Sinorhizobium meliloti exists either in a free-living state in the soil or in symbiosis within legume nodules, where the bacteria differentiate into nitrogen-fixing bacteroids. Expression of genes involved in nitrogen fixation and associated respiration is governed by two intermediate regulators, NifA and FixK, respectively, which are controlled by a two-component regulatory system FixLJ in response to low-oxygen conditions. In order to identify the FixLJ regulon, gene expression profiles were determined in microaerobic free-living cells as well as during the symbiotic life of the bacterium for the wild type and a fixJ null-mutant strain. We identified 122 genes activated by FixJ in either state, including 87 novel targets. FixJ controls 74% of the genes induced in microaerobiosis (2% oxygen) and the majority of genes expressed in mature bacteroids. Ninetyseven percent of FixJ-activated genes are located on the symbiotic plasmid pSymA. Transcriptome profiles of a nifA and a fixK mutant showed that NifA activates a limited number of genes, all specific to the symbiotic state, whereas FixK controls more than 90 genes, involved in free-living and/or symbiotic life. This study also revealed that FixJ has no other direct targets besides those already known. FixJ is involved in the regulation of functions such as denitrification or amino acid/polyamine metabolism and transport. Mutations in selected novel FixJ targets did not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa roots. From these results, we propose an updated model of the FixJ regulon.

Sinorhizobium meliloti is a soil bacterium able to induce the formation of nodules on the root of specific legumes, including alfalfa (Medicago sativa). Bacteria colonize nodules through infection threads, invade the plant intracellularly, and ultimately differentiate into bacteroids capable of reducing atmospheric nitrogen to ammonia, which is directly assimilated by the plant. As a first step to describe global changes in gene expression of S. meliloti during the symbiotic process, we used whole genome microarrays to establish the transcriptome profile of bacteria from nodules induced by a bacterial mutant blocked at the infection stage and from wild-type nodules harvested at various timepoints after inoculation. Comparison of these profiles to those of cultured bacteria grown either to log or stationary phase as well as examination of a number of genes with known symbiotic transcription patterns allowed us to correlate global gene-expression patterns to three known steps of symbiotic bacteria bacteroid differentiation, i.e., invading bacteria inside infection threads, young differentiating bacteroids, and fully differentiated, nitrogen-fixing bacteroids. Finally, analysis of individual gene transcription profiles revealed a number of new potential symbiotic genes.