Regulation of the nap operon encoding the periplasmic nitrate reductase of Paracoccus pantotrophus: delineation of DNA sequences required for redox control.

Expression of the nap operon, encoding the periplasmic nitrate reductase in Paracoccus pantotrophus, is maximal when cells are grown aerobically, but not anaerobically, with butyrate. Two promoters, termed P1 and P2, control operon expression and the operon-proximal P2 promoter is primarily responsible for increased nap expression in the presence of butyrate. A near-perfect palindromic sequence is centred at +7, relative to the P2 transcription start site. Mutation of this palindrome demonstrated that it is important for regulation of nap operon expression in response to both the redox and the oxidation state of the carbon substrate. A 5' deletion analysis of the nap promoter fused to lacZ revealed that full redox control of expression was retained when the DNA sequence up to position -49 bp, relative to the operon-distal P1 transcription start site, was removed. Encroaching beyond this position resulted in an approximately 4-fold reduction in expression when cells were grown aerobically with butyrate. Additionally, point mutations at position -38 and -45 relative to P1 also resulted in a reduction in expression during aerobic growth with butyrate. A GC-rich region of nap promoter DNA, centred on position -41 relative to the P1 transcription start site is thus proposed as a second DNA motif that is important for efficient expression of the nap operon.

Characterization of the expression and activity of the periplasmic nitrate reductase of Paracoccus pantotrophus in chemostat cultures.

The periplasmic nitrate reductase (Nap) from Paracoccus pantotrophus has a role in cellular redox balancing. Previously, transcription from the nap promoter in P. pantotrophus was shown to be responsive to the oxidation state of the carbon substrate. During batch culture, expression was higher during growth on reduced substrates such as butyrate compared to more oxidized substrates such as succinate. In the present study the effect of growth rate on nap expression in succinate-, acetate- and butyrate-limited chemostat cultures was investigated. In all three cases transcription from the nap promoter and Nap enzyme activity showed a strong correlation. At the fastest growth rates tested for the three substrates nap expression and Nap activity were highest when growth occurred on the most reduced substrate (butyrate > acetate > succinate). However, in all three cases a bell-shaped pattern of expression was observed as a function of growth rate, with the highest levels of nap expression and Nap activity being observed at intermediate growth rates. This effect was most pronounced on succinate, where an approximately fivefold variation was observed, and at intermediate dilution rates nap expression and Nap activity were comparable on all three carbon substrates. Analysis of mRNA prepared from the succinate-grown cultures revealed that different transcription initiation start sites for the nap operon were utilized as the growth rate changed. This study establishes a new regulatory feature of nap expression in P. pantotrophus that occurs at the level of transcription in response to growth rate in carbon-limited cultures.

Rhodobacter capsulatus gains a competitive advantage from respiratory nitrate reduction during light-dark transitions.

Rhodobacter capsulatus N22DNAR(+) possesses a periplasmic nitrate reductase and is capable of reducing nitrate to nitrite under anaerobic conditions. In the absence of light this ability cannot support chemoheterotrophic growth in batch cultures. This study investigated the effect of nitrate reduction on the growth of R. capsulatus N22DNAR(+) during multiple light-dark cycles of anaerobic photoheterotrophic/dark chemoheterotrophic growth conditions in carbon-limited continuous cultures. The reduction of nitrate did not affect the photoheterotrophic growth yield of R. capsulatus N22DNAR(+). After a transition from photoheterotrophic to dark chemoheterotrophic growth conditions, the reduction of nitrate slowed the initial washout of a R. capsulatus N22DNAR(+) culture. Towards the end of a period of darkness nitrate-reducing cultures maintained higher viable cell counts than non-nitrate-reducing cultures. During light-dark cycling of a mixed culture, the strain able to reduce nitrate (N22DNAR(+)) outcompeted the strain which was unable to reduce nitrate (N22). The evidence indicates that the periplasmic nitrate reductase activity supports slow growth that retards the washout of a culture during anaerobic chemoheterotrophic conditions, and provides a protonmotive force for cell maintenance during the dark period before reillumination. This translates into a selective advantage during repeated light-dark cycles, such that in mixed culture N22DNAR(+) outcompetes N22. Exposure to light-dark cycles will be a common feature for R. capsulatus in its natural habitats, and this study shows that nitrate respiration may provide a selective advantage under such conditions.

Hierarchy of carbon source selection in Paracoccus pantotrophus: strict correlation between reduction state of the carbon substrate and aerobic expression of the nap operon.

Paracoccus pantotrophus can express a periplasmic nitrate reductase (Nap) during aerobic growth. A proposed role for this enzyme is the dissipation of excess redox energy during oxidative metabolism of reduced carbon substrates. To investigate the regulation of nap expression, a transcriptional fusion between the nap promoter region of P. pantotrophus and the lacZ gene was constructed. When this fusion was used, analyses showed that transcription from the nap promoter increases as the average reduction state of the carbon atoms increases. Thus, beta-galactosidase activities increase as the carbon source changes in the order succinate-acetate-butyrate. This result was obtained regardless of which of the three carbon sources was used for culture of the inoculum. If two carbon sources were presented together, the beta-galactosidase activity was always the same as it was when the least-reduced carbon source was added alone. This suggests that the regulation is dependent upon metabolism of the more-reduced carbon sources rather than just their presence in the medium. Analysis of culture medium by (1)H nuclear magnetic resonance showed that for aerobic growth P. pantotrophus strictly selected its carbon source in the order succinate-acetate-butyrate. This was reflected by diauxic growth kinetics on medium containing mixed carbon substrates. The regulatory mechanism underpinning such a selection is unknown but is likely to be related to the mechanism which controls the transcription of the nap operon.