Yu Xu and Donald Bryant
Graduate Student, Biochemistry and Molecular Biology
The Pennsylvania State University
The marine, unicellular cyanobacterium Synechococcus sp. PCC 7002 synthesizes a bidirectional [NiFe] hydrogenase encoded by 5 hox genes (hoxE, hoxF, hoxH, hoxU, hoxY). The maturation of this H2ase requires at least seven hyp/hox gene products (HypA, HypB, HypC, HypD, HypE, HypF and HoxW) . We have studied the expression of these genes by reverse-transcription PCR (RT-PCR) and we will present a summary of those conditions that lead to increased transcription of the bidirectional [NiFe] H2ase activity in Synechococcus sp. PCC 7002. Although secondary transcription initiation sites cannot be excluded, it is likely that the hox and hyp genes form one operon of 13 genes, hypE-hoxEFUY-hyp3-hoxHW-hypABFCD. The hyp3 gene, encoding a conserved hypothetical protein between hoxY and hoxH, is a candidate regulator protein, since a null mutant strain exhibited enhanced hox gene transcription. Treatments and mutations affecting the redox state of the plastoquinone pool can lead to higher levels of hox mRNA in cells. For example, treatment of cells with 2 µM DBMIB, micro-oxic growth conditions or inactivation of the psbB gene all lead to increased levels of hox transcripts in cells. Although LexA has been reported to be a regulator of hox gene transcription in Synechocystis sp. PCC 7002, hox transcript levels were unchanged in a lexA null mutant of Synechococcus sp. PCC 7002. Nickel supplementation of the growth medium did not enhance hox gene transcript levels. Inactivation of the pntA gene, encoding a subunit of the pyridine nucleotide transhydrogenase, and the sdhB gene, encoding a subunit of succinate dehydrogenase, had no effect on hox gene expression. Finally, inactivation of the ndhF1 gene, encoding a subunit of the thylakoid-localized Type I NADH dehydrogenase, produced a small increase in the transcription of the hox genes, which was stimulated considerably when glycerol was added to the growth medium. It is possible that ferredoxin is the principal electron transfer partner of the bidirectional hydrogenase.
A shuttle vector based upon pAQ1, the smallest and most numerous plasmid in Synechococcus sp. PCC 7002, has been constructed and tested for protein over-production. The Clostridium acetobutylicum hydA gene, encoding the [FeFe] hydrogenase, and an nfu gene, encoding a putative scaffold protein for the biogenesis of iron-sulfur clusters, have been overproduced successfully. This shuttle vector system will be a powerful tool for protein overexpression or for characterizing gene functions through complementation assays. Finally, Hyp3, a potential regulator of the hox-hyp operon, has been overproduced with a C-terminal His-tag and purified from Escherichia coli. In vitro assays using Ni-NTA column pull-downs show that two unidentified proteins may interact with Hyp3. The identification of these two proteins could provide hints to the biochemical role of Hyp3 in hydrogenase structure, function, and/or gene expression.