Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon

Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ. Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.     

Isolation, characterization and complementation analysis of nirB mutants of Escherichia coli deficient only in NADH-dependent nitrite reductase activity

Mutants have been isolated which lack NADH-dependent nitrite reductase activity but retain NADPH-dependent sulphite reductase and formate hydrogenlyase activities. These NirB- strains synthesize cytochrome c552 and grow normally on anaerobic glycerol-fumarate plates. The defects map in a gene, nirB, which is extremely close to cysG, the gene order being crp, nirB, cysG, aroB. Complementation studies established that nirB+ and cysG+ can be expressed independently. The data strongly suggest that nirB is the structural gene for the 88 kDal NADH-dependent nitrite oxidoreductase apoprotein (EC 1.6.6.4). The nirB gene is apparently defective in the previously described nirD mutant, LCB82. The nirH mutant, LCB197, was unable to use formate as electron donor for nitrite reduction, but NADH-dependent nitrite reductase was extremely active in this strain and a normal content of cytochrome c552 was detected. Strains carrying a nirE, nirF or nirG mutation gave normal rates of nitrite reduction by glucose, formate or NADH.     

Characterization of a chromosomal nickel resistance determinant from Klebsiella oxytoca CCUG 15788

Klebsiella oxytoca CCUG 15788 is resistant to Ni2+ at a concentration of 10 mM and grows in an inducible manner when exposed to lower concentrations of Ni2+. The complete genomic sequence of a 4.2-kb HindIII-digested fragment of this strain was determined from genomic DNA. It was shown to contain four nickel resistance genes (nirA, nirB, nirC, and nirD) encoding transporter and transmembrane proteins for nickel resistance. When the plasmid pKOHI4, encoding nirABCD, was transformed into Escherichia coli JM109, the cells were able to grow in Tris-buffered mineral medium containing 3 mM nickel. TnphoA'-1 insertion mutants in the four nickel genes nirA, nirB, nirC, and nirD showed nickel sensitivity. The nir genes were heterogeneously expressed in E. coli, suggesting functional roles of these genes in nickel resistance.     

High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor.

The flavoprotein and hemoprotein components of Escherichia coli B NADPH-sulfite reductase are encoded by cysJ and cysI, respectively. Plasmids containing these two genes overexpressed flavoprotein catalytic activity and apohemoprotein by 13- to 35-fold, but NADPH-sulfite reductase holoenzyme activity was increased only 3-fold. Maximum overexpression of holoenzyme activity was achieved by the inclusion in such plasmids of Salmonella typhimurium cysG, which encodes a uroporphyrinogen III methyltransferase required for the synthesis of siroheme, a cofactor for the hemoprotein. Thus, cofactor deficiency, in this case siroheme, can limit overexpression of a cloned enzyme. Catalytically active holoenzyme accounted for 10% of total soluble protein in a host containing cloned cysJ, cysI, and cysG. A 5.3-kb DNA fragment containing S. typhimurium cysG was sequenced, and the open reading frame corresponding to cysG was identified by subcloning and by identifying plasmid-encoded peptides in maxicells. Comparison with the sequence reported for the E. coli cysG region (J. A. Cole, unpublished data; GenBank sequence ECONIRBC) indicates a gene order of nirB-nirC-cysG in the cloned S. typhimurium fragment. In addition, two open reading frames of unknown identity were found immediately downstream of cysG. One of these contains 11 direct repeats of 33 nucleotides each, which correspond to the consensus amino acid sequence Asp-Asp-Val-Thr-Pro-Pro-Asp-Asp-Ser-Gly-Asp.     

Biochemical and genetic characterization of nirB mutants of Escherichia coli K 12 pleiotropically defective in nitrite and sulphite reduction

Mutants of Escherichia coli K12 defective in the nirB gene lack NADH-dependent nitrite reductase activity and reduce nitrite slowly during anaerobic growth. With one exception these mutants require cysteine for growth. Cytochrome C552 synthesis and the assimilation of ammonia are unaffected by the nirB mutation. The defective gene is located between the crp and aroB genes at minute 73 on the E. coli chromosome. Mapping and reversion studies indicate the nirB is identical to the previously described cysG gene. It is suggested that the product of the cysG+ (nirB+)?gene is an enzyme required for the synthesis of sirohaem, a prosthetic group of enzymes which catalyse the six-electron reduction of nitrite to ammonia and sulphite to sulphide.     

