071 - Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins

This study looked at the genetics of the two open reading frames (ORFs) near the hydrogenase structural genes (hoxZM).

What They Saw
They sequenced the ORFs and compared to known genes. hoxZ seemed to have most homology with genes encoding proteins called rubredoxin from other species. These are typically small proteins that play roles in electron transport, which makes sense. And that's all.

Reference:

Chen, J. & Mortenson, L. Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins. Biochim Biophys Acta 1131, 122–124 (1992).

067 - Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii

This study looks more closely into the genes discovered in previous studies (518,050,051,052) to be required for hydrogenase in Azotobacter vinelandii.

What They Saw
They knocked out different genes (or the whole operon) in the hyp operons of A. vinelandii CA by inserting resistance + lacZ cassettes, then tested these strains for hydrogen oxidation and expression of the genes (via beta-galactosidase activity). The medium they used had a lot more trace elements than typical Burk medium, including nickel.

They found that when lacZ was inserted in the same direction as the gene, they saw expression in all cases, whether fixing nitrogen or not, but if it was inserted in the opposite direction, they didn't. So apparently the genes are expressed to some extent even when not fixing nitrogen.

When they measured hydrogen oxidation, there was about 6x more when fixing nitrogen though, in the wild-type, and none in the mutants. The growth rates of the mutants were similar to the wild-type though, or so they claim without reporting any details.

Finally they tried growing the strains with extra added nickel, because the hydrogenase is a nickel-containing enzyme. This didn't have much effect on the wild-type, but the hypB mutant actually showed some hydrogenase activity in nitrogen-fixing conditions with the extra nickel, and activity was higher with more nickel added.

What This Means
It seems like extra added nickel can substitute for the lack of HypB, so maybe the enzyme has a role in nickel cofactor processing somehow. This makes sense considering the multiple histidines it contains. It's interesting that nickel didn't help activity in non-fixing conditions; maybe there are multiple hydrogenases, active in different conditions, and hypB is required for all but nickel doesn't help some of them.

Reference:

Chen, J. C., Mortenson, L. E. & Seefeldt, L. C. Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii. Curr. Microbiol. 30, 351–355 (1995).

050 - Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism

This study looked into which genes in Azotobacter vinelandii are required for its uptake hydrogenase other than the structural genes, hoxKG.

What They Saw
They sequenced almost 6 kilobases somewhat downstream of the structural genes and found 6 open reading frames (ORFs), the last incomplete, so they sequenced some more to complete it. All 8 seemed to be a single operon. These genes are now called hoxV and hypABFCD (based on homology to E. coli genes).

They predicted that the middle 3 and last ORF (now called hypABF and hypD) would produce proteins good at binding Fe-S clusters. HypB has a histidine-rich region that might be good for binding nickel, and HypF has a zinc finger-like region. They have good homology to genes related to hydrogen metabolism in other organisms. They could be related to hydrogenase regulation or assembly, especially related to Ni and Fe; some (HypBF) have homology to other Ni-related proteins, such as urease.

Reference:

Chen, J. C. & Mortenson, L. E. Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism. Biochim. Biophys. Acta 1131, 199–202 (1992).