title: Discovery of [NiFe] hydrogenase genes in metagenomic DNA: cloning and heterologous expression in Thiocapsa roseopersicina.
abstract: Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O2-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.
authors.affiliation: J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA.
authors.forename: Gergely
authors.initials: G
authors.lastname: Maróti
authors.forename: Yingkai
authors.initials: Y
authors.lastname: Tong
authors.forename: Shibu
authors.initials: S
authors.lastname: Yooseph
authors.forename: Holly
authors.initials: H
authors.lastname: Baden-Tillson
authors.forename: Hamilton O
authors.initials: HO
authors.lastname: Smith
authors.forename: Kornél L
authors.initials: KL
authors.lastname: Kovács
authors.forename: Marvin
authors.initials: M
authors.lastname: Frazier
authors.forename: J Craig
authors.initials: JC
authors.lastname: Venter
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
mesh_terms: D020591:Alteromonas / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test; D006864:Hydrogenase / Q000235:genetics*; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D020616:Thiocapsa roseopersicina / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; C043296:nickel-iron hydrogenase; D006864:Hydrogenase