Purple non-sulfur (PNS) bacteria produce hydrogen under photoheterotrophic conditions. The produced hydrogen, which is catalyzed by Mo-nitrogenase, is re-oxidized by a membrane-bound uptake hydrogenase. To improve hydrogen-producing efficiency, the hupSL gene that encodes the uptake hydrogenase, and its positive regulator gene hupR were knocked out in Rhodobacter sphaeroides 6016. Compared to the wild-type strain, the hydrogen yields of the hupSL(-) and hupR(-) mutants each increased by 11%. When the nifA gene, which encodes a specific transcriptional activator for all other nif genes was over-expressed in the hupR(-) mutant, the hydrogen yield increased by 20%. In addition, hydrogen production was detected in the nifA/hupR(-) mutant in the presence of 14 mM NH(4)(+), whereas the wild-type strain produced no hydrogen under the same conditions. Crown Copyright (C) 2010 Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu. All rights reserved.