genome Announcements. PMC3695436
Exiguobacterium chiriqhucha RW2, built for almost anything
Co-isolated with RW1 from the same cold microbialite, strain RW2 grows from 4 to 50 °C and from pH 5 to 11, the widest ranges known for the genus. Its single 3.0 million base-pair chromosome encodes cold- and heat-shock systems, osmolyte uptake and synthesis, and ion antiporters. It is a robust biofilm former and carries genes that convert amino acids to ammonia, which could help drive carbonate precipitation. It was first described as E. pavilionensis.
White RA III, Soles SA, Gavelis G, Gosselin E, Slater GF, Lim DSS, Leander B, Suttle CA (2019). Frontiers in Microbiology 9:3189. doi:10.3389/fmicb.2018.03189. Draft genome: White RA III, Grassa CJ, Suttle CA (2013), Genome Announcements. PMC3738901
Shark Bay mats and stromatolites
Two decades of work by the Burns group, with Visscher and White as collaborators, have made Shark Bay one of the best-characterised microbial ecosystems on Earth.
Stromatolites growing in Hamelin Pool, Shark Bay.Photo: Paul Harrison, CC BY-SA 3.0, via Wikimedia Commons
Early surveys found unexpected diversity
Molecular surveys of Hamelin Pool stromatolites and mats revealed a high diversity of bacteria and salt-loving archaea, including candidate divisions then known only from DNA sequences, and described how specific populations were distributed through the stromatolite ecosystem.
Burns BP, Goh F, Allen M, Neilan BA (2004). Environmental Microbiology 6:1096–1101. doi. Allen MA, Goh F, Burns BP, Neilan BA (2009). Geobiology 7:82–96. doi. Goh F, Allen MA, Leuko S, Kawaguchi T, Decho AW, Burns BP, Neilan BA (2009). The ISME Journal. Find.
The first shotgun metagenomes
Across smooth mats, pustular mats and columnar stromatolites, three phyla dominated: proteobacteria, cyanobacteria and bacteroidetes. Shark Bay communities and their metabolic potential differed from those of Highborne Cay in the Bahamas. Alternative carbon-fixation routes, the reductive TCA cycle and the 3-hydroxypropionate/4-hydroxybutyrate pathway, were well represented in Shark Bay but not in Highborne Cay mats, and archaea were abundant enough to suggest important roles in ecosystem function.
Ruvindy R, White RA III, Neilan BA, Burns BP (2016). Unravelling core microbial metabolisms in the hypersaline microbial mats of Shark Bay using high-throughput metagenomics. The ISME Journal 10:183–196. doi:10.1038/ismej.2015.87
Niches at the millimetre scale
Slicing mats into 2 mm layers and pairing sequencing with chemical measurements showed how communities change with depth. Smooth mats had more diverse archaea, dominated by Parvarchaeota, with hydrogen- and methyl-using methanogens; pustular mats were enriched in Halobacteria. Oxygen turnover and sulfate reduction ran up to four times faster in smooth mats, and methane production peaked in the oxygenated layers, pointing to oxygen-free micro-niches near the surface.
Wong HL, Smith D-L, Visscher PT, Burns BP (2015). Scientific Reports 5:15607. doi. Wong HL, Visscher PT, White RA III, Smith D-L, Patterson MM, Burns BP (2017). Scientific Reports 7:46160. doi.
Genomes reconstructed from the mat
The first millimetre-scale functional metagenomes of Shark Bay mats produced 87 medium- to high-quality metagenome-assembled genomes, including potentially novel members of the Asgard archaea (Thorarchaeota and Lokiarchaeota). The data support models in which biogeochemical cycles and adaptive responses to salinity above 60, drying and ultraviolet light are partitioned among layers and taxa.
Wong HL, White RA III, Visscher PT, Charlesworth JC, Vázquez-Campos X, Burns BP (2018). Disentangling the drivers of functional complexity at the metagenomic level in Shark Bay microbial mat microbiomes. The ISME Journal 12:2619–2639. doi:10.1038/s41396-018-0208-8
Microbial dark matter fills the niches
The first detailed look at microbial dark matter in hypersaline mats reconstructed 115 genomes spanning 42 phyla. Despite small genomes, these microbes can ferment and degrade organic carbon, and many have a high capacity for producing hydrogen. The authors propose hydrogen, ribose and carbon monoxide or dioxide as the main energy currencies of this community, and describe novel eukaryote signature proteins.
Wong HL, MacLeod FI, White RA III, Visscher PT, Burns BP (2020). Microbial dark matter filling the niche in hypersaline microbial mats. Microbiome 8:135. doi:10.1186/s40168-020-00910-0
What is switched on, and when
Metagenomes show potential; RNA shows activity. Metatranscriptomes from mats at Nilemah followed how the active community, its most abundant transcripts and its photosynthetic and chemosynthetic capacity changed with mat type, the day–night cycle and season.
