Regulation ofnapGene Expression and Periplasmic Nitrate Reductase Activity in the Phototrophic BacteriumRhodobacter sphaeroidesDSM158

Abstract

Bacterial periplasmic nitrate reductases (Nap) can play different physiological roles and are expressed under different conditions depending on the organism.Rhodobacter sphaeroidesDSM158 has a Nap system, encoded by thenapKEFDABCgene cluster, but nitrite formed is not further reduced because this strain lacks nitrite reductase. Nap activity increases in the presence of nitrate and oxygen but is unaffected by ammonium. Reverse transcription-PCR and Northern blots demonstrated that thenapKEFDABCgenes constitute an operon transcribed as a single 5.5-kb product. Northern blots andnap-lacZfusions revealed thatnapexpression is threefold higher under aerobic conditions but is regulated by neither nitrate nor ammonium, although it is weakly induced by nitrite. On the other hand, nitrate but not nitrite causes a rapid enzyme activation, explaining the higher Nap activity found in nitrate-grown cells. Translationalnap′-′lacZfusions reveal that thenapKandnapDgenes are not efficiently translated, probably due to mRNA secondary structures occluding the translation initiation sites of these genes. Neither butyrate nor caproate increasesnapexpression, although cells growing phototrophically on these reduced substrates show a very high Nap activity in vivo (nitrite accumulation is sevenfold higher than in medium with malate). Phototrophic growth on butyrate or caproate medium is severely reduced in the NapA⁻mutants. Taken together, these results indicate that nitrate reduction inR. sphaeroidesis mainly regulated at the level of enzyme activity by both nitrate and electron supply and confirm that the Nap system is involved in redox balancing using nitrate as an ancillary oxidant to dissipate excess reductant.