Arsenite as a source of energy and arsenate as respiratory terminal electron acceptor in Haloarchaea Biofilms of Diamante Lake revealed by metagenomic analysis

RASCOVAN NICOLAS; TONEATTI DIEGO; JAVIER MALDONADO; MARTIN VAZQUEZ; FARIAS MARIA EUGENIA

ROSARIO

Congreso; 4to Congreso Argentino de Bioinformática y Biología Computacional; 2013

Asociación Argentina de Bioinformática y Biología Computacional

Arsenite [As(III)] has been proposed as an ancient source of energy used by the last universal common ancestor (LUCA) based on arsenite oxidase phylogenetic analysis. However, a limited number of Archaea sequences were used to arrive to this conclusion with no experimental support for activity.  Materials and methods  We collected samples of a red biofilm found in the bottom of calcareous rocks in the multi-extreme Diamante Lake and extracted total DNA. We sequenced DNA samples using 454 technology by shotgun metagenome sequencing generating 1 Mi reads. We generated custom databases for all genes related to the arsenic metabolism and other genes of interest and performed stringent BLAST searches to identify these genes in the Diamante metagenome. We also assemble the metagenomic reads to get full-length sequences used for phylogenetic analysis. To give a better support to the hypothesis raised from the bioinformatics analysis, we performed growth experiments in presence of arsenite and arsenate with a Halorubrum strain obtained from Diamante Lake.  Results  We found microbial biofilms composed mainly by Haloarchaea (93%) according to taxonomic analysis of 16S rRNA sequenced recovered by shotgun sequencing. They were forming under extreme conditions such as high arsenic concentration, high pH, high salinity, high UV radiation, low oxygen pressure at 4500 m.a.s.l. in Diamante Lake, Argentina. Metagenomic analysis indicated a high abundance of arsenite oxidases (Aio) and respiratory arsenate [As(V)] reductases (ArrA) coded by Haloarchaea. Phylogenetic analysis revealed a new clade of Aio enzymes in this group of archaea that gives stronger support to the pre-LUCA hypothesis. Our results also showed that ArrA genes in haloarchaea were probably obtained by horizontal gene transfer from sulfate reducing proteobacteria. A pure culture of an Halorubrum strain obtained from this lake showed a better growth in presence of As(III) than in no-arsenic control under light and mostly under dark conditions with an effective As(III) to As(V) transformation, indicating that Aio enzymes are functional.  Conclusions  Our results demonstrate that Archaea, in particular, the Haloarchaea, can benefit from arsenic through As(III) oxidation and give a strong support to the use of As(III) as a primary source of energy in early forms of life.