Adaptation to mercury stress by nitrogen-fixing bacteria is driven by horizontal gene transfer and enhanced gene expression of the Mer operon

Abstract

Abstract Background: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. We sequenced the genomes of Sinorhizobium medicae and Rhizobium leguminosarum with high variation in Hg-tolerance to identify differences between low and high Hg-tolerant strains. While independent mercury reductase A (merA) genes are prevalent in a-proteobacteria, Mer operons are rare and often vary in their gene organization. Results: Our analyses identified multiple structurally conserved merA homologs in the genomes of S. medicae, but only the strains that possessed a Mer operon exhibited hypertolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg2+ from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while inside of nodules showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant, indicating a genotype-by-genotype interaction that influences nitrogen-fixation under stress conditions. Transfer of the Mer operon to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the operon is solely necessary to confer hypertolerance to Hg, despite paralogous merA genes present elsewhere in the genome. Conclusions: Mercury reductase operons (Mer) have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.