Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3

Abstract

A gene cluster which includes genes required for the expression of nitric oxide reductase in Rhodobacter sphaeroides 2.4.3 has been isolated and characterized. Sequence analysis indicates that the two proximal genes in the cluster are the Nor structural genes. These two genes and four distal genes apparently constitute an operon. Mutational analysis indicates that the two structural genes, norC and norB, and the genes immediately downstream, norQ and norD, are required for expression of an active Nor complex. The remaining two genes, nnrT and nnrU, are required for expression of both Nir and Nor. The products of norCBQD have significant identity with products from other denitrifiers, whereas the predicted nnrT and nnrU gene products have no similarity with products corresponding to other sequences in the database. Mutational analysis and functional complementation studies indicate that the nnrT and nnrU genes can be expressed from an internal promoter. Deletion analysis of the regulatory region upstream of norC indicated that a sequence motif which has identity to a motif in the gene encoding nitrite reductase in strain 2.4.3 is critical for nor operon expression. Regulatory studies demonstrated that the first four genes, norCBQD, are expressed only when the oxygen concentration is low and nitrate is present but that the two distal genes, nnrTU, are expressed constitutively.Denitrification is the reduction of nitrate (NO 3 Ϫ ) to gaseous intermediates, principally nitrogen gas. Nitric oxide (NO), an obligatory intermediate during denitrification, is generated from the one-electron reduction of nitrite (NO 2 Ϫ ) (41). NO reduction is coupled to energy generation (18,29). NO is also a well-known cytotoxic compound, so its production during denitrification has the potential of causing significant cell damage. To mitigate the toxicity of NO, its steady-state concentration during denitrification is maintained at low-nanomolar levels (11). The protein responsible for NO reduction, NO reductase (Nor), catalyzes the reaction 2NO ϩ 2H ϩ 3N 2 O ϩ H 2 O. Nitrous oxide (N 2 O) is an inert, nontoxic intermediate that is frequently the terminal product of denitrification (42). Nor has been purified and shown to be a heterodimeric membrane protein (9,14,16). Metal analysis has shown that it contains only iron in stoichiometric amounts. Recently, the Nor structural genes have been characterized, and sequence analysis revealed that Nor was related to the cytochrome c oxidase superfamily (36). In particular, Nor is most closely related to the heme b-containing oxidases, which are expressed under conditions of low oxygen concentration. It has been suggested that Nor was the original member of this family and that the other members arose by modifying the Nor structure (26).The genetic organization of the region of the chromosome encoding the nor structural genes varies among denitrifiers. In Pseudomonas stutzeri, the two structural genes form a distinct transcriptional unit (43). In Pseudomonas aeruginosa, the two structural genes ...

Characterization of a member of the NnrR regulon in Rhodobacter sphaeroides 2.4.3 encoding a haem–copper protein The GenBank accession number for nnrS is U62403.

“…Previously, only nirK and nor operon expression had been shown to be dependent on NnrR in R. sphaeroides 2.4.3 (Bartnikas et al, 1997 ;Tosques et al, 1997). The expression pattern of nnrS was nearly identical to what has been observed for nirK and nor.…”

“…The pattern of expression of nnrS is very similar to the pattern of expression of both nirK and the nor operon (Bartnikas et al, 1997 ;Tosques et al, 1997). All three genes show limited expression aerobically, a significant increase under microaerobic conditions and maximal expression under microaerobic conditions in medium amended with nitrate.…”

“…The procedure used to generate R. sphaeroides strain 3T19A, which contains a Tn-lacZ insertion in nnrS, has been described previously (Bartnikas et al, 1997). The exact site of insertion was determined by cloning a fragment of DNA containing the site of insertion and sequencing from the Tn region into the nnrS region.…”

“…E. coli S17-1 was used as donor for matings (Simon et al, 1983). E. coli strain CC170, which contains a single chromosomal copy of Tn-lacZ (Manoil, 1990), was used to mutagenize a fragment of DNA containing nnrS (Bartnikas et al, 1997). R. sphaeroides 2.4.3 (ATCC 17025) is the wild-type strain.…”

“…Studies on two other members of the NnrR regulon, nirK and nor, have demonstrated that a sequence about 70 bases upstream of the start of translation and similar to the consensus Fnr-binding site, 5h-TTGAT(N % )AT-CAA-3h (Spiro, 1994), is required for transcriptional activation (Bartnikas et al, 1997 ;Tosques et al, 1997). In nirK this sequence is 5h-TTGCG(N % )CGCAA-3h and in nor this sequence is the same except the C in the first putative half-site is a T. Centred 186 bp upstream of the putative nnrS translation start is the sequence 5h-TTGCG(N % )CACAA-3h, which is similar to the sequences identified upstream of nirK and nor.…”