Alternative pathways for siroheme synthesis in Klebsiella aerogenes

Siroheme, the cofactor for sulfite and nitrite reductases, is formed by methylation, oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen III (Uro-III). The CysG protein performs all three steps of siroheme biosynthesis in the enteric bacteria Escherichia coli and Salmonella enterica. In either taxon, cysG mutants cannot reduce sulfite to sulfide and require a source of sulfide or cysteine for growth. In addition, CysG-mediated methylation of Uro-III is required for de novo synthesis of cobalamin (coenzyme B(12)) in S. enterica. We have determined that cysG mutants of the related enteric bacterium Klebsiella aerogenes have no defect in the reduction of sulfite to sulfide. These data suggest that an alternative enzyme allows for siroheme biosynthesis in CysG-deficient strains of Klebsiella. However, Klebsiella cysG mutants fail to synthesize coenzyme B(12), suggesting that the alternative siroheme biosynthetic pathway proceeds by a different route. Gene cysF, encoding an alternative siroheme synthase homologous to CysG, has been identified by genetic analysis and lies within the cysFDNC operon; the cysF gene is absent from the E. coli and S. enterica genomes. While CysG is coregulated with the siroheme-dependent nitrite reductase, the cysF gene is regulated by sulfur starvation. Models for alternative regulation of the CysF and CysG siroheme synthases in Klebsiella and for the loss of the cysF gene from the ancestor of E. coli and S. enterica are presented.     

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.     

Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding

The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.     

The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis.

Bacillus subtilis can use either nitrate or nitrite as a sole source of nitrogen. The isolation of the nasABCDEF genes of B. subtilis, which are required for nitrate/nitrite assimilation, is reported. The probable gene products include subunits of nitrate/nitrite reductases and an enzyme involved in the synthesis of siroheme, a cofactor for nitrite reductase.     

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.     

Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin

Plant sulfite reductase contains the siroheme and the [4Fe-4S] cluster as catalytically active redox centers and catalyzes the six-electron reductions of sulfite and nitrite using electrons donated from ferredoxin. A heterologous expression of a cDNA for maize sulfite reductase in E. coli has enabled us to produce the wild-type and mutant enzymes. Putative substrate-binding basic residues, located at the siroheme distal side, have been substituted for other residues with neutral or negatively charged side chains. Kinetic studies of the resulting mutant enzymes have demonstrated that substrate specificity for the two anions is remarkably changed by amino acid substitutions at a single site. We have also produced two classes of ferredoxin mutants with less ability to donate electrons to sulfite reductase: one with a defect in the recognition of the partner enzyme and the other with an unfavorable redox property. This article summarizes our knowledge about the structure function relationships of plant sulfite reductase.     

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.     

Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.     

A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis.

The role of cbiK, a gene found encoded within the Salmonella typhimurium cob operon, has been investigated by studying its in vivo function in Escherichia coli. First, it was found that cbiK is not required for cobalamin biosynthesis in the presence of a genomic cysG gene (encoding siroheme synthase) background. Second, in the absence of a genomic cysG gene, cobalamin biosynthesis in E. coli was found to be dependent upon the presence of cobA(P. denitrificans) (encoding the uroporphyrinogen III methyltransferase from Pseudomonas denitrificans) and cbiK. Third, complementation of the cysteine auxotrophy of the E. coli cysG deletion strain 302delta a could be attained by the combined presence of cobA(P. denitrificans) and the S. typhimurium cbiK gene. Collectively these results suggest that CbiK can function in fashion analogous to that of the N-terminal domain of CysG (CysG(B)), which catalyzes the final two steps in siroheme synthesis, i.e., NAD-dependent dehydrogenation of precorrin-2 to sirohydrochlorin and ferrochelation. Thus, phenotypically CysG(B) and CbiK have very similar properties in vivo, although the two proteins do not have any sequence similarity. In comparison to CysG, CbiK appears to have a greater affinity for Co2+ than for Fe2+, and it is likely that cbiK encodes an enzyme whose primary role is that of a cobalt chelatase in corrin biosynthesis.     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