Amino acid sequence of the [4Fe-4S] ferredoxin isolated from Desulfovibrio desulfuricans Norway

The complete amino acid sequence of the [4Fe-4S] ferredoxin from Desulfovibrio desulfuricans Norway was determined by repetitive Edman degradation of the whole protein and peptides derived from tryptic digestion. The protein has 59 residues. Four of the six cysteine residues are involved in the binding of the [4Fe-4S] cluster in the same arrangement as in clostridial ferredoxins. This sequence is compared to various Desulfovibrio ferredoxin sequences and to the sequence and three-dimensional structure of Peptococcus aerogenes ferredoxin. Evidence of gene duplication is indicated. The requirement of some sequence features in the ferredoxin for an interaction process with its electron transfer partner, cytochrome c3, is postulated in the discussion.     

Isolation and characterization of polyferredoxin from Methanobacterium thermoautotrophicum. The mvhB gene product of the methylviologen-reducing hydrogenase operon.

The methylviologen-reducing hydrogenase operon of Methanobacterium thermoautotrophicum contains an open reading frame, mvhB, the product of which was predicted to have a molecular weight of 44 kDa and to contain as many as 48 iron atoms in 12 [4Fe-4S] clusters, and was therefore suggested to be a polyferredoxin. We have now, for the first time, isolated this polyferredoxin. Its identity with the mvhB gene product was evidenced by a comparison of the N-terminal amino acid sequence. The dark-brown protein of apparent molecular weight 44 kDa was found to contain 53 mol Fe and 43 mol acid-labile sulfur per mol. The UV/visible spectrum showed two maxima at 280 nm and 390 nm, and a shoulder at 308 nm. The A390/A280 ratio was 0.73. The molar extinction coefficient at 390 nm was 170,000 M-1.cm-1. In the dithionite reduced state the protein displayed an EPR spectrum like that of [4Fe-4S] clusters. The results indicate that the mvhB gene product is indeed a polyferredoxin.     

Iron-sulphur clusters with labile metal ions.

A study has been carried out of the redox-linked metal ion uptake processes of the iron-sulphur cluster [3Fe-4S] in the bacterial ferredoxin, Fd III from Desulphovibrio africanus using a combination of electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectroscopy and direct, unmediated electrochemistry of the Fd in a film deposited at a pyrolytic graphite electrode. Reduction of the three-iron cluster is required before a divalent metal ion becomes bound as in the reaction sequence [formula: see text] The redox potentials of these processes and the metal binding constants have been determined. The affinities of the [3Fe-4S]0 cluster for divalent ions lie in the sequence Cd greater than Zn much greater than Fe. In addition, specific binding of a monovalent ion, Thallium(I), is detected for [3Fe-4S]1+ as well as for [3Fe-4S]0. The results provide a clear and quantitative demonstration of the capability of the open triangular tri-mu 2-sulphido face of a [3Fe-4S] cluster to bind a variety of metal ions if the protein environment permits. In each case the entering metal ion is coordinated by at least one additional ligand which may be from solvent (H2O or OH-) or from a protein side chain (e.g., carboxylate from aspartic acid). Hence the [3Fe-4S] core can be a redox-linked sensor of divalent metal ions, Fe(II) or Zn(II), that may trigger conformational change.     

Does ferredoxin I (Azotobacter) represent a novel class of DNA-binding proteins that regulate gene expression in response to cellular iron(II)?

Azotobacter vinelandii (Av) and chroococcum (Ac) ferredoxin I contain [3Fe-4S]1 + 0 and [4Fe-4S]2+1+ clusters, when isolated aerobically, which undergo one-electron redox cycles at potentials of -460 +/- 10 mV (vs SHE) at pH 8.3 and -645 +/- 10 mV, respectively. The X-ray structure of Fd I (Av) reveals that the N-terminal half of the polypeptide folds as a sandwich of beta-strands which enclose the iron-sulphur clusters. The C-terminal sequence contains an amphiphilic alpha-helix of four turns which lies on the surface of the beta-barrel. Fd I (Av) controls expression of an unknown protein of Mr approximately 18,000. Fd I (Ac) will complex iron(II) avidly above pH approximately 8.0 only when the [3Fe-4S] cluster is reduced and provided that cellular nucleic acid is bound. Fd I (Ac) rigorously purified from nucleic acid does not undergo iron(II) uptake. These facts, together with recent evidence that the interconversion process [3Fe-4S]0 + Fe2+----[4Fe-4S]2+ in the iron-responsive element binding protein (IRE-BP) of eukaryotic cells is controlling protein expression at the level of mRNA [1991, Cell 64, 4771; 1991, Nucleic Acid Res. 19, 1739] leads to the following hypothesis. Fd I is a DNA-binding protein which interacts by single alpha-helix binding in the wide groove of DNA. The binding is regulated by iron(II) levels in the cell. The 7Fe form binds to DNA and represses gene expression. Only the DNA-bound form of the 7Fe Fd I will take up iron(II), not the form free in solution. Iron(II) becomes bound when the [3Fe-4S] cluster is reduced. The 8Fe Fd I thus generated no longer binds DNA and the gene is de-repressed. Sequence comparisons and the crystal structure suggests that the two central turns of the alpha-helix are important elements of the DNA-recognition process and that residues Gln69 and Glu73, which lie on the outer surface of the helix, hydrogen-bond with specific base pairs.     

