Purification of the dissimilative nitrate reductase of pseudomonas fluorescens and the cloning and sequencing of its corresponding genes* 1 (original) (raw)
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Nitrate reductases inEscherichia coli
Antonie van Leeuwenhoek, 1994
Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems. The two enzymes are encoded by distinct operons located within two different loci on the E. coli chromosome. The narGHJl operon, encoding nitrate reductaseA, is located in the chiC locus at 27 minutes, along with several functionally related genes: narK, encoding a nitrate/nitrite antiporter, and the narXL operon, encoding a nitrate-activated, two component regulatory system. The narZYWVoperon, encoding nitrate reductase Z, is located in the chlZlocus located at 32.5 minutes, a region which includes a narK homologue, narU, but no apparent homologue to the narXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both the narGHJI operon and the narK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while the narZYWVoperon and the narU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase in E. coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.
The Bacterial Nitrate Reductases
Nitrate reductase of Escherichia coli has been solubilized from particle fractions by a double treatment : first an alkali-acetone precipitation and then a solubilization in a buffered sodium deoxycholate. The enzyme has been purified 50-fold with a yield of 1 to 2O/,. Polyacrylamide-gel electrophoresis and ultracentrifugation show the preparation to be nearly homogeneous. The protein has a molecular weight of 320000 and an iso-electric point a t pH 4.2. The absorbance which increases continuously from 600 to 280 nm does not reveal the presence of a heme or a flavin group but the spectrum resembles that of some bacterial ferroproteins. The estimation of metals indicates 1.5 atoms Mo and 20 atoms Fe per mole. Approximately one labile sulfide is found per iron atom. It is likely that nitrate reductase A is an iron-sulfur protein containing molybdenum. The purified protein uses as substrates NO,-and C10,and as electron donors reduced benzyl-and methyl-viologens, FMNH, and FADH, but not NADH or NADPH. It should be pointed out that the solubilization does not modify the enzymatic properties of nitrate reductase. CN-and N,are strong inhibitors. Azide is a competitive inhibitor and the nitrate reductase affinity for this inhibitor is 1000 times greater than for nitrate. The type of inhibition observed and the metal chelating nature of the inhibitor suggest that a metal, Fe or Mo, or both, play a role in the formation of enzyme-substrate complex.
Molecular & general genetics : MGG, 1990
The structural genes for NRZ, the second nitrate reductase of Escherichia coli, have been sequenced. They are organized in a transcription unit, narZYWV, encoding four subunits, NarZ, NarY, NarW and NarV. The transcription unit is homologous (73% identity) to the narGHJI operon which encodes the genes for NRA, the better characterized nitrate reductase of this organism. The level of homology between the corresponding polypeptides ranges from 69% for the NarW/NarJ pair to 86% for the NarV/NarI pair. The NarZ polypeptide contains the five conserved regions present in all other known molybdoproteins of E. coli and their relative order is the same. The NarY polypeptide, which contains the same four cysteine clusters in the same order as NarH, is probably an electron transfer unit of the complex. Upstream of narZ, an open reading frame, ORFA, is present which could encode a product which has homology (73% identity) with the COOH-terminal end of NarK. The ORFA-narZ intergenic region, howe...
1999
A nested PCR primed by four degenerate oligonucleotides was developed for the specific amplification of sequences from the napA gene encoding the periplasmic nitrate reductase. This approach was used to amplify fragments of the napA gene from 10 Pseudomonas species and one Moraxella sp., previously shown to be able to express the periplasmic nitrate reductase activity, from Rhodobacter capsulatus and from community DNA extracted from a freshwater sediment. Amino acid sequences encoded by the napA fragments were compared to one another and to the corresponding regions of related enzymes. This comparison indicates that the amplification protocol is specific for its intended target. The napA sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment community. All tested Gram-negative strains capable of aerobic nitrate respiration were found to have periplasmic nitrate reductase genes. However, some strains which have and express the genes are incapable of aerobic nitrate respiration. The PCR primers and amplification protocols described will be useful in future studies of nitrate respiring populations.
Journal of Bacteriology, 1999
Bacterial denitrification is expressed in response to the concurrent exogenous signals of low-oxygen tension and nitrate or one of its reduction products. The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium Pseudomonas stutzeri ATCC 14405. We have identified and isolated from this organism the chromosomal region encoding the two-component sensor-regulator pair NarXL and found that it is linked with the narG operon for respiratory nitrate reductase. The same region encodes two putative nitrate or nitrite translocases, NarK and NarC (the latter shows the highest similarity to yeast [Pichia] and plant [Nicotiana] nitrate transporters), and the nitrate-regulated transcription factor, DnrE, of the FNR family. The roles of NarX and NarL in nitrate respiration were studied with deletion mutants. NarL activated the transcription of narG, narK, anddnrE but did not affect the denitrification regulons for the respiratory substrate...
Journal of Bacteriology, 2000
By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase, luxAB, to genes of Pseudomonas putida KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking nasB suggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction of nasB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of the gltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.
Canadian Journal of Microbiology, 2009
Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. Inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The role of the two dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expression were examined by using a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 hr. In contrast, nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 hr and reached early stationary phase in liquid culture after 120 hr.
Molecular evolution of the dissimilatory nitrate reductase: a survey to assess its diversity
The narG gene, which codes for the a subunit of Escherichia coli's dissimilatory nitrate reductase (NR), was utilized to construct two DNA probes which were subsequently used in a survey of nitrate-reducing bacteria. Both of these probes had previously been reported to share homology with the genomic DNA of prominent denitrifiers such as Pseudomonas aeruginosa and P. stutzeri. Of the 15 species surveyed which contain a respiratory NR (or enzyme analogous to typical dissimilatory NRs), 9 demonstrated homology with the probes (6 different genera). The NR enzyme appears to be genetically diverse like other NOX reductases that have been characterized. These molecular probes have also identified a second narG-\ike gene in some organisms, several of which are known to contain forms of NR which appear to have no physiological function. The current survey suggests that narG gene is distributed over a wide variety of nitrate-reducing bacteria, thus implicating its potential use in conjunction with other genetic probes to assess environmental samples.