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 O 2 -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.

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli

“…SDS-PAGE was performed as described by Sambrook & Russell (2001). Gels were either stained with Coomassie using the SimplyBlue SafeStain reagent (Invitrogen) or transferred to nitrocellulose and Western blotting was performed using polyclonal rabbit antibodies specific for T. roseopersicina HynL and HynS as the primary antibodies (Sambrook & Russell, 2001;Maró ti et al, 2009 To heterologously express the AltDE [NiFe] hydrogenase in E. coli, we sought to clone the HynSL gene cluster in an expression vector, pTrc-NSI, which contains an IPTG/ lactose-inducible promoter P Trc . The entire 13 kb operon including hynSL and the 11 surrounding genes was amplified by PCR from AltDE genomic DNA in four consecutive and overlapping pieces (Fig.…”

“…These experiments involved either the transfer of a hydrogenase gene cluster including the entire set of accessory genes (Friedrich et al, 1984;Yagi et al, 1986;Báscones et al, 2000;Lenz et al, 2005) or the transfer of the structural genes and a small subset of the accessory genes with maturation relying upon the accessory proteins of the heterologous host (Rousset et al, 1998;Maró ti et al, 2009). Despite the successful transfer of hydrogenases between several species, there has been limited success in heterologously expressing active [NiFe] hydrogenases in E. coli.…”

“…A number of T. roseopersicina genes encoding accessory proteins have been identified by mutagenesis including hynD, hupK, hypC1, hypC2, hypD, hypE and hypF (Fodor et al, 2001;Kovács et al, 2002). HynD is an endoprotease involved in cleaving the large subunit HynL, while HypCDEF are thought to function in assembly of the active [NiFe] catalytic site (Maró ti et al, 2003;Maró ti et al, 2009).…”

[NiFe] Hydrogenase from Alteromonas macleodii with Unusual Stability in the Presence of Oxygen and High Temperature

“…The putative [NiFe] hydrogenase HynSL from AltDE displayed 60% identity to the large subunit and 64% to the small subunit of the stable [NiFe] hydrogenase (HynSL) from the purple sulfur bacterium Thiocapsa roseopersicina (12,20,30,54). In a previous study, we identified an Alteromonas [NiFe] hydrogenase from the Sargasso Sea, which is 99% identical to HynSL in AltDE (30).…”

“…In a previous study, we identified an Alteromonas [NiFe] hydrogenase from the Sargasso Sea, which is 99% identical to HynSL in AltDE (30). The expression of its genes cloned from the Sargasso Sea in the foreign host T. roseopersicina generated an active hydrogenase capable of producing H 2 (30).…”