Metagenomic Sequencing Unravels Gene Fragments with Phylogenetic Signatures of O(2)-Tolerant NiFe Membrane-Bound Hydrogenases in Lacustrine Sediment

Many promising hydrogen technologies utilising hydrogenase enzymes have been slowed by the fact that most hydrogenases are extremely sensitive to O(2). Within the group 1 membrane-bound NiFe hydrogenase, naturally occurring tolerant enzymes do exist, and O(2) tolerance has been largely attributed to changes in iron–sulphur clusters coordinated by different numbers of cysteine residues in the enzyme’s small subunit. Indeed, previous work has provided a robust phylogenetic signature of O(2) tolerance [1], which when combined with new sequencing technologies makes bio prospecting in nature a far more viable endeavour. However, making sense of such a vast diversity is still challenging and could be simplified if known species with O(2)-tolerant enzymes were annotated with information on metabolism and natural environments. Here, we utilised a bioinformatics approach to compare O(2)-tolerant and sensitive membrane-bound NiFe hydrogenases from 177 bacterial species with fully sequenced genomes for differences in their taxonomy, O(2) requirements, and natural environment. Following this, we interrogated a metagenome from lacustrine surface sediment for novel hydrogenases via high-throughput shotgun DNA sequencing using the Illumina™ MiSeq platform. We found 44 new NiFe group 1 membrane-bound hydrogenase sequence fragments, five of which segregated with the tolerant group on the phylogenetic tree of the enzyme’s small subunit, and four with the large subunit, indicating de novo O(2)-tolerant protein sequences that could help engineer more efficient hydrogenases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00284-015-0846-2) contains supplementary material, which is available to authorized users.