Transcriptome Response to Heavy Metals in Sinorhizobium meliloti CCNWSX0020 Reveals New Metal Resistance Determinants That Also Promote Bioremediation by Medicago lupulina in Metal-Contaminated Soil

Abstract

The symbiosis of the highly metal-resistant <i>Sinorhizobium meliloti</i> CCNWSX0020 and <i>Medicago lupulina</i> has been considered an efficient tool for bioremediation of heavy metal-polluted soils. However, the metal resistance mechanisms of <i>S. meliloti</i> CCNWSX00200 have not been elucidated in detail. Here we employed a comparative transcriptome approach to analyze the defense mechanisms of <i>S. meliloti</i> CCNWSX00200 against Cu or Zn exposure. Six highly upregulated transcripts involved in Cu and Zn resistance were identified through deletion mutagenesis, including genes encoding a multicopper oxidase (CueO), an outer membrane protein (Omp), sulfite oxidoreductases (YedYZ), and three hypothetical proteins (a CusA-like protein, a FixH-like protein, and an unknown protein), and the corresponding mutant strains showed various degrees of sensitivity to multiple metals. The Cu-sensitive mutant (Δ<i>cueO</i>) and three mutants that were both Cu and Zn sensitive (Δ<i>yedYZ</i>, Δ<i>cusA</i>-like, and Δ<i>fixH</i>-like) were selected for further study of the effects of these metal resistance determinants on bioremediation. The results showed that inoculation with the Δ<i>cueO</i> mutant severely inhibited infection establishment and nodulation of <i>M. lupulina</i> under Cu stress, while inoculation with the Δ<i>yedYZ</i> and Δ<i>fixH</i>-like mutants decreased just the early infection frequency and nodulation under Cu and Zn stresses. In contrast, inoculation with the Δ<i>cusA</i>-like mutant almost led to loss of the symbiotic capacity of <i>M. lupulina</i> to even grow in uncontaminated soil. Moreover, the antioxidant enzyme activity and metal accumulation in roots of <i>M. lupulina</i> inoculated with all mutants were lower than those with the wild-type strain. These results suggest that heavy metal resistance determinants may promote bioremediation by directly or indirectly influencing formation of the rhizobium-legume symbiosis.<b>IMPORTANCE</b> Rhizobium-legume symbiosis has been promoted as an appropriate tool for bioremediation of heavy metal-contaminated soils. Considering the plant-growth-promoting traits and survival advantage of metal-resistant rhizobia in contaminated environments, more heavy metal-resistant rhizobia and genetically manipulated strains were investigated. In view of the genetic diversity of metal resistance determinants in rhizobia, their effects on phytoremediation by the rhizobium-legume symbiosis must be different and depend on their specific assigned functions. Our work provides a better understanding of the mechanism of heavy metal resistance determinants involved in the rhizobium-legume symbiosis, and in further studies, genetically modified rhizobia harboring effective heavy metal resistance determinants may be engineered for the practical application of rhizobium-legume symbiosis for bioremediation in metal-contaminated soils.