Derepressive effect of NH4+ on hydrogen production by deleting the glnA1 gene in Rhodobacter sphaeroides.

Purple non-sulfur (PNS) bacteria produce hydrogen by photofermentation of organic acids in wastewater. However, NH(4)(+) in wastewater may inhibit hydrogen synthesis by repressing the expression and activity of nitrogenase, the enzyme catalyzing hydrogen production in PNS bacteria. In this study, the Rhodobacter sphaeroides 6016 glnA gene encoding glutamine synthetase (GS) was knocked out by homologous recombination, and the effects on hydrogen production and nitrogenase activity were examined. Using 3 mM glutamine as the nitrogen source, hydrogen production (1,245-1,588 mL hydrogen/L culture) and nitrogenase activity were detected in the mutant in the presence of relatively high NH(4)(+) concentrations (15-40 mM), whereas neither was detected in the wild-type strain under the same conditions. Further analysis indicated that high NH(4)(+) concentrations greatly inhibited the expression of nifA and nitrogenase gene in the wild-type strain but not in the glnA1(-) mutant. These observations suggest that GS is essential to NH(4)(+) repression of nitrogenase and that deletion of glnA1 results in the complete derepression of nitrogenase by preventing NH(4)(+) assimilation in vivo, thus relieving the inhibition of nifA and nitrogenase gene expression. Knocking out glnA1 therefore provides an efficient approach to removing the inhibitory effects of ammonium ions in R. sphaeroides and possibly in other hydrogen-producing PNS bacteria.