Analysis of an Abundantly Expressed Novel Member of the Nifa Regulon in Rhizobium Etli

During the development of the mutualistic association between rhizobia and legumes, the rhizobia encounter a succession of different environments to which they respond through differential gene expression. A major difference was identified in this study in gene expression between Rhizobium etli CE3 cultured in tryptone-yeast medium and bacteroids (the nitrogen fixing form of the bacteria) from bean nodules. The difference is the presence of an abundant bacteroid protein (BacS) with an apparent molecular mass of 15.6kd. This protein has not been studied previously. Expression of the protein was induced ex planta by growth at low oxygen concentration, which is the normal condition within the nodule tissue. Rhizobial nitrogen fixation proteins and other proteins necessary for bacteroid development and function are known to be induced in response to microaerobic conditions. This protein could be a component of these processes. There were three potential copies of the gene encoding the protein. All resided on the symbiotic plasmid of this bacterial strain. This endogenous plasmid is 390 kb in size and carries most of the genes necessary for nodulation and nitrogen fixation. The major function of bacteroids, nitrogen fixation, is microaerobically regulated by a protein called NifA, and expression of the BacS protein ex planta required NifA. The open reading frame (ORF) of one copy of the gene for BacS was sequenced. The only homology in the sequence databases was to a hypothetical protein of unknown function encoded on the symbiotic plasmid in the broad-host range Rhizobium spp. strain NGR234. To determine if this protein is essential for bacteroid or nodule development, mutants with targeted disruptions of the gene were isolated. By insertion mutation of a conserved intragenic sequence, a mutant altered in one of the bacS ORFs was isolated. This mutant still produced BacS. To generate a complete knockout, an alternative approach utilized R. etli strain CFNX250 which has a modified symbiotic plasmid. This modified plasmid contains all the genes necessary for nodulation and nitrogen fixation but only one copy of the gene for the BacS protein. By in vitro disruption of this bacS copy within a 2.6kb PCR product and genetic recombination to replace wild-type DNA in strain CFNX250 with this mutant DNA, three mutant strains, CE461, CE462, and CE463, were isolated. All three were deficient in production of BacS ex planta under inducing conditions and in bacteroids from nitrogen-fixing nodules. Plants inoculated with these mutants showed a reproducible delay in nodulation and fewer, more erratically spaced nodules. However, nodules from all the plants fixed nitrogen at wild-type levels. Therefore, under the conditions tested, lack of BacS appeared to affect but not eliminate the establishment of symbiosis with Phaseolus vulgaris