Microbial dark matter filling the niche in hypersaline microbial mats

Abstract

Background: Shark Bay, Australia, harbours one of the most extensive and diverse systems of living microbial mats that are proposed to be analogs of some of the earliest ecosystems on Earth. These ecosystems have been shown to possess a substantial abundance of uncultivable microorganisms. These enigmatic microbes, jointly coined as 'microbial dark matter' (MDM), are hypothesised to play key roles in modern microbial mats.

Results: We reconstructed 115 metagenome-assembled genomes (MAGs) affiliated to MDM, spanning 42 phyla. This study reports for the first time novel microorganisms (Zixibacterial order GN15) putatively taking part in dissimilatory sulfate reduction in surface hypersaline settings, as well as novel eukaryote signature proteins in the Asgard archaea. Despite possessing reduced-size genomes, the MDM MAGs are capable of fermenting and degrading organic carbon, suggesting a role in recycling organic carbon. Several forms of RuBisCo were identified, allowing putative CO₂ incorporation into nucleotide salvaging pathways, which may act as an alternative carbon and phosphorus source. High capacity of hydrogen production was found among Shark Bay MDM. Putative schizorhodopsins were also identified in Parcubacteria, Asgard archaea, DPANN archaea, and Bathyarchaeota, allowing these members to potentially capture light energy. Diversity-generating retroelements were prominent in DPANN archaea that likely facilitate the adaptation to a dynamic, host-dependent lifestyle.

Conclusions: This is the first study to reconstruct and describe in detail metagenome-assembled genomes (MAGs) affiliated with microbial dark matter in hypersaline microbial mats. Our data suggests that these microbial groups are major players in these systems. In light of our findings, we propose H₂, ribose and CO/CO₂ as the main energy currencies of the MDM community in these mat systems. Video Abstract.

Fig. 1

Phylogenetic tree of novel MAGs of the MDM in Shark Bay microbial mats. Maximum likelihood phylogenetic tree of up to 16 concatenated ribosomal proteins (rpL2, 3, 4, 5, 6, 14, 15, 16, 18, 22, 24 and rpS3, 8, 10, 17, 19) was constructed. Bin_245 (Bathyarchaeota) is not included in the tree as it has less than 8 ribosomal proteins. MAGs assigned to different groups are highlighted in different colors on the outer circular stripe. Circles represent genes for various nutrient cycles present in the MAGs (detailed in Additional file 20: Table S3)

Fig. 2

Color-coded table indicating major functional genes identified in Shark Bay mat novel microbiome MAGs. X-axis indicates specific genes likely involved in either nutrient cycling or environmental adaptation and y-axis indicates the specific microbial dark matter MAGs. Key: Grey indicates the partial pathways identified in carbon, sulfur and nitrogen cycles; white indicates the genes and associated pathways are absent. Color panel on the left represents different groups of MDM MAGs according to Fig. 1. ASR assimilatory sulfate reduction, DSR dissimilatory sulfate reduction, SOX sulfur oxidation, DNRA dissimilatory nitrate reduction, ANR assimilatory nitrate reduction, rTCA reverse tricarboxylic cycle, WL pathway Wood-Ljungdahl pathway, Inorg P inorganic phosphorus, Alkal Phos alkaline phosphatase, copAB/cusAB copper efflux systems, opu osmoprotectant transport system, pro glycine betaine/proline transport system, bet choline/glycine/proline betaine transport protein, uvr exinuclease, cph cyanophycin, gltBD/glnAE ammonium assimilation, mtr tetrahydromethanopterin S-methyltransferase, hdr heterodisulfide reductase, mttB trimethylamine-corrinoid protein co-methyltransferase, DGR diversity-generating retroelements

Fig. 3

Putative involvement of Shark Bay MDM MAGs in carbon, sulfur, nitrogen and arsenic metabolisms. CAZy enzymes with different coloured arrows representing various groups of glycoside hydrolase corresponding to Additional file 6: Figure S5. Numbers indicate the quantity and percentage of MAGs encoding for the nitrogen/sulfur cycles and metabolic pathways

Fig. 4

Metabolic potential of Asgard archaea, DPANN archaea, Parcubacteria and Bathyarchaeota in the Shark Bay systems. A metabolic map summarising the genomic potential and metabolic capacities of MAGs affiliated with a Asgard archaea MAGs, b DPANN archaea MAGs, c Parcubacteria MAGs and d Bathyarchaeota (TACK) MAGs. Different colors represent different metabolisms; Purple: central carbon metabolisms; light green: nucleoside salvaging pathway; green: nitrogen metabolism; blue: sulfur metabolism; grey: Wood-Ljungdahl pathway; orange: isoprenoid biosynthesis pathway; hexagons: hydrogenases. Dashed arrows indicate genes that are absent. TCA tricarboxylic acid cycle, THF tetrahydrofolate, THMPT tetrahydromethanopterin, WL pathway Wood-Ljungdahl pathway, PAPS 3′-phosphoadenylyl sulfate, APS adenylyl sulfate, MVA mevalonate. More details can be found in Additional File 7-15: Figure S6-14

Fig. 5

Unrooted maximum-likelihood phylogenetic tree of RuBisCo genes in Shark Bay MDM MAGs. Maximum-likelihood phylogenetic tree constructed with RuBisCo gene found in the MDM MAGs with 1000 bootstrap replications. Archaeal and bacterial type III, type IIIa [64, 65], type IIIb [46], type IIIc [46] and type IV RuBisCo-like protein [66] were identified. Circular dots of different colors represent bootstrap values. RuBisCo sequences in this study and reference sequences are listed in Additional file 21: Table S4

Fig. 6

Proposed ecological roles of MDM in Shark Bay microbial mats. Orange rectangular indicates metabolic pathways, green oval represents MDM in Shark Bay microbial mats while blue circles represent genes found in both a previous study [24] and the current study. CO₂/CO, H₂ and ribose are proposed as main energy currencies of Shark Bay MDM, enclosed in red boxes. Dashed arrows indicate putative metabolic exchange/microbial interactions. Within the dashed box are genes involved in environmental adaptation. HM hydrogenotrophic methanogens, WL pathway Wood-Ljungdahl pathway, 3HP/4HB pathway 3-hydroxypropionate/4-hydroxybutyrate pathway, EPS extracellular polymeric substance, DGR diversity-generating retroelements. A more detailed proposed ecological model of Shark Bay microbial mats combining MDM with other community members is illustrated in Additional File 17: Figure S16