Barbara Reinhold-Hurek

Transcriptional fusions of gusA and gfp to the nifH gene as well as immunogold labeling with antibodies against the iron protein of nitrogenase revealed high nitrogenase gene expression levels of the endophyte Azoarcus sp. strain BH72 inside infected rice roots (Oryza sativa) in the laboratory. Thus, environmental conditions inside rice roots are permissive for endophytic nitrogen fixation in bacterial microcolonies in the aerenchyma.

A gfp (green fluorescent protein) cassette for transcriptional fusions has been developed to study gene expression in Azoarcus sp. BH72 in association with plant roots. The bacteria expressed nitrogenase genes (nifHDK) in the rhi-zosphere, on root tips, and in epidermal cells of rice seedlings. Green fluorescent protein fusions also visualized promoter activity of single cells in soil.

Nitrogenase is a functionally constant protein catalyzing N2 reduction, which is found in many phylogenetic lineages of Archaea and Bacteria. A phylogenetic analysis of nif genes may provide insights into the evolution of the bacterial genomes. Moreover, it may be used to study diazotrophic communities, when classical isolation techniques may fail to detect all contributing populations. Among six species of the genus Azoarcus, diazotrophic Proteobacteria of the beta subclass, the deduced amino acid sequences of nifH genes of two species were unusually divergent from each other. Nitrogenases of the "authentic" Azoarcus branch formed a monophyletic unit with those of gamma Proteobacteria, thus being in accordance with 16S ribosomal DNA phylogeny. The nitrogenase proteins of the two aberrant strains clustered within the alpha proteobacterial clade with rhizobial nitrogenases. This relationship was supported by bootstrap values of 87 to 98% obtained by various distance and maximum parsimony methods. Phylogenetic distances of NifH proteins indicate a possible lateral gene transfer of nif genes to Azoarcus from a common donor of the alpha subclass at the time of species diversification or several more recent, independent transfers. Application of the phylogenetic analysis to DNA isolated from environmental samples demonstrated novel habitats for Azoarcus: in guts of termites and rice grown in Japan, nifH genes belonging to the authentic Azoarcus branch were detected. This is the first evidence suggesting the occurrence of Azoarcus spp. in a plant other than its originally described host, Kallar grass. Moreover, evidence for expression of nif genes inside grass roots was obtained by in situ hybridization studies with antisense nifH probes.

© 1997 Federation of European Biochemical Societies.