Reversible inactivation of the O2-labile hydrogenases from Azotobacter vinelandii and Rhizobium japonicum

Hydrogenases catalyze the reversible activation of dihydrogen. The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes. Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases. These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases. The reversible inactivation required the presence of an electron acceptor. The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate. The rate of inactivation was also dependent on the pH. However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation. Thus, the reversible inactivation was not a result of high pH alone. The inactive enzyme could not be reactivated by H2 or other reductants at high pH. Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme. However, if reductants were present during this titration, the enzyme could be fully reactivated. The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1. Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation. We propose a four-state model to describe this reversible inactivation.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Two nifA-like genes required for expression of alternative nitrogenases by Azotobacter vinelandii.

Two nifA-like genes, designated anfA and vnfA, have been identified in Azotobacter vinelandii. The anfA gene is located upstream from the nitrogenase-3 structural gene cluster (anfHDGK) and is preceded by a sequence that is potentially part of a ntrA-dependent promoter. The product of anfA appears to be required for expression of nitrogenase-3, since cells of the anfA deletion strain CA66 were unable to synthesize this nitrogenase when derepressed in N-free, Mo- and V-deficient medium. The vnfA gene was identified after determination of the nucleotide sequence of DNA flanking the Tn5 insertion in mutant strain CA46. Two open reading frames (ORF1 and ORF2) were found located upstream from the vnfA gene, and a nifE-like ORF, preceded by a possible ntrA-dependent promoter, was found downstream from this gene. It is not known whether vnfA is expressed only under N2-fixing conditions. However, potential ntrA-dependent promoters were found immediately upstream from vnfA (within the 3' end of ORF2) and immediately downstream from ORF1. The region spanning ORF1 and ORF2 contained an A + T-rich sequence that was also found immediately upstream from the potential ntrA-dependent promoter of anfA. The product of vnfA appears to be required for the synthesis of nitrogenase-2, since cells of strain CA46 synthesized only nitrogenase-1 and -3 but not nitrogenase-2 when grown in the presence of vanadium. The product of nifA, which is required for synthesis of nitrogenase-1, is not required for synthesis of either nitrogenase-2 or nitrogenase-3. However, growth data indicate that nifA is required for a factor (or factors) necessary for maximal diazotrophic growth under Mo- and V-deficient conditions.     

Beta-ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Mutants of Rhizobium japonicum defective in nodulation

Several mutants defective in nodulation were isolated from Rhizobium japonicum strains 3I1b110 and 61A 76. Mutants of class I do not form nodules after incubation with soybean [Glycine max (L.) Merrill] for 17 days, but will do so by 28 days. When host plants other than G. max are infected with several of these strains, there is no detectable difference in the time of nodulation or size of nodules as compared to the wild type. Two mutants of class I (i. e., SM1 and SM2) have been shown previously to be altered in the lipopolysaccharide portion of their cell wall. Mutants of class II are not slow to nodulate but form fewer nodules than the wild type on all the host plants tested. Mutants of class III are unable to form nodules. Some bacteriophage-resistant mutants, altered in cell surface structure, fall into this class. Two mutants of class III do not bind to soybean roots.     

DNA sequence of the Rhizobium leguminosarum nodulation genes nodAB and C required for root hair curling

A 3.2kb fragment of DNA cloned from Rhizobium leguminosarum has been shown to contain the genes necessary for the induction of root hair curling, the first observed step in the infection of leguminous plants by R. leguminosarum. The DNA sequence of this region has been determined and three open reading frames were identified: genes corresponding to these open reading frames have been called nodA, nodB and nodC and are transcribed in that order. Mutations within the nodC gene completely blocked root hair curling. However, a subcloned fragment containing only the nodC gene did not induce normal root hair curling (although some branching was observed), indicating that the nodA and B genes may also be required for normal root hair curling. From an analysis of the predicted amino acid sequences of the nodAB and C genes it appeared unlikely that their products are secreted; therefore it is concluded that the induction of root hair curling could be due to a secreted metabolite.     