Molecular biology, genetics and regulation of nitrite reduction in higher plants

Nitrite reductase (ferredoxin:nitrite oxidoreductase, EC 1.6.6.1) carries out the six-electron reduction of nitrite to ammonium ions in the chloroplasts/plastids of higher plants. The complete or partial nucleotide sequences of a number of nitrite reductase apoprotein genes or cDNAs have been determined. Deduced amino acid sequence comparisons have identified conserved regions, one of which probably is involved in binding the sirohaem/4Fe4S centre and another in binding the electron donor, reduced ferredoxin. The nitrite reductase apoprotein is encoded by the nuclear DNA and is synthesised as a precursor carrying an N-terminal extension, the transit peptide, which acts to target the protein to, and within, the chloroplast/plastid. In those plants examined the number of nitrite reductase apoprotein genes per haploid genome ranges from one (barley, spinach) to four ( Nicotiana tabacum ). Mutants defective in the nitrite reductase apoprotein gene have been isolated in barley. During plastidogenesis in etiolated plants, synthesis of nitrite reductase is regulated by nitrate, light (phytochrome), and an uncharacterised 'plastidic factor' produced by functional chloroplasts. In leaves of green, white-light-grown plants up-regulation of nitrite reductase synthesis is achieved via nitrate and light and down-regulation by a nitrogenous end-product of nitrate assimilation, perhaps glutamine. A role for phytochrome has not been demonstrated in green, light-grown plants. Light regulation of nitrite reductase genes is related more closely to that of photosynthetic genes than to the nitrate reductase gene. In roots of green, white-light-grown plants nitrate alone is able to bring about synthesis of nitrite reductase, suggesting that the root may possess a mechanism that compensates for the light requirement seen in the leaf.     

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate-dependent anaerobic growth on a non-fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK-narU double mutant was defective in nitrate-dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH-dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH-dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+-NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in-dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH-dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex-cretion, not nitrite uptake.     

Ecological Physiology of Synechococcus sp. Strain SH-94-5, a Naturally Occurring Cyanobacterium Deficient in Nitrate Assimilation

Synechococcus sp. strain SH-94-5 is a nitrate assimilation-deficient cyanobacterium which was isolated from an ammonium-replete hot spring in central Oregon. While this clone could grow on ammonium and some forms of organic nitrogen as sole nitrogen sources, it could not grow on either nitrate or nitrite, even under conditions favoring passive diffusion. It was determined that this clone does not express functional nitrate reductase or nitrite reductase and that the lack of activity of either enzyme is not due to inactivation of the cyanobacterial nitrogen control protein NtcA. A few other naturally occurring cyanobacterial strains are also nitrate assimilation deficient, and phylogenetic analyses indicated that the ability to utilize nitrate has been independently lost at least four times during the evolutionary history of the cyanobacteria. This phenotype is associated with the presence of environmental ammonium, a negative regulator of nitrate assimilation gene expression, which may indicate that natural selection to maintain functional copies of nitrate assimilation genes has been relaxed in these habitats. These results suggest how the evolutionary fates of conditionally expressed genes might differ between environments and thereby effect ecological divergence and biogeographical structure in the microbial world.     

A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation.

By its inability to grow on sulfate as the sole sulfur source, a mutant strain (CTNUX8) of Rhizobium etli carrying Tn5 was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a cysG (siroheme synthetase)-homologous gene. By RNase protection assays, it was established that the cysG-like gene had a basal level of expression in thiosulfate- or cysteine-grown cells, which was induced when sulfate or methionine was used. Unlike its wild-type parent (strain CE3), the mutant strain, CTNUX8, was also unable to grow on nitrate as the sole nitrogen source and was unable to induce a high level of nitrite reductase. Despite its pleiotropic phenotype, strain CTNUX8 was able to induce pink, effective (N2-fixing) nodules on the roots of Phaseolus vulgaris plants. However, mixed inoculation experiments showed that strain CTNUX8 is significantly different from the wild type in its ability to nodulate. Our data support the notion that sulfate (or sulfite) is the sulfur source of R. etli in the rhizosphere, while cysteine, methionine, or glutathione is supplied by the root cells to bacteria growing inside the plant.     

Ecological physiology of Synechococcus sp. strain SH-94-5, a naturally occurring cyanobacterium deficient in nitrate assimilation.

Synechococcus sp. strain SH-94-5 is a nitrate assimilation-deficient cyanobacterium which was isolated from an ammonium-replete hot spring in central Oregon. While this clone could grow on ammonium and some forms of organic nitrogen as sole nitrogen sources, it could not grow on either nitrate or nitrite, even under conditions favoring passive diffusion. It was determined that this clone does not express functional nitrate reductase or nitrite reductase and that the lack of activity of either enzyme is not due to inactivation of the cyanobacterial nitrogen control protein NtcA. A few other naturally occurring cyanobacterial strains are also nitrate assimilation deficient, and phylogenetic analyses indicated that the ability to utilize nitrate has been independently lost at least four times during the evolutionary history of the cyanobacteria. This phenotype is associated with the presence of environmental ammonium, a negative regulator of nitrate assimilation gene expression, which may indicate that natural selection to maintain functional copies of nitrate assimilation genes has been relaxed in these habitats. These results suggest how the evolutionary fates of conditionally expressed genes might differ between environments and thereby effect ecological divergence and biogeographical structure in the microbial world.