Community genomic analysis of an extremely acidophilic sulfur-oxidizing biofilm

Abstract

Highly acidic (pH 0-1) biofilms, known as 'snottites', form on the walls and ceilings of hydrogen sulfide-rich caves. We investigated the population structure, physiology and biogeochemistry of these biofilms using metagenomics, rRNA methods and lipid geochemistry. Snottites from the Frasassi cave system (Italy) are dominated (>70% of cells) by Acidithiobacillus thiooxidans, with smaller populations including an archaeon in the uncultivated 'G-plasma' clade of Thermoplasmatales (>15%) and a bacterium in the Acidimicrobiaceae family (>5%). Based on metagenomic evidence, the Acidithiobacillus population is autotrophic (ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), carboxysomes) and oxidizes sulfur by the sulfide-quinone reductase and sox pathways. No reads matching nitrogen fixation genes were detected in the metagenome, whereas multiple matches to nitrogen assimilation functions are present, consistent with geochemical evidence, that fixed nitrogen is available in the snottite environment to support autotrophic growth. Evidence for adaptations to extreme acidity include Acidithiobacillus sequences for cation transporters and hopanoid synthesis, and direct measurements of hopanoid membrane lipids. Based on combined metagenomic, molecular and geochemical evidence, we suggest that Acidithiobacillus is the snottite architect and main primary producer, and that snottite morphology and distributions in the cave environment are directly related to the supply of C, N and energy substrates from the cave atmosphere.

Figure 1

(a, b) Field photographs of the collection site for snottite sample RS24. Note elemental sulfur (black arrows) occurring in close association with biofilm surfaces. (c) Schematic diagram depicting the formation of sulfidic cave snottites. Snottites form on subaerial cave surfaces in areas exposed to H₂S(g) degassing from circumneutral cave streams. Snottites have extremely acidic pH values (0–1), because they are isolated from limestone cave walls and gypsum corrosion residues that would otherwise buffer the pH >2.

Figure 2

Comparison of RS24 community composition based on FISH and metagenomic data. (a) Taxonomic classification and binning of all metagenomic reads. Using the criteria described in the methods, 40.5% of total metagenome reads were assigned to taxa. ^*groups include reads that cannot be assigned to a more specific taxonomic group. Taxa that make up <0.5% of all matches are omitted from the figure. (b) Community composition based on taxonomic classification of all metagenomic reads, after removing reads assigned to non-specific groups (for example, ‘other bacteria'). (c–e) Community composition based on phylogenetic markers from the metagenome: (c) 31 universal genes (Ciccarelli et al., 2006), (d) 16S rRNA genes and (e) RNA polymerase-β subunit sequences. (f) Community composition determined from FISH cell counts. Numbers in parentheses represent one s.d. FISH probes used to generate the data were THIO1, genus Acidithiobacillus, ACM732, Acidimicrobiaceae family, EUBMIX, bacteria and ARCH915, archaea.

Figure 3

Conceptual model of the biogeochemistry of Frasassi snottite biofilms, based on evidence from metagenomic, rRNA, lipid and geochemical analyses. Carbon fixation, expolymeric substance (EPS) production and acid generation occur largely due to the metabolism of lithoautotrophic, sulfide-oxidizing Acidithiobacillus. Energy substrates (H₂S, O₂) and C and N for primary production (CO₂, NH₃) are from gasses in the cave atmosphere. Trace metals and other non-volatile nutrients such as phosphorous ultimately derive from limestone cave walls, corrosion residues or downward percolating groundwater solutions.

Figure 4

(a) Proportion of reads assigned to COG categories. COG categories are listed to the right. Categories marked by a + or a − symbol are strongly over- or underrepresented in the snottite metagenome, respectively. (b) Standardized abundance scores of COGs classified as P-type ATPases according to the TransportDB (Ren et al., 2004). The COG id, category, description and gene name if available are provided in the legend.