Campbell MA, Grice K, Visscher PT, Morris T, Wong HL, White RA III, Burns BP, Coolen MJL (2020). Functional gene expression in Shark Bay hypersaline microbial mats: adaptive responses. Frontiers in Microbiology 11:560336. doi:10.3389/fmicb.2020.560336
Gypsum mats, natural products and sunscreens
Supratidal mats at Blue Holes yielded 117 genomes, including DPANN and Asgard archaea, with antiviral defences, osmoprotection and metal and ultraviolet resistance. Other studies found an abundance of biosynthetic gene clusters and traced the genes and chemistry of the ultraviolet-screening compounds scytonemin and mycosporine-like amino acids.
Kindler GS et al. (2022). FEMS Microbiology Ecology 98:fiab158. doi. Chen R et al. (2020). Frontiers in Microbiology 11:1950. doi. D’Agostino PM et al. (2019). Environmental Microbiology 21:702–715. doi.
Viruses
Viruses were almost absent from the microbialite literature until recently. They now have their own page.
The first viral metagenome from Shark Bay stromatolites
Direct sequencing of the viral fraction showed single-stranded DNA viruses with a diversity similar to that at Highborne Cay, and two putative genomes related to the Genomoviridae. The cellular fraction was enriched in antiviral defence genes, CRISPR-Cas, BREX and DISARM. This was the first evidence for viruses in Shark Bay stromatolites.
White RA III, Wong HL, Ruvindy R, Neilan BA, Burns BP (2018). Viral communities of Shark Bay modern stromatolites. Frontiers in Microbiology 9:1223. doi:10.3389/fmicb.2018.01223
A viral role in turning mats to stone
White, Visscher and Burns proposed that viruses, directly or indirectly, affect the microbial metabolisms that govern the transition from microbial mat to stromatolite. Read the full hypothesis.
White RA III, Visscher PT, Burns BP (2021). Between a rock and a soft place: the role of viruses in lithification of modern microbial mats. Trends in Microbiology 29:204–213. doi:10.1016/j.tim.2020.06.004
Biogeochemistry and lithification
Sequencing says what microbes could do. Measuring what they actually do, at the scale of a millimetre, is the specialty of the Visscher group.
Sulfur cycling builds the hard layers
In Bahamian stromatolites, soft and hard laminae alternate. Microscale measurements tied the hard, micritic laminae to sulfur cycling, with the highest sulfate-reduction rates in the layers that lithify.
Stromatolites at Highborne Cay grow by alternating between burial under fresh sediment and pauses in which microbes harden the surface. That alternation is what produces their millimetre-scale layering. See the growth cycle.
A mat turns to stone only when minerals form faster than they dissolve. Two coupled components decide this. One is the alkalinity engine: the combined effect of microbial metabolism and environmental conditions on how saturated the water is with calcium carbonate. The other is the EPS matrix, which first binds calcium and later serves as a template on which crystals can nucleate.
Dupraz C, Visscher PT (2005). Trends in Microbiology 13:429–438. Find. Dupraz C, Reid RP, Braissant O, Decho AW, Norman RS, Visscher PT (2009). Earth-Science Reviews 96:141–162. doi.
Mats that never see oxygen
At Laguna La Brava in Chile’s Salar de Atacama, purple mats live in permanently oxygen-free, arsenic-laden and sulfidic water, under intense ultraviolet light and wide temperature swings. Visscher and colleagues measured how these mats cycle elements and whether they can build rock, and proposed these arsenic- and sulfur-based phototrophic mats as a link to life in the anoxic Archean.
Visscher PT, Gallagher KL, Bouton A, Farias ME, Kurth D, Sancho-Tomás M, et al. (2020). Modern arsenotrophic microbial mats provide an analogue for life in the anoxic Archean. Communications Earth & Environment 1:24. doi:10.1038/s43247-020-00025-2
Methods in plain language
Amplicon sequencing
Reading one marker gene, usually the 16S rRNA gene, from every cell in a sample. It gives a census of who is present.
Shotgun metagenomics
Sequencing all the DNA in a sample. It shows which genes, and therefore which capabilities, the community has.
Metagenome-assembled genome (MAG)
A genome rebuilt by sorting assembled DNA fragments into bins that belong to one population. MAGs let researchers study microbes that have never been grown in the lab.
Metatranscriptomics
Sequencing RNA instead of DNA, to see which genes are actually switched on at a given moment.
Viral metagenomics
Separating virus particles from cells and sequencing their genomes.
Microelectrodes
Needle-thin sensors that measure oxygen, sulfide and pH at sub-millimetre resolution, revealing the chemistry each layer experiences.
