Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti

Abstract

The complete nos region essential for dissimilatory nitrous oxide reduction by the endosymbiotic diazotroph Rhizobium meliloti was identified in a cosmid (pYC7) carrying a 10.1-kb EcoRI fragment of the nod megaplasmid. This gene region was localized by Southern hybridization and Tn5 mutagenesis to within 8 kb downstream from the fixGHIS cluster. Nucleotide sequence determination of a 4.6-kb DNA segment including the structural gene nosZ and its flanking regions showed sequence homology and similarity in genetic organization with the nosRZDFY genes of Pseudomonas stutzeri Zobell. The genes were arranged in three complementation groups, comprising the nosZ structural gene, the nosR regulatory gene, and the nosDFY copper-processing genes. The derived amino acid sequence of the R. meliloti nosZ product (a multi-copper nitrous oxide reductase) was more similar to those of the analogous gene products of Paracoccus and Pseudomonas species than to that of Alcaligenes eutrophus. The nosZ gene was preceded by nosR, which encodes a regulatory protein containing C-terminal cysteine clusters similar to those present in the 4Fe-4S binding region of bacterial ferredoxins. The nosDFY genes, located downstream from nosZ, were identified as copper-processing genes encoding a periplasmic protein, an ATP/GTP-binding protein, and a membrane protein, presumably forming a copper-processing system. A consensus sequence for an Anr-or Fnr-binding site similar to that in the upstream sequence of nosZ in Paracoccus denitrificans or P. stutzeri was absent in R. meliloti. No rpoN-binding site preceding the nos genes was detected, and none of the Tn5 insertions in the nos gene region affected symbiotic N 2 -fixing ability.

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

“…The NosR protein has a periplasmic domain of about 390 amino acids (of a total of 689 residues) and a hydrophobic domain of five transmembrane helices. The Cterminal region features distinct cysteine patterns, the most conspicuous of which make up a polyferredoxin-like structure (12,13,20,26,43). In parallel with our study, it was noted recently by use of a comparative bioinformatics approach that part of NosR has structural similarity to a bacterial flavin mononucleotide (FMN)-binding protein domain (49).…”

“…An important role for N 2 O respiration is attributed to the NosR protein, which is consistently encoded in the nos gene cluster of N 2 O-respiring bacteria. nosR mutants are unable to utilize N 2 O (13,20,43). In most cases, the nosR gene is located adjacent to and upstream of nosZ.…”

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid

“…Other proteins that are potentially important in denitrification are also here: NnrU (SMA1283), required for expression of nir and nor genes; Azu1, a blue copper protein associated with periplasmic nitrite reductase; HemN (SMA1266), which is involved in heme maturation; the Crp͞Fnr-like regulator y protein NnrR (SMA1245); and the regulator NnrS (SMA1252). The previously identified genes encoding nitrous oxide reductase (nos) are also located here (13). From similarity searches, M. loti apparently lacks genes encoding denitrification enzymes.…”

“…Previously discovered nif and fix genes for symbiotic nitrogen fixation also lie within the 275-kb region (12), as do genes encoding nitrous oxide reductase (nos; ref. 13), a functional copy of the groESL chaperonin operon (14), and genes needed for catabolism of betaines (15). However, except for syrB and a locus that inf luences symbiotic effectiveness (16), little was known outside this 275-kb region.…”

Mitigation of postharvest nitrous oxide emissions from soybean ecosystems: a review

“…Cueto et al 1996;Holloway et al 1996;Yamasaki and Sakihama 2000). Very few studies have been conducted on the nosZ gene activity in nodules of legumes other than soybeans.…”