The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration

Abstract

The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66?5 bp downstream of the centre of a putative FNR-like binding site.

Functional characterization of the Bradyrhizobium japonicum modA and modB genes involved in molybdenum transport

“…described by Delgado et al (2003). Nitrite was estimated after diazotization by adding the sulphanilamide/naphthylethylene diamine dihydrochloride reagent (Nicholas & Nason, 1957).…”

“…In addition to assimilating nitrate (NO À 3 ) to NH z 4 (Bergersen, 1977), B. japonicum is also capable of denitrification, that is, the reduction of NO À 3 or nitrite (NO À 2 ) via nitric oxide (NO) and nitrous oxide (N 2 O) to N 2 , when cultured under oxygen-limiting conditions (Bedmar et al, 2005). The first step of denitrification, the anaerobic reduction of nitrate to nitrite, is carried out by the periplasmic Mo-containing nitrate reductase (Delgado et al, 2003).…”

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

“…This was concluded from the fact that its maximal expression varies between the species and no transcription promoter or conserved DNA consensus related to the nitrogen metabolism have been found in all the operons that encode them [1]. Therefore, physiological functions such as minimization of the reducing power under certain conditions of growth [7][8][9][10][11][12], denitrification [13][14][15][16], and scavenging nitrate when it is limited in the medium [17] have been proposed for these enzymes. In contrast to most denitrifying bacteria, the sulfate reducer Desulfovibrio desulfuricans ATCC 27774 (Dd) has Nap as the only NR activity.…”

Comprehensive Assessment of the Regulons Controlled by the FixLJ-FixK2-FixK1Cascade inBradyrhizobium japonicum

“…Until now, the expression from their promoters of 14 genes or operons was known to be controlled either directly or indirectly by FixK 2 . Microaerobically induced targets of FixK 2 include the operons fixNOQP (as mentioned above) and fixGHIS (58), both essential for microaerobic respiration; several heme biosynthesis genes (hemA, hemB, hemN 1 , and hemN 2 ) (15,27,55); denitrification genes (napEDABC, nirK, norCBQD, and nosRZDFYLX) (18,50,60,67,68); and some hydrogen oxidation genes (hup genes) (21). In a cell-free transcription system (in vitro), RNA polymerase, together with purified FixK 2 , was shown to directly activate transcription from the fixNOQP, fixGHIS, and hemN 2 promoters (49).…”