nai

Project Progress

Fieldwork and sampling for geochemistry and microbial genomics was conducted in four sulfidic cave systems hosting extreme acidophile communities, the Frasassi Caves and Grotta Nuova del Rio Garaffo in Italy, and Cueva de Villa Luz and Cueva Luna Azufre in Mexico.

{{ 1 }}

Snottites (pH 0-1 biofilms) were collected from several locations in each cave system. Metagenome sequencing of a snottite sample from the Frasassi Caves is currently in progress.

{{ 2 }}

We have retrieved approximately 20 megabase pairs of sequence to date. At least 70% of the 16S rDNA sequences in the metagenome are most closely related to Acidithiobacillus species, consistent with the dominance of Acidithiobacillus strains in the original acidic biofilm sample measured using fluorescence in situ hybridization. The majority of other sequences in the metagenome are most closely related to genes from Acidithiobacillus ferroxidans, the nearest relative of Frassassi snottite Acidithiobacillus strains that has a complete genome in public databases. Initial assembly of the 20 Mb of short (100 bp) reads from the metagenome produced 1000 contiguous sequences of between 400-4000 base pairs, of which the majority are most closely related to A. ferroxidans. Among the sequences represented in the preliminary metagenome are genes which code for functions relevant to metabolism and survival in the extremely acidic, sulfur-rich environment. These genes include RuBisCO (carbon dioxide fixation), squalene-hopane cyclase (hopanoid production decreases membrane proton permeability), and a gene essential for the metabolism of hydrophobic, solid elemental sulfur. A second group of sequences in the metagenome are most closely related to genes found in the archaeal acidophile lineage Thermoplasmales, consistent with the presence of ~20% Ferroplamsa cells in the sample. Due to the simplicity of the microbial community in the snottite samples, it has been possible to get almost complete coverage of the genome of the dominant population (Acidithiobacillus) at low cost. Assuming a genome size of 3 Mb, the preliminary metagenome data are equivalent to 4.3x coverage of the genome, leaving only an estimated 5% of the genome unsequenced.