Nucleotide sequence of Rhizobium meliloti nodulation genes

A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively. We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively. Comparison of the R. meliloti nodA, B, C nucleotide and amino acid sequences with those from R. leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.     

Involvement of Rhizobium japonicum O antigen in soybean nodulation.

Non-nodulating mutant strains of Rhizobium japonicum lacked a surface antigen that was present on the wild type. This surface antigen is associated with the O antigen portion of the lipopolysaccharide. Paper chromatography of hydrolyzed lipopolysaccharide and O antigen revealed three major component differences between the non-nodulating strains and the wild type.     

Inosine nucleosidase from Azotobacter vinelandii

An enzyme catalyzing the hydrolysis of purine nucleosides was found to occur in the extract of Azotobacter vinelandii, strain 0, and was highly purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography and gel filtration on Sephadex G-150. A strict substrate specificity of the purified enzyme was shown with respect to the base components. The enzyme specifically attacked the nucleosides without amino groups in the purine moiety: inosine gave the maximum rate of hydrolysis and xanthosine was hydrolyzed to a lesser extent. The pH optimum of inosine hydrolysis was observed from pH 7 to 9, while xanthosine was hydrolyzed maximally at pH 7. The K _m values of the enzyme for inosine were 0.65 and 0.85 mM at pH 7.1 and 9.0, respectively, and the value for xanthosine was 1.2 mM at pH 7.1.Several nucleotides inhibited the enzyme: the phosphate portions of the nucleotides were suggested to be responsible for the inhibition by nucleotides. Although the inhibition of the enzyme by nucleotides was apparently non-competitive type with respect to inosine, allosteric (cooperative) binding of the substrate was suggested in the presence of the inhibitor. The physiological significance of the enzyme was discussed in connection with the degradation and salvage pathways of purine nucleotides.     

Transfer of nitrate reductase genes of the cyanobacterium Nostoc muscorum into Rhizobium japonicum

Transformation of Rhizobium japonicum CB1809 was studied using DNA from the cyanobacterium Nostoc muscorum ATCC 27893. A spontaneous nitrate reductase deficient (Nar-) mutant (NR-6) of R. japonicum CB1809 was isolated with a frequency of 8.4 X 10(-7). Streptomycin (Sm) and Neomycin (Neo) resistance markers were introduced into strain NR-6, and the resulting strain was designated NR-6 SmR NeoR. Experiments with cyanobacterial DNA and live cells of strain NR-6 SmR NeoR indicated transformation of nitrate reductase (nar) genes of N. muscorum into this strain. This conclusion was supported by the reversion frequency of strain NR-6 SmR NeoR to Nar+ and the transformation frequency when recipient cells were exposed to N. muscorum DNA (with heat-treated DNA as control). Comparisons of growth, nitrate uptake, assimilatory nitrate reductase activity and nodulation of parent CB1809, NR-6 SmR NeoR and five transformant clones (Nar+) suggest that there may be considerable homology between the nar genes of R. japonicum CB1809 and N. muscorum.     

Photo-lability of CO bound to Mo-nitrogenase from Azotobacter vinelandii

In the presence of CO and under turnover conditions, Mo-nitrogenase generates three different electron paramagnetic resonance (EPR) signals. One of the signals, lo-CO, is an S=1/2 signal and occurs under low CO concentrations. The other two signals, hi-CO (S=1/2) and hi(5)-CO (S=3/2) displace the lo-CO as the CO concentration is raised above 0.05 atm. Irradiation of hi-CO with visible light at 12 K converts it into lo-CO. Using a series of color filters, the corrected action spectrum is determined and shown to contain 2-3 broad maxima in the region 350-730 nm. The conversion of lo-CO back into hi-CO is accomplished by warming the sample to 77 K for 5 min. Using this temperature cycle, the rate constant for the re-association of CO with lo-CO to form hi-CO is determined in the range 12-90 K. From these data, the activation energy for this reaction is calculated to be 3.9 kJ/mol. Identical irradiation of either lo-CO or hi(5)-CO induces no spectral change, showing that both of these states are photo-stable. The photo-stability of hi(5)-CO demonstrates that it is structurally different from hi-CO.