Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier <i>Paracoccus denitrificans</i>

Abstract

Transcriptional adaptation to nitrate-dependent anabolism by <i>Paracoccus denitrificans</i> PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including <i>nasTSABGHC</i> and <i>ntrBC</i> genes. The <i>nasT</i> and <i>nasS</i> genes were cotranscribed, although <i>nasT</i> was more strongly induced by nitrate than <i>nasS</i> The <i>nasABGHC</i> genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The <i>nasTS</i> and <i>nasABGHC</i> transcripts were detected at similar levels with nitrate or glutamate as N-source, but <i>nasABGHC</i> transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The <i>nasT</i> mutant lacked both <i>nasABGHC</i> transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the <i>nasS</i> mutant showed similar levels of the <i>nasABGHC</i> transcript to the wild-type strain and displayed NasG protein and NADH-nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of <i>nasABGHC</i> was dependent on the Ntr system. The <i>ntrBC</i> and <i>ntrYX</i> genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the <i>ntrBCYX</i> genes indicated that while <i>ntrBC</i> genes are required for nitrate assimilation, <i>ntrYX</i> genes can only partially restore growth on nitrate in the absence of <i>ntrBC</i> genes. The existence of a regulation mechanism for nitrate assimilation in <i>P. denitrificans</i>, by which nitrate induction operates at both transcriptional and translational levels, is proposed.