Discovery of a [NiFe]-hydrogenase In Metagenomic Sargasso Sea DNA: Cloning and Functional Analysis In Thiocapsa Roseopersicina
Appl Environ Microbiol. 2009 Jul 24;
Using a metagenomic approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes a [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 metagenomically identified hydrogenase 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 co-expressing only two accessory genes hyaD and hupH, without the need to express any of its 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 between 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 HynL large hydrogenase subunit of T. roseopersicina, and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropicity 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.