A fegA knockout, constructed by insertion of an omega spectinomycin/streptomycin resistance cassette into the coding region, is incapable of utilizing the hydroxamate siderophore ferrichrome and produces a dramatic phenotype in planta, failing to develop a nitrogen-fixing symbiosis. Ten putative fegA mutant suppressors were isolated from wild type-like nodules infrequently observed on soybeans inoculated with the fegA knockout strain. These suppressor strains have not regained the ability to use ferrichrome but are able to establish a nitrogen-fixing symbiosis. We are currently creating genomic libraries from the suppressor strains to complement the fegA knockout.
The gene encoding the second iron-regulated OMP in B. japonicum 61A152, hemR, is predicted to encode a protein with significant similarity to heme receptors from other gram-negative bacteria (Genco, Dixon 2001). We have also sequenced two ORFs downstream of hemR. Recently, sequences encoding a heme uptake system in B. japonicum strain USDA 110 were deposited in Genbank. The heme receptor genes from USDA110 and 61A152 are 60% identical and 66% similar. The two downstream ORFs, orfllO and orfl65, are 84% and 92% identical and 85% and 95% similar, respectively. There are 19 transmembrane domains predicted in the 61A152 heme receptor.
Using a simple plate assay, we have established that B. japonicum is able to utilize heme and leghemoglobin as iron sources. We are currently constructing a stable knockout of the hemR gene and the resulting mutant will be characterized in both free-living conditions and in planta. We have also cloned a predicted heme receptor from S. meliloti strain Rm 1021, and currently we are making a knockout of the heme receptor gene. We have shown that Rml021 cells are able to utilize heme and leghemoglobin as iron sources. A third iron-regulated OMP fron B. japonicum 61A152 is currently being isolated using 2D-gel SDS-PAGE analysis to facilitate the cloning of the gene.
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