Identification and Deletion of The Genes Responsible for Hydrogen Production in Thermoanaerobacter Ethanolicus JW200

Background Thermoanaerobacter ethanolicus produces a considerable amount of ethanol from a range of carbohydrates and is an attractive candidate for applications in bioconversion processes. Due to the coupling of hydrogenase activity with fermentation product distribution, understanding hydrogen production of T. ethanolicus, particularly the genes responsible, is valuable for metabolic engineering of the species. Results Utilizing the hydrogenases reported in Thermoanaerobacterium saccharolyticum and Pyrococcus furiosus as templates, BLAST search identified five hydrogenase gene clusters, including two membrane-bound [NiFe] hydrogenases ech and mbh, two cytoplasmic [FeFe] hydrogenases hyd and hydII, and one cytoplasmic [NiFe] hydrogenase shi. The combined deletion of ech, mbh, shi and hydG resulted in a strain that did not produce hydrogen and showed no methyl viologen hydrogenase activity in cell extracts. Strains with deletions of all the hydrogenases except one showed normal hydrogen production. Methyl viologen hydrogenase activity was greatly reduced in all combined deletion strains except the strain with an intact hydG gene. Conclusion High hydrogen production and hydrogenase activities have been observed for T. ethanolicus. Five hydrogenases have been identified. Hydrogen production was eliminated by deleting genes required for all five hydrogenases. Each individual hydrogenase was verified to be capable of producing hydrogen during fermentation, indicating a high degree of redundancy and flexibility in the hydrogenase systems of T. ethanolicus. A large portion of hydrogenase activity is encoded by the [Fe-Fe] hydrogenases.

The Activity of the Stress Modulated Arabidopsis Ubiquitin Ligases PUB46 and PUB48 is Partially Redundant

The Arabidopsis ubiquitin ligases PUB46, PUB47 and PUB48 are encoded by paralogus genes; pub46 and pub48 mutants display increased drought sensitivity compared to wild type (WT). Although the phenotype displayed in the single gene mutants, suggest that each has specific biological activity, PUB46 and PUB48 activity may be also redundant. To test functional redundancy between two gene products requires a double mutant. Unfortunately, the close proximity of the PUB46 and PUB48 gene loci precludes obtaining a double mutant by crossing the available single mutants. We thus applied microRNA technology to reduce the activity of all three gene products of the PUB46-48 subfamily by constructing an artificial microRNA (aMIR) targeted to this subfamily. Expressing aMIR46-48 in WT plants resulted in increased drought-sensitivity, a phenotype resembling that of the single pub46 and pub48 mutants, and enhanced sensitivity to methyl viologen, similar to that observed for the pub46 mutant. Furthermore, the WT plants expressing aMIR46-48 also revealed reduced inhibition by ABA at seed germination, a phenotype not evident in the single mutants. Expressing aMIR46-48 in pub46 and pub48 mutants further enhanced the drought sensitivity of each parental single mutant and of WT expressing aMIR46-48. Thus, whereas the gene-specific activity of the PUB46 and PUB48 E3s is partially redundant in that absence of either E3 leads to drought sensitivity, our ability to eliminate the activity of both PUB46 and PUB48 in the same plant reveals additional gene specific facets of their activity in the reaction to abiotic stress.

Arabidopsis iron superoxide dismutase 1 protects against methyl viologen-induced oxidative stress in a copper-dependent manner

Iron superoxide dismutase 1 (FSD1) was recently characterized as a plastidial, cytoplasmic, and nuclear superoxide dismutase with osmoprotective and antioxidative functions. However, its role in oxidative stress tolerance is not well understood. Here, we characterized the role of FSD1 in response to methyl viologen (MV)-induced oxidative stress in Arabidopsis thaliana. The findings demonstrated that the antioxidative function of FSD1 depends on the availability of Cu2+ in growth media. Prolonged MV exposure led to a decreased accumulation rate of superoxide, higher levels of hydrogen peroxide production, and higher protein carbonylation in the fsd1 mutants and transgenic plants lacking a plastidial pool of FSD1, compared to the wild type. MV led to a rapid increase in FSD1 activity, followed by a decrease. Chloroplastic localization of FSD1 is necessary for these changes. Proteomic analysis showed that the sensitivity of the fsd1 mutants coincided with decreased abundance of ferredoxin and light PSII harvesting complex proteins, with altered levels of signaling proteins. Collectively, the study provides evidence for the conditional antioxidative function of FSD1 and its possible role in signaling.

Significance of AtMTM1 and AtMTM2 for Mitochondrial MnSOD Activation in Arabidopsis

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. We confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMYM1 as they can revive yeast MnSOD activity in yMTM1-mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2-double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1, AtMTM2, and AtMnSOD respond positively to methyl viologen (MV) and metal stress. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. Transient expression of chloroplast-destined AtMnSOD revealed that an evolutionarily conserved activation mechanism, like the chloroplastic-localized MnSOD in some algae, still exists in Arabidopsis chloroplasts. This study strengthens the proposition that AtMTM1 and AtMTM2 participate in the AtMnSOD activation and ion homeostasis.