The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio desulfuricans (ATCC 27774).

The gene encoding a protein containing a novel iron sulfur cluster ([6Fe-6S]) has been cloned from Desulfovibrio desulfuricans ATCC 27774 and sequenced. An open reading frame was found encoding a 545 amino acid protein (M(r) 58,496). The amino acid sequence is highly homologous with that of the corresponding protein from D. vulgaris (Hildenborough) and contains a Cys-motif that may be involved in coordination of the Fe-S cluster.     

Biosynthesis of tetrahydrofolate. Sequence of GTP cyclohydrolase I from Escherichia coli.

The sequence of the gene coding for GTP cyclohydrolase I of Escherichia coli and of the adjacent regions was determined. The open reading frame contains 669 nucleotides. The deduced amino-acid sequence represents a protein consisting of 223 amino-acid residues with a molecular mass of 24,873 Da. Partial amino-acid sequences of the N-terminal region and of 5 peptides obtained by trypsin and BrCN cleavage were determined by Edman degradation and were in full agreement with the sequence deduced from the nucleotide sequence. The starting methionine is removed by posttranslational modification. The protein shows extensive homology to the recently reported GTP cyclohydrolase from rats.     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

A partial cDNA for a novel protein which has a typical E-F hand structure

We cloned a partial cDNA which includes an open reading frame of 459 bp long from a mouse fibroblast cDNA library. The deduced amino-acid sequence showed a partial homology with several calcium-binding proteins. The clone possibly encodes a novel calcium-binding protein whose domain adopts the E-F hand structure.     

Characterization of the genome region encoding an fdxH-type ferredoxin and a new 2[4Fe-4S] ferredoxin from the nonheterocystous, nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110.

A genomic DNA region with four consecutive open reading frames, including an fdxH-type gene, has been sequenced and initially characterized for the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110. The fdxH gene encodes a [2Fe-2S]-type ferredoxin, 98 amino acids in length, with a deduced molecular mass of 10.9 kDa. Conserved residues include two characteristic lysines at positions 10 and 11, shown recently to be important for interaction with nitrogenase reductase (S. Schmitz, B. Schrautermeier, and H. Böhme, Mol. Gen. Genet. 240:455-460, 1993). The gene is transcribed only under anaerobic nitrogenase-inducing conditions, whereas the Plectonema petF gene, encoding a different (type 1) [2Fe-2S] ferredoxin, is only transcribed in cultures growing with combined nitrogen. The fdxH gene was expressed in Escherichia coli as a holoprotein. The purified protein was able to effectively donate electrons to cyanobacterial nitrogenase, whereas PetF from the same organism was not. The occurrence of FdxH in the nonheterocystous genus Plectonema demonstrates for the first time that FdxH-type ferredoxins are not exclusively expressed within heterocysts, as is true for cyanobacteria differentiating these cells for nitrogen fixation under aerobic growth conditions. Two open reading frames that precede fdxH have high similarity to those found at a corresponding location in Anabaena sp. strain PCC 7120. In the latter organism, they are transcribed only under nitrogen-fixing conditions, but the functions of their gene products remain unclear (D. Borthakur, M. Basche, W. J. Buikema, P. B. Borthakur, and R. Haselkorn, Mol. Gen. Genet. 221:227-234, 1990). An fdxB-type gene encoding a 2[4Fe-4S] ferredoxin not previously identified in cyanobacteria is located immediately downstream of fdxH in P. boryanum.