Kaistella yananensis sp. nov., a Novel Indoleacetic Acid-Producing Bacterium Isolated from the Root Nodules of Sophora davidii (Franch.) Skeels.

A novel endophytic bacterium, designated strain BT6-1-3T, was isolated from the root nodules of a leguminous shrub named Sophora davidii (Franch.) Skeels, found growing wild in Yan'an, Shaanxi Province, China. Cells were Gram-staining-negative, non-motile, catalase-positive, oxidase-positive, and did not produce H2S. Strain BT6-1-3T grew at 15-40 °C (optimum 30 °C), at pH 6.0-10.0 (optimum pH 9.0), and with 0-1% (w/v) NaCl (optimum 0.5%). The quinone system was menaquinone 6. The major fatty acids present in BT6-1-3T were iso-C11:0, iso-C15:0, and C16:0. The G+C content of genomic DNA was 39.4 mol% by whole genome sequencing. According to the analysis of 16S rRNA gene sequence, the closest relative was Kaistella montana WG4 (nucleotide identity was 97.6%). The genome of strain BT6-1-3T was sequenced, and the genome similarity was calculated using average nucleotide identity and genome-to-genome distance analysis with the genomes of other strains of Kaistella. Both strongly supported that the strain BT6-1-3T belonged to the genus Kaistella as a representative of a new species. Based on phylogenetic analysis, chemotaxonomic data, and physiological and biochemical characteristics, strain BT6-1-3T represents a new species of the genus Kaistella and is named as Kaistella yananensis sp. nov. Type strain is BT6-1-3T (= NBRC 115452T = CGMCC 1.60032T).

The bacterial consortia promote plant growth and secondary metabolite accumulation in Astragalus mongholicus under drought stress.

Astragalus mongholicus is a widely used Traditional Chinese Medicine. However, cultivated A. mongholicus is often threatened by water shortage at all growth stage, and the content of medicinal compounds of cultivated A. mongholicus is much lower than that of wild plants. To alleviate drought stress on A. mongholicus and improve the accumulation of medicinal components in roots of A. mongholicus, we combined different bacteria with plant growth promotion or abiotic stress resistance characteristics and evaluated the role of bacterial consortium in helping plants tolerate drought stress and improving medicinal component content in roots simultaneously. Through the determination of 429 bacterial strains, it was found that 97 isolates had phosphate solubilizing ability, 63 isolates could release potassium from potash feldspar, 123 isolates could produce IAA, 58 isolates could synthesize ACC deaminase, and 21 isolates could secret siderophore. Eight bacterial consortia were constructed with 25 bacterial isolates with more than three functions or strong growth promoting ability, and six out of eight bacterial consortia significantly improved the root dry weight. However, only consortium 6 could increase the root biomass, astragaloside IV and calycosin-7-glucoside content in roots simultaneously. Under drought challenge, the consortium 6 could still perform these functions. Compared with non-inoculated plants, the root dry weight of consortium inoculated-plants increased by 120.0% and 78.8% under mild and moderate drought stress, the total content of astragaloside IV increased by 183.83% and 164.97% under moderate and severe drought stress, calycosin-7-glucoside content increased by 86.60%, 148.56% and 111.45% under mild, moderate and severe drought stress, respectively. Meanwhile, consortium inoculation resulted in a decrease in MDA level, while soluble protein and proline content and SOD, POD and CAT activities increased. These findings provide novel insights about multiple bacterial combinations to improve drought stress responses and contribute to accumulate more medicinal compounds.

Beneficial bacteria activate nutrients and promote wheat growth under conditions of reduced fertilizer application.

BACKGROUND: Excessive application of chemical fertilizer has exerted a great threat to soil quality and the environment. The inoculation of plants with plant-growth-promoting rhizobacteria (PGPR) has emerged as a great prospect for ecosystem recovery. The aim of this work to isolate PGPRs and highlights the effect of bacterial inoculants on available N/P/K content in soil and on the growth of wheat under conditions of reduced fertilizer application. RESULTS: Thirty-nine PGPRs were isolated and tested for their growth-promoting potential. Thirteen isolates had nitrogen fixation ability, of which N9 (Azotobacter chroococcum) had the highest acetylene reduction activity of 156.26 nmol/gh. Eleven isolates had efficient phosphate solubilizing ability, of which P5 (Klebsiella variicola) released the most available phosphorus in liquid medium (231.68 mg/L). Fifteen isolates had efficient potassium solubilizing ability, of which K13 (Rhizobium larrymoorei) released the most available potassium in liquid medium (224.66 mg/L). In culture medium supplemented with tryptophan, P9 (Klebsiella pneumoniae) produced the greatest amount of IAA. Inoculation with the bacterial combination K14 + 176 + P9 + N8 + P5 increased the alkali-hydrolysed nitrogen, available phosphorus and available potassium in the soil by 49.46, 99.51 and 19.38%, respectively, and enhanced the N, P, and K content of wheat by 97.7, 96.4 and 42.1%, respectively. Moreover, reducing fertilizer application by 25% did not decrease the available nitrogen, phosphorus, and potassium in the soil and N/P/K content, plant height, and dry weight of wheat. CONCLUSIONS: The bacterial combination K14 + 176 + P9 + N8 + P5 is superior candidates for biofertilizers that may reduce chemical fertilizer application without influencing the normal growth of wheat.

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.

Role of exopolysaccharide in salt stress resistance and cell motility of Mesorhizobium alhagi CCNWXJ12-2T.

Mesorhizobium alhagi, a legume-symbiont soil bacterium that forms nodules with the desert plant Alhagi sparsifolia, can produce large amounts of exopolysaccharide (EPS) using mannitol as carbon source. However, the role of EPS in M. alhagi CCNWXJ12-2T, an EPS-producing rhizobium with high salt resistance, remains uncharacterized. Here, we studied the role of EPS in M. alhagi CCNWXJ12-2T using EPS-deficient mutants constructed by transposon mutagenesis. The insertion sites of six EPS-deficient mutants were analyzed using single primer PCR, and two putative gene clusters were found to be involved in EPS synthesis. EPS was extracted and quantified, and EPS production in the EPS-deficient mutants was decreased by approximately 25 times compared with the wild-type strain. Phenotypic analysis revealed reduced salt resistance, antioxidant capacity, and cell motility of the mutants compared with the wild-type strain. In conclusion, our results indicate that EPS can influence cellular Na+ content and antioxidant enzyme activity, as well as play an important role in the stress adaption and cell motility of M. alhagi CCNWXJ12-2T.

Functional analysis of PrkA - a putative serine protein kinase from Mesorhizobium alhagi CCNWXJ12-2 - in stress resistance.

BACKGROUND: Serine/threonine protein kinases are highly conserved kinases with a wide distribution in microbes and with multiple functions. Mesorhizobium alhagi CCNWXJ12-2, a α-proteobacterium which could be able to form symbiosis with Alhagi sparsifolia in northwest of China, contains a putative PrkA-family serine protein kinase, PrkA. In our previous study, the expression of prkA was found to be downregulated in high-salt conditions. To elucidate the function of M. alhagi PrkA, a prkA deletion mutant was constructed and the phenotypes of the mutant were analyzed. RESULTS: The salt and alkaline tolerance and antioxidant capacity of the wild-type strain and the prkA deletion mutant was measured. Our results showed that the deletion mutant had higher salt and alkaline tolerance than the wild-type strain. The total cellular Na+ content was measured and showed no significant difference between the wild-type strain and the mutant. The prkA deletion mutant also showed a higher H2O2 tolerance than the wild-type strain. Therefore the activities of antioxidant enzymes were measured. Catalase activity was similar in the wild-type and the deletion mutant, while the superoxide dismutase activity in the mutant was higher than that in the wild-type. CONCLUSIONS: We firstly analyze the function of a serine protein kinase, PrkA, in M. alhagi. Our data indicate that PrkA could reduce the survival of M. alhagi under environmental stress and deletion of prkA dramatically improved the salt and alkaline tolerance and antioxidant capacity of M. alhagi.

Genes conferring copper resistance in Sinorhizobium meliloti CCNWSX0020 also promote the growth of Medicago lupulina in copper-contaminated soil.

Sinorhizobium meliloti CCNWSX0020, isolated from root nodules of Medicago lupulina growing in gold mine tailings in the northwest of China, displayed both copper resistance and growth promotion of leguminous plants in copper-contaminated soil. Nevertheless, the genetic and biochemical mechanisms responsible for copper resistance in S. meliloti CCNWSX0020 remained uncharacterized. To investigate genes involved in copper resistance, an S. meliloti CCNWSX0020 Tn5 insertion library of 14,000 mutants was created. Five copper-sensitive mutants, named SXa-1, SXa-2, SXc-1, SXc-2, and SXn, were isolated, and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Both SXa-1 and SXa-2 carried a transposon insertion in lpxXL (SM0020_18047), encoding the LpxXL C-28 acyltransferase; SXc-1 and SXc-2 carried a transposon insertion in merR (SM0020_29390), encoding the regulatory activator; SXn contained a transposon insertion in omp (SM0020_18792), encoding a hypothetical outer membrane protein. The results of reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that SM0020_05862, encoding an unusual P-type ATPase, was regulated by the MerR protein. Analysis of the genome sequence showed that this P-type ATPase did not contain an N-terminal metal-binding domain or a CPC motif but rather TPCP compared with CopA from Escherichia coli. Pot experiments were carried out to determine whether growth and copper accumulation of the host plant M. lupulina were affected in the presence of the wild type or the different mutants. Soil samples were subjected to three levels of copper contamination, namely, the uncontaminated control and 47.36 and 142.08 mg/kg, and three replicates were conducted for each treatment. The results showed that the wild-type S. meliloti CCNWSX0020 enabled the host plant to grow better and accumulate copper ions. The plant dry weight and copper content of M. lupulina inoculated with the 5 copper-sensitive mutants significantly decreased in the presence of CuSO4.

Comparing the Potential of Silicon Nanoparticles and Conventional Silicon for Salinity Stress Alleviation in Soybean (Glycine max L.): Growth and Physiological Traits and Rhizosphere/Endophytic Bacterial Communities.

In this study, 20-day-old soybean plants were watered with 100 mL of 100 mM NaCl solution and sprayed with silica nanoparticles (SiO2 NPs) or potassium silicate every 3 days over 15 days, with a final dosage of 12 mg of SiO2 per plant. We assessed the alterations in the plant's growth and physiological traits, and the responses of bacterial microbiome within the leaf endosphere, rhizosphere, and root endosphere. The result showed that the type of silicon did not significantly impact most of the plant parameters. However, the bacterial communities within the leaf and root endospheres had a stronger response to SiO2 NPs treatment, showing enrichment of 24 and 13 microbial taxa, respectively, compared with the silicate treatment, which led to the enrichment of 9 and 8 taxonomic taxa, respectively. The rhizosphere bacterial communities were less sensitive to SiO2 NPs, enriching only 2 microbial clades, compared to the 8 clades enriched by silicate treatment. Furthermore, SiO2 NPs treatment enriched beneficial genera, such as Pseudomonas, Bacillus, and Variovorax in the leaf and root endosphere, likely enhancing plant growth and salinity stress resistance. These findings highlight the potential of SiO2 NPs for foliar application in sustainable farming by enhancing plant-microbe interactions to improve salinity tolerance.

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.

Draft genome sequence of Sinorhizobium meliloti CCNWSX0020, a nitrogen-fixing symbiont with copper tolerance capability isolated from lead-zinc mine tailings.

Sinorhizobium meliloti CCNWSX0020 was isolated from Medicago lupulina plants growing in lead-zinc mine tailings, which can establish a symbiotic relationship with Medicago species. Also, the genome of this bacterium contains a number of protein-coding sequences related to metal tolerance. We anticipate that the genomic sequence provides valuable information to explore environmental bioremediation.

Sulfuric acid leaching of ball-milling activated FePO4 residue after lithium extraction from spent lithium iron phosphate cathode powder.

A mechanical-chemical process is proposed to recover the iron phosphate residue(IPR)of spent lithium iron phosphate(LFP)after lithium extraction. In this process, the IPR was pretreated by ball-milling and leached with the sulfuric acid solution. The results showed that, under the optimized ball-milling conditions (a mass ratio of the stainless-steel-ball to material to water of 2:1:2.5, a milling time of 20 min), the maximum particle size of IPR decreased from 34.265 um to 13.102 um, the specific surface increased from 11.41 m2/g to 13.74 m2/g, and the cell volume distortion rate could reach 0.331 %. Under the optimized leaching conditions (a temperature of 333 K, a concentrated acid-to-material ratio of 0.46 mL/g, a liquid-to-solid ratio of 5:1 mL/g, and a stirring speed of 600 rpm), the leaching efficiency of iron phosphate could reach 98 %. The kinetic study indicated that the leaching was controlled by diffusion and chemical reaction with the apparent activation energy of 29 kJ/mol. The dissolution-precipitation phase transition of IPR was also found at high temperatures. This study illustrates that such a mechanical-chemical process is an effective way to improve the leaching efficiency of IPR with a lower sulfuric acid dosage, which has great potential in industrial applications.

1-[4-(4-Chloro-but-oxy)-2-hy-droxy-phen-yl]ethanone.

In the title compound, C(12)H(15)ClO(3), the eth-oxy group is nearly coplanar with the benzene ring, making a dihedral angle of 9.03 (4)°, and is involved in an intra-molecular O-H⋯O hydrogen bond to the neighbouring hy-droxy group.

The Composition of Root-Associated Bacteria and Fungi of Astragalus mongholicus and Their Relationship With the Bioactive Ingredients.

Astragalus membranaceus (Fisch.) Bge. var. mongholicus, which is used in traditional Chinese medicine, contains several bioactive ingredients. The root-associated microbial communities play a crucial role in the production of secondary metabolites in plants. However, the correlation of root-associated bacteria and fungi with the bioactive ingredients production in A. mongholicus has not been elucidated. This study aimed to examine the changes in soil properties, root bioactive ingredients, and microbial communities in different cultivation years. The root-associated bacterial and fungal composition was analyzed using high-throughput sequencing. The correlation between root-associated bacteria and fungi, soil properties, and six major bioactive ingredients were examined using multivariate correlation analysis. Results showed that soil properties and bioactive ingredients were distinct across different cultivation years. The composition of the rhizosphere microbiome was different from that of the root endosphere microbiome. The bacterial community structure was affected by the cultivation year and exhibited a time-decay pattern. Soil properties affected the fungal community composition. It was found that 18 root-associated bacterial operational taxonomic units (OTUs) and four fungal OTUs were positively and negatively correlated with bioactive ingredient content, respectively. The abundance of Stenotrophomonas in the rhizosphere was positively correlated with astragaloside content. Phyllobacterium and Inquilinus in the endosphere were positively correlated with the calycosin content. In summary, this study provided a new opportunity and theoretical reference for improving the production and quality of in A. mongholicus, which thus increase the pharmacological value of A. mongholicus.

A simplified synthetic community rescues Astragalus mongholicus from root rot disease by activating plant-induced systemic resistance.

BACKGROUND: Plant health and growth are negatively affected by pathogen invasion; however, plants can dynamically modulate their rhizosphere microbiome and adapt to such biotic stresses. Although plant-recruited protective microbes can be assembled into synthetic communities for application in the control of plant disease, rhizosphere microbial communities commonly contain some taxa at low abundance. The roles of low-abundance microbes in synthetic communities remain unclear; it is also unclear whether all the microbes enriched by plants can enhance host adaptation to the environment. Here, we assembled a synthetic community with a disease resistance function based on differential analysis of root-associated bacterial community composition. We further simplified the synthetic community and investigated the roles of low-abundance bacteria in the control of Astragalus mongholicus root rot disease by a simple synthetic community. RESULTS: Fusarium oxysporum infection reduced bacterial Shannon diversity and significantly affected the bacterial community composition in the rhizosphere and roots of Astragalus mongholicus. Under fungal pathogen challenge, Astragalus mongholicus recruited some beneficial bacteria such as Stenotrophomonas, Achromobacter, Pseudomonas, and Flavobacterium to the rhizosphere and roots. We constructed a disease-resistant bacterial community containing 10 high- and three low-abundance bacteria enriched in diseased roots. After the joint selection of plants and pathogens, the complex synthetic community was further simplified into a four-species community composed of three high-abundance bacteria (Stenotrophomonas sp., Rhizobium sp., Ochrobactrum sp.) and one low-abundance bacterium (Advenella sp.). Notably, a simple community containing these four strains and a thirteen-species community had similar effects on the control root rot disease. Furthermore, the simple community protected plants via a synergistic effect of highly abundant bacteria inhibiting fungal pathogen growth and less abundant bacteria activating plant-induced systemic resistance. CONCLUSIONS: Our findings suggest that bacteria with low abundance play an important role in synthetic communities and that only a few bacterial taxa enriched in diseased roots are associated with disease resistance. Therefore, the construction and simplification of synthetic communities found in the present study could be a strategy employed by plants to adapt to environmental stress. Video abstract.

Promotion of growth and metal accumulation of alfalfa by coinoculation with Sinorhizobium and Agrobacterium under copper and zinc stress.

The Legume-Rhizobium symbiosis has been proposed as a promising technique for the phytoremediation of contaminated soils due to its beneficial activity in symbiotic nitrogen fixation. However, numerous studies have shown that excessive heavy metals reduce the efficiency of symbiotic nodulation with Rhizobium and inhibit plant growth. In this study, we aimed to evaluate the synergistic effects of IAA-producing bacteria and Rhizobium on Medicago lupulina growth under Cu and Zn stress. Pot experiments showed that 400 mg kg-1 Cu2 + and Zn2 + greatly inhibited plant growth, but dual inoculation of Medicago lupulina with Sinorhizobium meliloti CCNWSX0020 and Agrobacterium tumefaciens CCNWGS0286 significantly increased the number of nodules and plant biomass by enhancing antioxidant activities. Under double stress of 400 mg kg-1 Cu2 + and Zn2 +, the nodule number and nitrogenase activities of dual-inoculated plants were 48.5% and 154.4% higher, respectively, than those of plants inoculated with Sinorhizobium meliloti. The root and above-ground portion lengths of the dual-inoculated plants were 32.6% and 14.1% greater, respectively, than those of the control, while the root and above-ground portion dry weights were 34.3% and 32.2% greater, respectively, than those of the control. Compared with S. meliloti and A. tumefaciens single inoculation, coinoculation increased total Cu uptake by 39.1% and 47.5% and increased total Zn uptake by 35.4% and 44.2%, respectively, under double metal stress conditions. Therefore, coinoculation with Sinorhizobium meliloti and Agrobacterium tumefaciens enhances metal phytoextraction by increasing plant growth and antioxidant activities under Cu/Zn stress, which provides a new approach for bioremediation in heavy metal-contaminated soil.

Functional Analysis of a Putative Type III Secretion System in Stress Adaption by Mesorhizobium alhagi CCNWXJ12-2T.

