Crystallization and preliminary X-ray analysis of the periplasmic nitrate reductase (NapA-NapB complex) from Rhodobacter sphaeroides f. sp. denitrificans.

The periplasmic nitrate reductase of Rhodobacter sphaeroides f. sp. denitrificans is a heterodimer responsible for the first step of reduction in the denitrification process by the conversion of nitrate to nitrite. It consists of a 91 kDa molybdenum-containing catalytic subunit (NapA) and a 17 kDa dihaem cytochrome c (NapB). Crystals of the NapA-NapB complex were obtained by the vapour-diffusion method using ammonium sulfate as precipitant. They belong to the P6(1)22 space group, with unit-cell parameters a = b = 151.9, c = 255.8 A, and contain a single complex in the asymmetric unit. A complete native data set was collected at a synchrotron source to 3.1 A resolution.

Characterization of the reduction of selenate and tellurite by nitrate reductases.

Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V(max) and K(m) were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 micromol x min(-1) x mg(-1) and 0.12 mM were obtained for V(max) and K(m), respectively, whereas the V(max) values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.

Nitrite and nitrous oxide reductase regulation by nitrogen oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106.

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.

Plasmid content and localization of the genes encoding the denitrification enzymes in two strains of Rhodobacter sphaeroides.

Plasmid content and localization of the genes encoding the reductases of the denitrification pathway were determined in the photosynthetic bacterium Rhodobacter sphaeroides forma sp. denitrificans by transverse alternating-field electrophoresis (TAFE) and hybridization with digoxigenin-labeled homologous probes. Two large plasmids of 102 and 115 kb were found. The genes encoding the various reductases are not clustered on a single genetic unit. The nap locus (localized with a napA probe), the nirK gene and the norCB genes encoding the nitrate, nitrite and nitric oxide reductases, respectively, were found on different AseI and SnaBI digested chromosomal DNA fragments, whereas the nos locus (localized with a nosZ probe), encoding the nitrous oxide reductase, was identified on the 115-kb plasmid. Furthermore, the genes encoding two proteins of unknown function, one periplasmic and the other cytoplasmic, but whose synthesis is highly induced by nitrate, were found on a different chromosomal fragment. For comparison, the same experiments were carried out on the well-characterized strain Rhodobacter sphaeroides 2.4.1.

mgpS, a complex regulatory locus involved in the transcriptional control of the puc and puf operons in Rhodobacter sphaeroides 2.4.1.

A new method has been developed in order to select mutants showing decreased puc operon transcription in Rhodobacter sphaeroides 2.4.1. A transcriptional fusion of a promoterless fragment derived from the sacB gene, encoding the levansucrase from Bacillus subtilis, to the upstream regulatory region of the puc operon has been constructed. With appropriate levels of exogenous sucrose, survivors of a sucrose killing challenge have been isolated. Subsequent analysis revealed the presence of both cis- and trans-acting "down" mutations in relation to puc operon expression. One of the trans-acting regulatory mutations was chosen for further study. The original mutation showed less than 2% of the level of puc operon transcription compared with the wild type under aerobic conditions and an 86% reduction under dark dimethyl sulfoxide conditions. This mutation can be complemented by a 3.9-kb BamHI DNA fragment derived from a cosmid contained within a genomic cosmid bank. DNA sequence analysis of this fragment revealed the presence of a 2.8-kb open reading frame, designated mgpS, which would encode a 930-amino-acid protein. The N-terminal portion of the putative protein product presents homologies to proteins of the RNA helicase family. Disruption of the chromosomal mgpS resulted in decreased transcription of both puc and puf, while the presence of mgpS in multicopy in the wild type, 2.4.1., increased puc expression by a factor of 2 under aerobic conditions. Structural analysis of the mgpS locus revealed that expression of mgpS was likely to be complex. A smaller protein containing the 472 C-terminal amino acids of MgpS is able to act by itself as an activator of puc transcription and is expressed independently of the large open reading frame in which it is contained.

Induction by nitrate of cytoplasmic and periplasmic proteins in the photodenitrifier Rhodobacter sphaeroides forma sp. denitrificans under anaerobic or aerobic condition.

The synthesis of nitrate, nitrite, and nitrous oxide reductases is highly enhanced by the addition of nitrate during growth of Rhodobacter sphaeroides forma sp. denitrificans. Contrary to what is observed in many denitrifiers, the synthesis of these enzymes is not repressed by oxygen at concentrations as high as 37% air saturation. When oxygen concentration is increased up to 100% air saturation, the synthesis of nitrite and nitrous oxide reductases is repressed while the nitrate reductase is still synthesized. Two proteins, one periplasmic (35 kDa) and the other cytoplasmic (32 kDa), are also induced by nitrate, but not by trimethylamine-N-oxide or oxygen. Although their function is not yet known, these two proteins appear to be specifically linked to the denitrification pathway. The amino acid sequences of tryptic peptides and of the N-terminal ends of these proteins indicate no significant similarity with the sequences in the Swiss Prot Data Bank. However, a very good alignment is obtained between the amino acid sequences of the periplasmic nitrate reductase of Alcaligenes eutrophus H16 and those of various tryptic peptides of the nitrate reductase of R. sphaeroides forma sp. denitrificans.