Cloning of Azorhizobium caulinodans nicotinate catabolism genes and characterization of their importance in N2 fixation.
Twenty Azorhizobium caulinodans vector insertion (Vi) mutants unable to catabolize nicotinate (Nic- phenotype) were identified and directly cloned as pVi plasmids. These pVi plasmids were used as DNA hybridization probes to isolate homologous wild-type sequences. From subsequent physical mapping experiments, the nic::Vi mutants defined four distinct loci. Two, possibly three, of these loci are physically linked. A. caulinodans nic loci II and III encode the structural genes for nicotinate catabolism; nic loci I and IV encode nicotinate-driven respiratory chain components. Recombinant lambda bacteriophages corresponding to three of these loci were subcloned in pRK293; resulting plasmids were used for complementation tests with resolved nic::IS50 derivatives of the nic::Vi mutants. When wild-type A. caulinodans was cultured in defined liquid medium under 3% O2, nicotinate catabolism stimulated N2 fixation 10-fold. In these exponentially growing cultures, the entire (300 microM) nicotinate supplement was exhausted within 10 h. While nic::Vi mutants retained the ability to fix some N2, they did so at rates only 10% of that of the wild type: nitrogenase activity by nic::Vi mutants was not stimulated by 300 microM added nicotinate. Higher-level (5 mM) nicotinate supplementation inhibited N2 fixation. Because 5 mM nicotinate repressed nitrogenase induction in all nic::Vi mutants as well, this repression was independent of nicotinate catabolism. During catabolism, nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase which drives a respiratory chain to O2. In A. caulinodans wild-type cultures, added 300 microM 6-OH-nicotinate stimulated N2 fixation twofold better than did added 300 microM nicotinate. Likewise, nic::Vi mutant 61302, defective in nicotinate hydroxylase, fixed N2 at wild-type levels when supplemented with 300 microM 6-OH-nicotinate. Therefore, nicotinate catabolism stimulates N2 fixation not by nicotinate hydroxylase-driven respiration but rather by some subsequent aspect(s) of nicotinate catabolism.
Buckmiller
L M
LM
Department of Biology, University of California, Santa Cruz 95064.
Lapointe
J P
JP
Ludwig
R A
RA
D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D009525:Niacin / Q000378:metabolism*; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012230:Rhizobiaceae / Q000235:genetics* / Q000378:metabolism
D009525:Niacin; C021862:nicotinate dehydrogenase; D009591:Nitrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors