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.

The symbiosis of the highly metal-resistant Sinorhizobium meliloti CCNWSX0020 and Medicago lupulina has been considered an efficient tool for bioremediation of heavy metal-polluted soils. However, the metal resistance mechanisms of S. meliloti CCNWSX00200 have not been elucidated in detail. Here we employed a comparative transcriptome approach to analyze the defense mechanisms of S. meliloti 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 (ΔcueO) and three mutants that were both Cu and Zn sensitive (ΔyedYZ, ΔcusA-like, and ΔfixH-like) were selected for further study of the effects of these metal resistance determinants on bioremediation. The results showed that inoculation with the ΔcueO mutant severely inhibited infection establishment and nodulation of M. lupulina under Cu stress, while inoculation with the ΔyedYZ and ΔfixH-like mutants decreased just the early infection frequency and nodulation under Cu and Zn stresses. In contrast, inoculation with the ΔcusA-like mutant almost led to loss of the symbiotic capacity of M. lupulina to even grow in uncontaminated soil. Moreover, the antioxidant enzyme activity and metal accumulation in roots of M. lupulina 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.IMPORTANCE 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.

Factors influencing cadmium accumulation in plants after inoculation with rhizobacteria: A meta-analysis.

Rhizobacteria have the potential to enhance phytoremediation by generating substances that stimulate plant development and influence the effectiveness of cadmium (Cd) remediation by adjusting Cd availability via metal solubilization. Furthermore, rhizobacterial inoculation affects plants' metal tolerance and uptake by controlling the expression of several metal transporters, channels, and metal chelator genes. A meta-analysis was conducted to quantitatively assess the effects of rhizobacteria on Cd accumulation in plants using 207 individual observations from 47 articles. This meta-analysis showed an average Cd concentration increase of 8.09 % in plant cells under rhizobacteria treatment. The effects of different plant-microbial interactions on the bioaccumulation of Cd in plants varied. Selecting the proper rhizobacteria-plant association is essential to affect Cd buildup in plant roots and shoots. A more extended planting period (>30 days) and a suitable soil pH (<6, 7-8) would aid in the phytoextraction of Cd from the soil. This study comprehensively and quantitatively investigated the effects of plants, rhizobacteria, soil pH, planting period, experimental sites, and plant organs on plant Cd accumulation. According to the analysis of explanatory factors, plant species, planting period, soil pH, and rhizobacteria species have a more decisive influence on Cd accumulation than other factors. The results provide information for future research on the successful remediation of soils contaminated with Cd. More investigations are required to elucidate the intricate interactions between plant roots and microorganisms.

An omp gene enhances cell tolerance of Cu(II) in Sinorhizobium meliloti CCNWSX0020.

The main aim of this work was to study molecular characterization of a DNA fragment conferring resistance to Cu(II) in Sinorhizobium meliloti CCNWSX0020. The strain CCNWSX0020, resistant to 1.4 mmol l(-1) Cu(II) in tryptone-yeast extract medium was isolated from Medicago lupulina growing in mine tailings of Fengxian County, China. The availability of the complete genome sequence of S. meliloti CCNWSX0020 provides an opportunity for investigating genes that play significant roles in Cu(II) resistance. A copper resistance gene, with a length of 1,445 bp, encoding 481 amino acids, designated omp, was identified by cDNA-amplified fragment length polymorphism from S. meliloti CCNWSX0020. The expression of omp gene strongly increased in the presence of Cu(II). The omp-defective mutants display sensitivities to Cu(II) compared with their wild types. The Cu(II)-sensitive phenotype of the mutant was complemented by a 1.5-kb DNA fragment containing omp gene. BLAST analysis revealed that this gene encoded a hypothetical outer membrane protein with 75 % similarity to outer membrane efflux protein in Rhizobium leguminosarum bv. viciae 3841. These studies suggested that the omp product was involved in the Cu(II) tolerance of S. meliloti CCNWSX0020.

Distinct succession patterns of abundant and rare bacteria in temporal microcosms with pollutants.

