Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.
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The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed. (
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Nitrate removal in closed-system aquaculture by columnar denitrification.
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The columnar denitrification method of nitrate-nitrogen removal from high-density, closed system, salmonid aquaculture was investigated and found to be feasible. However, adequate chemical monitoring was found to be necessary for the optimization and quality control of this method. When methanol-carbon was not balanced with inlet nitrate-nitrogen, the column effluent became unsatisfactory for closed-system fish culture due to the presence of excess amounts of nitrite, ammonia, sulfide, and dissolved organic carbon. Sulfide production was also influenced by column maturity and residence time. Methane-carbon was found to be unsatisfactory as an exogenous carbon source. Endogenous carbon could not support high removal efficiencies. Freshwater columns adpated readily to an artificial seawater with a salinity of 18% without observable inhibition. Scanning electron microscopy revealed that the bacterial flora was mainly rod forms with the Peritricha (protozoa) dominating as the primary consumers. Denitrifying bacteria isolated from freshwater columns were tentatively identified as species of Pseudomonas and Alcaligenes. A pilot plant column was found to behave in a manner similar to the laboratory columns except that nitrite production was never observed. (
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Biochemical characterization and solution structure of nitrous oxide reductase from Alcaligenes xylosoxidans (NCIMB 11015).
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Nitrous oxide reductase (N2OR) is the terminal enzyme involved in denitrification by microbes. No three-dimensional structural information has been published for this enzyme. We have isolated and characterised N2OR from Alcaligenes xylosoxidans (AxN2OR) as a homodimer of M(r) 134,000 containing seven to eight copper atoms per dimer. Comparison of sequence and compositional data with other N2ORs suggests that AxN2OR is typical and can be expected to have similar domain folding and subunit structure to other members of this family of enzymes. We present synchrotron X-ray-scattering data, analysed using a model-independent method for shape restoration, which gave a approximately 20 A resolution structure of the enzyme in solution, providing a glimpse of the structure of any N2OR and shedding light on the molecular architecture of the molecule. The specific activity of AxN2OR was approximately 6 mumol of N2O reduced.min-1. (mg of protein)-1; N2OR activity showed both base and temperature activation. The visible spectrum exhibited an absorption maximum at 550 nm with a shoulder at 635 nm. On oxidation with K3Fe(CN)6, the absorption maximum shifted to 540 nm and a new shoulder at 480 nm appeared. Reduction under anaerobic conditions resulted in the formation of an inactive blue form of the enzyme with a broad absorption maximum at 650 nm. As isolated, the enzyme shows an almost featureless EPR spectrum, which changes on oxidation to give an almost completely resolved seven-line hyperfine signal in the gII region, g = 2.18, with AII = 40 G, consistent with the enzyme being partially reduced as isolated. Both the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a CuA centre. (
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PCR detection of genes encoding nitrite reductase in denitrifying bacteria.
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Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222. (
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The blue copper-containing nitrite reductase from Alcaligenes xylosoxidans: cloning of the nirA gene and characterization of the recombinant enzyme.
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The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed in Escherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein. (
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Transcriptional organization of the czc heavy-metal homeostasis determinant from Alcaligenes eutrophus.
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The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. DNA sequencing of the region upstream of the czcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5' end of czcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of czc. Additional open reading frames upstream of czc had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide czcN message and a 400-nucleotide czcI message were found, and the presence of 6, 200-nucleotide czcCBA message (D. Van der Lelie et al., Mol. Microbiol. 23:493-503, 1997) was confirmed. Induction of czcN, czcI, czcCBA, and czcDRS followed a similar pattern: transcription was induced best by 300 microM zinc, less by 300 microM cobalt, and only slightly by 300 microM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from czcN to czcC and from czcD to czcS, but not between czcA and czcD nor between czcS and a 131-amino-acid open reading frame following czcS. The CzcR putative response regulator was purified and shown to bind in the 5' region of czcN. A reporter strain carrying a czcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were DeltaczcR and DeltaczcS mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn2+, (ii) induction of the beta-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of czcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation. (
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Re-evaluation of the primary structure of Ralstonia eutropha phasin and implications for polyhydroxyalkanoic acid granule binding.
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Sequence analysis of several cDNAs encoding the phasin protein of Ralstonia eutropha indicated that the carboxyl terminus of the resulting derived protein sequence is different from that reported previously. This was confirmed by: (1) sequencing of the genomic DNA; (2) SDS-PAGE and peptide analysis of wild-type and recombinant phasin; and (3) mass spectrometry of wild-type phasin protein. The results have implications for the model proposed for the binding of this protein to polyhydroxyalkanoic acid granules in the bacterium. (
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Phospholipid bound to the flavohemoprotein from Alcaligenes eutrophus.
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The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipid-binding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the -sc/beta/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1- and sn-2-fatty acid chains. The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipid-binding protein. Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively. (
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