Characterization of hydrogen production by an ammonium insensitive Pseudo-revertant of a CBB deficient Rhodobacter capsulatus strain

Abstract

Rhodobacter capsulatus uses nitrogenase or the Calvin–Benson–Bassham (CBB) cycle to balance the redox state of cell by removing excess electrons either through hydrogen production or by fixing CO2. A phosphoribulokinase (PRK) inactivated CBB deficient R. capsulatus strain (YO7) cannot grow properly under photoheterotrophic conditions in the presence ammonium which represses nitrogenase, due to inefficient redox balancing. However this strain gave spontaneous photoheterotrophically competent pseudo-revertants in which ammonium repression of nitrogenase was overcome and that provide redox balance by hydrogen production. YO7-R3, one of these pseudo-revertants, was characterized as to hydrogen production capacity, productivity and the molecular basis of reversion. The results indicate that although the total hydrogen production was decreased by 10% in nitrogen-limited hydrogen producing medium, the maximum hydrogen production rate was increased by 56% comparing to the parental strain YO7. Moreover, in the presence of ammonium, YO7-R3 produced 0.43 ml hydrogen/ml culture (0.015 ml/ml culture × h) as compared to the wild type strains MT1131 and YO7 which fail to produce hydrogen when ammonium is available. Molecular genetic studies revealed that one of the suppressor mutations was an A35V substitution at the N-terminal part of the transcriptional regulatory protein NifA2. The expression of nitrogenase in the YO7-R3 pseudo-revertant was approximately 3.5 times higher than in the wild type strains MT1131 and YO7 in the presence of ammonium.