297 - The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum

And finally, seeming to complete our journey back in time through the discovery of hydrogenase genetics in Azotobacter chroococcum, this study looks at the structural genes, hupSL.

What They Saw
The sequences were similar to A. vinelandii's hoxKG structural genes. They tried knocking each out, then measuring hydrogen oxidation (with methylene blue) and hydrogen production (with methyl viologen). As expected, hydrogen oxidation in mutants was no higher than negative controls. Surprisingly, they did see hydrogen production in the some of the different mutants with a strong electron donor, but it was less than in the wild-type. Only the mutant with an insertion very close to the start of the hupS gene had no hydrogen production.

What This Means
It seems like a fragment of HupS is sufficient to produce hydrogen with a strong electron donor, but not as much as with both HupS and HupL completely intact.

Reference:

Ford, C. M. et al. The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H₂-uptake hydrogenase of Azotobacter chroococcum. Mol Microbiol 4, 999–1008 (1990).

295 - Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster

This study focuses on the hydrogenase genes in Azotobacter chroococcum. They had already found hupSL encoding the structural genes (equivalent to A. vinelandii's hoxKG I guess), and then the accessory genes hupABYCDE further downstream (homologous to A. vinelandii's hypABFCDE). Now, in between, are the hupZMNOQRTV genes, completing the 16-gene operon.

What They Saw
This set of genes seems to be homologous to A. vinelandii's hoxZMLOQRTV string, making the whole operon very similar in both organisms.

As in previous studies, HupZ (and its analog, HoxZ) seems to be an electron carrier in the membrane, possibly a cytochrome. When they knocked it out, they observed similar results to 070: hydrogenase could oxidize hydrogen with methylene blue as an electron acceptor, but not with oxygen, so HupZ seems to be part of the transport chain to oxygen.

Based on comparison with homologs in other organisms, HupM may help the hydrogenase attach to the membrane, or with processing the subunits. It's unclear. The other genes may be involved in processing or metal stuff. HupR may be another electron-carrying protein. Further study is required.

Reference:

Du, L., Tibelius, K. H., Souza, E. M., Garg, R. P. & Yates, M. G. Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster. Journal of Molecular Biology 243, 549–557 (1994).

051 - The hypE Gene Completes the Gene Cluster for H2-oxidation in Azotobacter vinelandii

As a followup to 050, this study completed the hyp operon by sequencing hypE. They suspected its existence because other sequenced operons always had a hypE after the hypD, so they purified a fragment from Azotobacter vinelandii that hybridized hypD and contained some downstream region.

What They Saw
The open reading frame (ORF) in the downstream region had good homology to hypE genes in other organisms. A few bases of hypD and hypE genes overlap, suggesting that they're translated together so there should be equal amounts present so they can work together equally.

They knocked out hypE by inserting a lacZ and kanamycin resistance cassette, which also let them detect expression (the product of lacZ can break down certain compounds to make color, so color intensity can be measured as a proxy for enzyme activity). It seemed like transcription of all the hox and hyp genes was in the same direction, and knocking out hypE meant that A. vinelandii couldn't oxidize hydrogen anymore.

They also noticed that there was a higher proportion of unprocessed hydrogenase structural component, so HypE might be involved in processing; this seems true in other organisms. It also affects the localization of hydrogenase, soluble or membrane-bound. It may affect pre-protein folding.

What This Means
This study completes the discovery of the whole uptake hydrogenase gene set in A. vinelandii. It's pretty similar to the gene sets in other organisms, including A. chroococcum, and sequencing A. vinelandii's genome confirmed the results.

Reference:

Garg, R. P., Menon, A. L., Jacobs, K., Robson, R. M. & Robson, R. L. The hypE Gene Completes the Gene Cluster for H₂-oxidation in Azotobacter vinelandii. J. Mol. Biol. 236, 390–396 (1994).