Elucidating the driving forces behind the temporal dynamics of abundant and rare microbes is essential for understanding the assembly and succession of microbial communities. Here, we explored the successional trajectories and mechanisms of abundant and rare bacteria via soil-enrichment subcultures in response to various pollutants (phenanthrene, n-octadecane, and CdCl2) using time-series Illumina sequencing datasets. The results reveal different successional patterns of abundant and rare sub-communities in eighty pollutant-degrading consortia and two original soil samples. A temporal decrease in α-diversity and high turnover rate for ß-diversity indicate that deterministic processes are the main drivers of the succession of the abundant sub-community; however, the high cumulative species richness indicates that stochastic processes drive the succession of the rare sub-community. A functional prediction showed that abundant bacteria contribute primary functions to the pollutant-degrading consortia, such as amino acid metabolism, cellular responses to stress, and hydrocarbon degradation. Meanwhile, rare bacteria contribute a substantial fraction of auxiliary functions, such as carbohydrate-active enzymes, fermentation, and homoacetogenesis, which indicates their roles as a source of functional diversity. Our study suggests that the temporal succession of microbes in polluted microcosms is mainly associated with abundant bacteria rather than the high proportion of rare taxa. The major forces (i.e., stochastic or deterministic processes) driving microbial succession could be dependent on the low- or high-abundance community members in temporal microcosms with pollutants.

Zinc Resistance Mechanisms of P1B-type ATPases in Sinorhizobium meliloti CCNWSX0020.

The Sinorhizobium meliloti (S. meliloti) strain CCNWSX0020 displayed tolerance to high levels exposures of multiple metals and growth promotion of legume plants grown in metal-contaminated soil. However, the mechanism of metal-resistant strain remains unknown. We used five P1B-ATPases deletions by designating as ∆copA1b, ∆fixI1, ∆copA3, ∆zntA and ∆nia, respectively to investigate the role of P1B-ATPases in heavy metal resistance of S. meliloti. The ∆copA1b and ∆zntA mutants were sensitive to zinc (Zn), cadmium (Cd) and lead (Pb) in different degree, whereas the other mutants had no significant influence on the metal resistance. Moreover, the expression of zntA was induced by Zn, Cd and Pb whereas copA1b was induced by copper (Cu) and silver (Ag). This two deletions could led to the increased intracellular concentrations of Zn, Pb and Cd, but not of Cu. Complementation of ∆copA1b and ∆zntA mutants showed a restoration of tolerance to Zn, Cd and Pb to a certain extent. Taken together, the results suggest an important role of copA1b and zntA in Zn homeostasis and Cd and Pb detoxification in S. meliloti CCNWSX0020.

Functional characterization of a csoR-cueA divergon in Bradyrhizobium liaoningense CCNWSX0360, involved in copper, zinc and cadmium cotolerance.

Random mutagenesis in a symbiotic nitrogen-fixing Bradyrhizobium liaoningense CCNWSX0360 (Bln0360) using Tn5 identified five copper (Cu) resistance-related genes. They were functionally sorted into three groups: transmembrane transport (cueA and tolC); oxidation (copA); and protection of the membrane barrier (lptE and ctpA). The gene cueA, together with the upstream csoR (Cu-sensitive operon repressor), constituted a csoR-cueA divergon which plays a crucial role in Cu homeostasis. Deletion of cueA decreased the Cu tolerance of cells, and complementation of this mutant restored comparable Cu resistance to that of the wild-type. Transcriptional and fusion expression analysis demonstrated that csoR-cueA divergon was up-regulated by both the monovalent Cu+ and divalent Zn2+/Cd2+, and negatively regulated by transcriptional repressor CsoR, via a bidirectional promoter. Deletion of csoR renders the cell hyper-resistant to Cu, Zn and Cd. Although predicted to encode a Cu transporting P-type ATPase (CueA), cueA also conferred resistance to zinc and cadmium; two putative N-MBDs (N-terminal metal binding domains) of CueA were required for the Cu/Zn/Cd tolerance. Moreover, cueA is needed for nodulation competitiveness of B. liaoningense in Cu rich conditions. Together, the results demonstrated a crucial role for the csoR-cueA divergon as a component of the multiple-metal resistance machinery in B. liaoningense.