Periplasmic nitrate-reducing system of the phototrophic bacterium Rhodobacter sphaeroides DSM 158: transcriptional and mutational analysis of the napKEFDABC gene cluster

Abstract

The phototrophic bacterium Rhodobacter sphaeroides DSM 158 is able to reduce nitrate to nitrite by means of a periplasmic nitrate reductase which is induced by nitrate and is not repressed by ammonium or oxygen. Recently, a 6.8 kb PstI DNA fragment carrying the napABC genes coding for this periplasmic nitrate-reducing system was cloned [Reyes, Roldán, Klipp, Castillo and Moreno-Vivián (1996) Mol. Microbiol. 19, 1307-1318]. Further sequence and genetic analyses of the DNA region upstream from the napABC genes reveal the presence of four additional nap genes. All these R. sphaeroides genes seem to be organized into a napKEFDABC transcriptional unit. In addition, a partial open reading frame similar to the Azorhizobium caulinodans yntC gene and the Escherichia coli yjcC and yhjK genes is present upstream from this nap gene cluster. The R. sphaeroides napK gene codes for a putative 6.3 kDa transmembrane protein which is not similar to known proteins and the napE gene codes for a 6.7 kDa transmembrane protein similar to the Thiosphaera pantotropha NapE. The R. sphaeroides napF gene product is a 16.4 kDa protein with four cysteine clusters that probably bind four [4Fe-4S] centres. This iron-sulphur protein shows similarity to the NapF and NapG proteins of E. coli and Haemophilus influenzae. Finally, the napD gene product is a 9.4 kDa soluble protein which is also found in E. coli and T. pantotropha. The 5' end of the nap transcript has been determined by primer extension, and a sigma70-like promoter has been identified upstream from the napK gene. The same transcriptional start site is found for cells growing aerobically or anaerobically with nitrate. Different mutant strains carrying defined polar and non-polar insertions in each nap gene were constructed. Characterization of these mutant strains demonstrates the participation of the nap gene products in the periplasmic nitrate reduction in R. sphaeroides.

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

“…Furthermore, nitrite accumulation did not match nitrate consumption, indicating that, as observed previously (Sears et al, 1997) have been undertaken in Rhodobacter sp. and Escherichia coli (Darwin et al, 1998;Dobao et al, 1994;Gavira et al, 2002;Liu et al, 1999;Reyes et al, 1998;Stewart et al, 2002;Wang et al, 1999). A notable feature of Nap systems is the variation, both within and between organisms, in the physiological functions, as well as the regulation and expression, of nap.…”

The periplasmic nitrate reductase in Shewanella: the resolution, distribution and functional implications of two NAP isoforms, NapEDABC and NapDAGHB

“…The napB gene encodes the di-c-haem subunit (NapB) that transfers electrons to NapA, with the napAB genes being accompanied by different combinations and arrangements of napCDEFGHKLM genes. The napDABC genes are found in a-, b-and c-proteobacteria (Berks et al, 1995; Delgado et al, 2003; Hettmann et al, 2004;Reyes et al, 1998) and encode what were once defined as the 'essential' NAP proteins (Potter & Cole, 1999), with NapC being a membrane-anchored tetra-haem c-type cytochrome of the NapC/NirT family that mediates electron transport from the quinol pool to NapB (Cartron et al, 2002; Roldan et al, 1998) and NapD being a maturation chaperone for NapA. The napDABC gene cluster is interrupted by genes encoding an iron-sulfurcluster-based NapGH complex in the napFDAGHBC operon of the c-proteobacteria are conserved across all NapAs in Shewanella (Fig.…”

The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA

“…Periplasmic nitrate reductases have been found in many different organisms, where they fulfil different physiological roles, depending on the species. For example, they can function as electron sinks during photosynthesis in Rhodobacter species (Reyes et al, 1996(Reyes et al, , 1998 and during aerobic growth of Paracoccus species on highly reduced carbon sources (Richardson & Ferguson, 1992;Sears et al, 2000), or they may be used during anaerobic respiration, as in E. coli Brondijk et al, 2002;Stewart et al, 2002). In spite of their different roles, there are several similarities between the periplasmic nitrate reductase systems of different species.…”