Mesorhizobium alhagi CCNWXJ12-2T, isolated from root nodules of the desert plant Alhagi sparsifolia, contains two type III secretion systems (T3SSs). T3SSs are specialized machinery with wide distribution in bacteria that inject effector proteins into target cells. Our previous study showed that the expression of M. alhagi T3SS1 is upregulated in high-salt conditions. Here, phylogenetic analysis of T3SS1 using the core protein RhcU suggested that T3SS1 belongs to the α-Rhc II subgroup of the Rhc T3SS family. To elaborate the function of M. alhagi CCNWXJ12-2T T3SS1 in stress adaption, two T3SS1 mutants (ΔrhcQ and ΔMA29250) were constructed and analyzed. ß-galactosidase transcriptional fusion assays showed that activity of the promoter of T3SS1 was induced by salts. Mutant ΔrhcQ was more sensitive to NaCl and LiCl than the wild-type, but ΔMA29250 was not. Both mutants were more sensitive to KCl than the wild-type. The intracellular Na+ concentration in ΔrhcQ in high-NaCl conditions (0.4 M) increased by 37% compared to that of the wild-type strain, while the Na+ concentration in ΔMA29250 increased by 13%. The K+ concentration in both mutants increased by 16% compared to the wild-type in high-KCl conditions (0.3 M). Strain ΔrhcQ showed decreased survival compared to the wild-type after treatment with H2O2, while the survival rate of ΔMA29250 was almost the same as that of the wild-type. Antioxidant enzyme activities in ΔrhcQ were lower than those in the wild-type strain, but this was not the case for ΔMA29250. Our data elucidate the beneficial effects of T3SS1 in the adaption of M. alhagi CCNWXJ12-2T to stress.

Nickel and cobalt resistance properties of Sinorhizobium meliloti isolated from Medicago lupulina growing in gold mine tailing.

Sinorhizobium meliloti CCNWSX0020, isolated from root nodules of Medicago lupulina growing in gold mine tailings in the northwest of China, displayed multiple heavy metal resistance and growth promotion of M. lupulina. In our previous work, the expression level of dmeR and dmeF genes were induced by Cu2+ through comparative transcriptome approach. Based on protein analysis, the dmeF encoded for a protein which showed a 37% similarity to the cation transporter DmeF of Cupriavidus metallidurans, whereas dmeR encoded transcriptional regulator which was highly homologous with DmeR belonging to RcnR/CsoR family metal-responsive transcriptional regulator. In addition to copper, quantitative real-time PCR analysis showed that dmeR and dmeF were also induced by nickel and cobalt. To investigate the functions of dmeR and dmeF in S. meliloti CCNWSX0020, the dmeR and dmeF deletion mutants were constructed. The dmeF mutant was more sensitive to Co2 + and Ni2 + than the wild type strain. Pot experiments were carried out to determine whether the growth of M. lupulina was affected when the dmeF gene was knocked out in the presence of nickel or cobalt. Results indicated that the nodule number of the host plant inoculated with the dmeF deletion mutant was significantly less than the S. meliloti CCNWSX0020 wild-type in the presence of Co2 + or Ni2 +. However, when standardized by nodule fresh weight, the nitrogenase activities of nodules infected by the dmeF deletion mutant was similar to nitrogenase activity of the wild type nodule.

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.

PS/CTAB/silica composites from room temperature polymerization of high internal phase emulsion gels.

Polystyrene (PS)/cetyltrimethylammonium bromide (CTAB)/silica composites were prepared by CTAB-catalyzed in-situ polymerization of high internal phase emulsion gels (HIPE gels) at room temperature. The room temperature approach in HIPE gels yielded mono-dispersed PS/CTAB/silica spherical composite particles with sizes of 121 and 155nm. The complex microscopic internal structures of these particles were determined with the use of temperature-modulated differential scanning calorimetry (TMDSC). CTAB, in particular, was identified in different environments within the composite particles based on its different crystallization temperatures observed on cooling: a thin layer at the surface of the fumed silica (TC,CTAB=74°C), multilayers (TC,CTAB=82°C), and a mixed PS/CTAB shell (TC,CTAB=65°C). The amounts of each component were estimated from the enthalpies of the crystallization. The PS/CTAB mixed shell contained CTAB on the surface of the particle plus internal CTAB domains.