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

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.

Molecular and Regulatory Properties of the Nitrate Reducing Systems of Rhodobacter

Phototrophic bacteria of the genus Rhodobacter possess several forms of nitrate reductase including assimilatory and dissimilatory enzymes. Assimilatory nitrate reductase from Rhodobacter capsulatus E1F1 is cytoplasmic, it uses NADH as the physiological electron donor and reduced viologens as artificial electron donors, and it is coupled to an ammonium-producing nitrite reductase. Nitrate reductase induction requires a high C/N balance and the presence of nitrate, nitrite, or nitroarenes. A periplasmic 47-kDa protein facilitates nitrate uptake, thus increasing nitrate reductase activity. Two types of dissimilatory nitrate reductases have been found in strains from Rhodobacter sphaeroides. One of them is coupled to a complete denitrifying pathway, and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power, thus improving photoanaerobic growth. Periplasmic nitrate reductase does not use NADH as the physiological electron donor and is a 100-kDa heterodimeric hemoprotein that receives electrons through an electron transport chain spanning the plasma membrane. This nitrate reductase is regulated neither by the intracellular C/N balance nor by O2 pressure. The enzyme also exhibits chlorate reductase activity, and both reaction products, nitrite and chlorite, are released almost stoichiometrically into the medium; this accounts for the high resistance to chlorate or nitrite exhibited by this bacterium. Nitrate reductases from both strains seem to be coded by genes located on megaplasmids.