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Five representative bacterial strains resembling Pseudomonas pseudoflava, chosen from among forty strains isolated by three different procedures, were characterized by phenotypic and genotypic analyses. They were shown to belong to the species P. pseudoflava, or to a distinct, but closely related, cluster. In contrast to the initial isolates of this species, most of the new strains were able to fix N2, as shown by their growth in nitrogen-free medium and by the acetylene reduction test. Moreover, DNA regions of these new strains were homologous to Klebsiella pneumomae nifHDK genes, confirming the presence of nitrogenase genes. The nif genes are probably chromosomally located, since no plasmid was detected in these strains. The presence of complete but defective nif genes in P. pseudoflava GA3 (the type strain, which does not fix N2) was excluded, as no homology was found between K. pneumoniae nif genes and P. pseudoflava GA3 total DNA. The isolation conditions seem to determine the selection of P. pseudoflava N2-fixing strains, since selection for H2-chemolithoautotrophs gave no N2-fixing isolates, whereas H2-chemolithoheterotrophic selection conditions (with combined nitrogen) yielded a majority of N2 fixers.
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