In order to mitigate the ammonia-repression effect of nitrogenase in photosynthetic bacteria, we screened a library of transposon insertion mutants that showed better hydrogen production performance under ammonia-repressed conditions, revealing a relevant regulatory gene. The transposon plasmid pRL27 was engaged for transposon mutagenesis in a cbbR nullmutant of photosynthetic bacteria that has poor growth in the presence of ammonia, and mutants with better growth characteristics were successfully isolated. The hydrogen production yield and rate of one mutant (WRR04) were improved by 14.16% and 31.38%, respectively. BLAST analysis revealed that the interrupted DNA sequence encodes trmB, a transcriptional regulator. Additionally, the mutants WTF05 (trmB(-)) and WTRO6 (trmB(-), cbbR(-)) were constructed, and their H-2 production rates were increased remarkably by 54.72% and 41.44% comparing with the wild strain, respectively. The enhancement of nitrogenase activity demonstrates that the rupture of trmB could partly de-repress the activity of nitrogenase inhibited by ammonium for Rhodobacter sphaeroides. (C) 2018 Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC.