nitrogenase and Chromosome-Deletion

We have constructed a series of small, chloramphenicol-resistance-encoding, lacZ alpha-complementing vectors with novel multiple cloning sites, based on the pACYC184 replicon. The sacB gene of Bacillus subtilis, which is lethal to Gram- organisms in the presence of sucrose, was cloned into one of these, giving the counterselectable vector pSG335. This was used to substitute a streptomycin-resistance-encoding cassette for the ntrBC genes in the Klebsiella pneumoniae chromosome.

Topics: Base Sequence; Chloramphenicol Resistance; Chromosome Deletion; Chromosomes, Bacterial; Cloning, Molecular; DNA, Bacterial; Genetic Complementation Test; Genetic Vectors; Hexosyltransferases; Klebsiella pneumoniae; Molecular Sequence Data; Nitrogenase; Plasmids

In Rhodobacter capsulatus there exists, in addition to a conventional Mo-containing nitrogenase, a second, Mo-indendent nitrogenase which was demonstrated in wild-type cells as well as in cells of a nifHDK- mutant. To construct this R. capsulatus mutant, a 4-kb BglII-HindIII fragment encompassing nifK, nifD and most of the nifH coding region was substituted by an interposon coding for kanamycin resistance. The alternative nitrogenase is repressed by molybdenum. Mo concentration greater than 1 ppb in the growth medium prevented diazotrophic growth of nifHDK- cells and the expression of nitrogenase activity. The Mo-independent nitrogenase was maximally derepressed in activated carbon-treated media which contained less than 0.05 ppb Mo, high concentrations of iron (1 mM ferric citrate) and serine as N source. Under N2-fixing and optimal Mo-deficient conditions, nifHDK- cells grew with a doubling time of 9 h. The highest activity achieved with whole cells was 1.2 nmol ethylene.min-1.mg protein-1. Vanadium neither stimulated nor inhibited growth and activity. The alternative nitrogenase reduced acetylene to both ethylene and ethane. With whole cells (nifHDK-) the proportion of ethane varied over 2-5% depending on the amount of residual traces of Mo in the medium. The addition of Mo to a growing, nitrogenase-active culture resulted in a slow decrease of total activity but also in a simultaneous increase of ethane production up to 40%. In contrast, cell-free extracts and the purified enzyme did not show any or only very little ethane formation (0-0.4%). Both enzyme components appeared to be very labile proteins. Component 2 lost almost all its activity during cell breakage. With component 1 in crude extracts, if complemented with the stable component 2 of the Mo-nitrogenase from Xanthobacter autotrophicus, a recovery of 50% of the original whole cell activity could be achieved. During purification, component 1 (from the nifHDK- mutant) remained remarkably stable. The partially purified component 1 had a pH optimum (acetylene reduction) of 7.8-8.0, relatively high affinity to acetylene (Km = 0.055 mM) and was analyzed to contain 20 mol Fe atoms/mol protein, 0.2 mol Mo atoms and negligible amounts of V, W and Re. The dithionite-reduced dinitrogenase appeared to be ESR-silent. The results indicate that the alternative nitrogenase of R. capsulatus is not a vanadium enzyme but rather a heterometal-free Fe-nitrogenase or a nitrogenase with an as-yet-unidentified heter

Topics: Chromatography, Gel; Chromatography, Ion Exchange; Chromosome Deletion; Genes, Bacterial; Isoenzymes; Kinetics; Metals; Molybdenum; Mutagenesis, Site-Directed; Nitrogen Fixation; Nitrogenase; Restriction Mapping; Rhodobacter capsulatus; Vanadium

Rhizobium leguminosarum biovar phaseoli CFN23 loses its ability to nodulate beans at a high frequency because of a deletion of part of its symbiotic (pSym) plasmid (Soberón-Chávez et al., 1986). We report here that at least 80 kb of pSym are deleted upon loss of the symbiotic phenotype; the deletion removes the nitrogenase structural nifHDK and the common nodABC genes. The size of the deleted pSym is not reduced, since it is accompanied by an amplification of other pSym plasmid sequences. This genetic rearrangement is similar to the deletion and amplification of yeast mitochondrial DNA leading to 'petite' mutations.

Topics: Blotting, Southern; Chromosome Deletion; DNA, Bacterial; Fabaceae; Gene Amplification; Genes, Bacterial; Nitrogen Fixation; Nitrogenase; Phenotype; Plants, Medicinal; Plasmids; Rhizobium; Symbiosis

A binary plasmid system was used to produce nitrogenase components in Escherichia coli and subsequently to define a minimum set of nitrogen fixation (nif) genes required for the production of the iron-molybdenum cofactor (FeMoco) reactivatable apomolybdenum-iron (apoMoFe) protein of nitrogenase. The active MoFe protein is an alpha 2 beta 2 tetramer containing two FeMoco clusters and 4 Fe4S4 P centers (for review see, Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419-459). The plasmid pVL15, carrying a tac-promoted nifA activator gene, was coharbored in E. coli with the plasmid pGH1 which contained nifHDKTYENXUSVWZMF' derived from the chromosome of the nitrogen fixing bacterium Klebsiella pneumoniae. The apoMoFe protein produced in E. coli by pGH1 + VL15 was identical to the apoprotein in derepressed cells of the nifB- mutant of K. pneumoniae (UN106) in its electrophoretic properties on nondenaturing polyacrylamide gels as well as in its ability to be activated by FeMoco. The constituent peptides migrated identically to those from purified MoFe protein during electrophoresis on denaturing gels. The concentrations of apoMoFe protein produced in nif-transformed strains of E. coli were greater than 50% of the levels of MoFe protein observed in derepressed wild-type K. pneumoniae. Systematic deletion of individual nif genes carried by pGH1 has established the requirements for the maximal production of the FeMoco-reactivatable apoMoFe protein to be the following gene products, NifHDKTYUSWZM+A. It appears that several of the genes (nifT, Y, U, W, and Z) are only required for maximal production of the apoMoFe protein, while others (nifH, D, K, and S) are absolutely required for synthesis of this protein in E. coli. One curious result is that the nifH gene product, the peptide of the Fe protein, but not active Fe protein itself, is required for formation of the apoMoFe protein. This suggests the possibility of a ternary complex of the NifH, D, and K peptides as the substrate for the processing to form the apoMoFe protein. We also find that nifM, the gene which processes the nifH protein into Fe protein (Howard, K.S., McLean, P.A., Hansen, F. B., Lemley, P.V., Kobla, K.S. & Orme-Johnson, W.H. (1986) J. Biol. Chem. 261, 772-778) can, under certain circumstances, partially replace other processing genes (i.e. nifTYU and/or WZ) although it is not essential for apoMoFe protein formation. It also appears that nifS and nifU, reported to play a role in

Topics: Amino Acid Sequence; Apoproteins; Bacterial Proteins; Chromosome Deletion; Escherichia coli; Ferredoxins; Genes, Bacterial; Genotype; Klebsiella pneumoniae; Molecular Sequence Data; Molecular Weight; Molybdoferredoxin; Mutation; Nitrogen Fixation; Nitrogenase; Phenotype; Plasmids; Recombinant Proteins

The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

Topics: Amino Acid Sequence; Azotobacter; Bacterial Proteins; Base Sequence; Chromosome Deletion; Codon; Escherichia coli; Genes; Genes, Bacterial; Isoenzymes; Molecular Sequence Data; Mutation; Nitrogen Fixation; Nitrogenase; Plasmids; Restriction Mapping

Nitrogenase is composed of two separately purified proteins called the Fe protein and the MoFe protein. In Azotobacter vinelandii the genes encoding these structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). The MoFe protein contains an ironmolybdenum cofactor (FeMo cofactor) whose biosynthesis involves the participation of at least five gene products, nifQ, nifB, nifN, nifE, and nifV. In this study an A. vinelandii mutant strain, which contains a defined deletion within the nifH (Fe protein) gene, was isolated and studied. This mutant is still able to accumulate significant amounts of MoFe protein subunits. However, extracts of this nifH deletion strain have only very low levels of MoFe protein acetylene reduction activity. Fully active MoFe protein can be reconstituted by simply adding isolated FeMo cofactor to the extracts. Fe protein is not necessary to stabilize or insert this preformed FeMo cofactor into the FeMo cofactor-deficient MoFe protein synthesized by the nifH deletion strain. Extracts of the nifH deletion strain can carry out molybdate and ATP-dependent in vitro FeMo cofactor biosynthesis provided Fe protein is added, demonstrating that they contain the products encoded by the FeMo cofactor biosynthetic genes. These data demonstrate that the Fe protein is physically required for the biosynthesis of FeMo cofactor in A. vinelandii.

Topics: Azotobacter; Chromosome Deletion; Ferredoxins; Genes; Genes, Viral; Macromolecular Substances; Metalloproteins; Molybdoferredoxin; Nitrogenase; Plasmids

The nitrogen fixation promoters of Klebsiella pneumoniae are atypical procaryotic promoters lacking the usual -10 and -35 elements, requiring instead conserved sequences around -12 and -24 for transcriptional activation. By constructing a set of five deletions between the -12 and -24 elements in the nifH promoter, the spacing between the conserved GC and GG motifs at -12 and -24, respectively, has been reduced from the wild-type 10 bases to 9, 8, 6, 5, and 4 bases. The deletion of a single nonconserved nucleotide was sufficient to eliminate transcriptional activation by either nifA or ntrC (glnG). All deletions relieved the multicopy inhibition of chromosomal nif expression normally shown by the nifH promoter. These results demonstrate a stringent requirement for the 10-base spacing found in ntr-activated promoters. In addition, specific sequences around the invariant GG at -24 were shown to be necessary for activation by either nifA or ntrC, with a minimal requirement for nucleotides through to position -27 for this activation.

Topics: Bacterial Proteins; Base Sequence; Chromosome Deletion; Enzyme Activation; Gene Expression Regulation; Klebsiella pneumoniae; Nitrogen Fixation; Nitrogenase; Promoter Regions, Genetic

Wild-type Azotobacter vinelandii strain UW was transformed with plasmid pDB12 to produce a species (LS10) unable to synthesize the structural proteins of component 1 and component 2 of native nitrogenase. A spontaneous mutant of this strain was isolated (LS15) which can grow by nitrogen fixation in the presence or absence of either Mo or W. It is proposed that LS15 fixes nitrogen solely by an alternative nitrogen-fixing system which previously has been hypothesized to exist in A. vinelandii. Under nitrogen-fixing conditions, LS15 synthesizes a protein similar to component 2 (Av2) of native nitrogenase in that it can complement native component 1 (Av1) for enzymatic activity. Isolation and characterization of this second component 2 shows it to be a 4Fe-4S protein of molecular mass about 62 kDa and is antigenically similar to Av2. This protein is also similar to Av2 in that in the reduced state it possesses a rhombic ESR spectrum in the g = 2 region, which changes to an axial spectrum upon addition of MgATP. It is suggested that this second Fe-protein is associated with the alternative nitrogen-fixing system in A. vinelandii.

Topics: Azotobacter; Chromosome Deletion; Electron Spin Resonance Spectroscopy; Iron; Mutation; Nitrogenase; Plasmids

The Azotobacter vinelandii genes encoding the nitrogenase structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). In this study various A. vinelandii mutant strains which contain defined deletions within the nitrogenase structural genes were isolated and studied. Mutants deleted for the nifD or nifK genes were still able to accumulate significant amounts of the unaltered MoFe protein subunit as well as active Fe protein. Extracts of such nifD or nifK deletion strains had no MoFe protein activity. However, active MoFe protein could be reconstituted by mixing extracts of the mutant strains. These results establish an approach for the purification of the individual MoFe protein subunits. Mutants lacking either or both of the MoFe protein subunits were still able to synthesize the iron-molybdenum cofactor (FeMo-cofactor), indicating that in A. vinelandii the FeMo-cofactor is preassembled and inserted into the MoFe protein. In contrast, a mutant strain lacking both the Fe protein and the MoFe protein failed to accumulate any detectable FeMo-cofactor. The further utility of specifically altered A. vinelandii strains for the study of the assembly, structure, and reactivity of nitrogenase is discussed.

Topics: Azotobacter; Chromosome Deletion; Genes; Nitrogenase; Oxidoreductases

Steady-state chemostat cultures of Azotobacter vinelandii strain CA11, carrying a deletion of genes encoding the structural polypeptides of nitrogenase nifHDK, were established in a simple defined medium chemically purified to minimize contamination by Mo. The medium contained no utilizable N source. Growth was dependent on N2 (1.1 X 10(8) viable cells X ml-1 at D = 0.176 h-1), and was inhibited by Mo (20 nM). DNA hybridization showed the deletion to be stable during prolonged (55 days) growth in the chemostat (132 doublings). Since batch cultures, using unsupplemented 'spent' chemostat medium, showed good growth (1.9 X 10(8) cells X ml-1), no requirement for subnanomolar concentrations of Mo was found. The biomass yield, as the dilution rate (D) was varied, showed that the N content of the culture, protein and dry wt. increased as D was decreased, indicating that neither N2 nor O2 were limiting growth. The limiting nutrient was not identified. Substantial amounts of H2 were evolved by the chemostat cultures, probably as the result of inhibition of O2-dependent hydrogenase activity by nitrilotriacetic acid present in the medium. Over a range of D values approx. 50% of the electron flux through the alternative system was allocated to H+ reduction. C2H2 was a poor substrate, being reduced at 0.14-0.1 times the rate of N2 fixation, calculated from the N content of the cells. SO4(2-)-limited steady-state continuous cultures of strain UW136 (wild-type for nifHDK) had a 2-fold greater biomass in the presence of MoO4(2-) (1 microM). The significance of this finding for 'Mo-limited' continuous cultures [Eady & Robson (1984) Biochem. J. 224, 853-862] is discussed.

Topics: Azotobacter; Chromosome Deletion; Electron Transport; Genes; Molybdenum; Nitrogen Fixation; Nitrogenase; Oxidation-Reduction; Sulfates