Multiple exposures to high concentrations of selenate significantly improve selenate tolerability, red elemental selenium (Se0) and selenoprotein biosynthesis in Herbaspirillum camelliae WT00C.

Herbaspirillum camelliae WT00C is a gram-negative endophyte isolated from the tea plant. It has an intact selenate metabolism pathway but poor selenate tolerability. In this study, microbiological properties of the strain WT00C were examined and compared with other three strains CT00C, NCT00C and NT00C, which were obtained respectively from four, six and eight rounds of 24-h exposures to 200 mM selenate. The selenate tolerability and the ability to generate red elemental selenium (Se0) and selenoproteins in H. camelliae WT00C has significantly improved by the forced evolution via 4-6 rounds of multiple exposures a high concentration of selenate. The original strain WT00C grew in 200 mM selenate with the lag phase of 12 h and 400 mM selenate with the lag phase of 60 h, whereas the strains CT00C and NCT00C grew in 800 mM selenate and showed a relatively short lag phase when they grew in 50-400 mM selenate. Besides selenate tolerance, the strains CT00C and NCT00C significantly improved the biosynthesis of red elemental selenium (Se0) and selenoproteins. Two strains exhibited more than 30% selenium conversion efficiency and 40% selenoprotein biosynthesis, compared to the original strain WT00C. These characteristics of the strains CT00C and NCT00C make them applicable in pharmaceuticals and feed industries. The strain NT00C obtained from eight rounds of 24-h exposures to 200 mM selenate was unable to grow in ≥ 400 mM selenate. Its selenium conversion efficiency and selenoprotein biosynthesis were similar to the strain WT00C, indicating that too many exposures may cause gene inactivation of some critical enzymes involving selenate metabolism and antioxidative stress. In addition, bacterial cells underwent obviously physiological and morphological changes, including gene activity, cell enlargement and surface-roughness alterations during the process of multiple exposures to high concentrations of selenate.

Selenate Reduction and Selenium Enrichment of Tea by the Endophytic Herbaspirillum sp. Strain WT00C.

Herbaspirillum sp. WT00C is a tea-plant-specific endophytic bacterium. A genomic survey revealed an intact pathway for selenocompound metabolism in the genome of this bacterium. When it was cultured with sodium selenate, Herbaspirillum sp. WT00C was able to turn the culture medium to red. Electron microscopy and energy-dispersive X-ray spectroscopy confirmed that Herbaspirillum sp. WT00C reduced selenite (Se6+) to elemental selenium (Se0), and selenium nanoparticles (SeNPs) were secreted outside bacterial cells and grew increasingly larger to form Se-nanospheres and finally crystallized to form selenoflowers. Biochemical assays showed that selenospheres contained proteins but not carbohydrates or lipids. The improvement of selenium enrichment of tea plants by Herbaspirillum sp. WT00C was also tested. After Herbaspirillum sp. WT00C was inoculated into tea seedlings via needle injection and soaking tea-cutting methods, this endophytic bacterium markedly enhanced selenium enrichment of tea. When the tea seedlings inoculated by soaking tea-cutting mode were cultivated in the selenium-containing soils, selenium contents of tea leaves in three experimental groups were more than twofold compared to those of control groups. Our study demonstrates that the endophytic bacterium Herbaspirillum sp. WT00C has the ability to reduce selenate and improve selenium enrichment of tea.

[Microbiological properties of two endophytic bacteria isolated from tea (Camellia sinensis L.)].

OBJECTIVE: We investigated biological activities, physiological and biochemical properties of two endophytic bacteria isolated from fresh leaves of tea plants. METHOD: We did morphological observation, biological activity test, physiological and biochemical assays, 16S rDNA analysis, and compared their genotype and phenotype with those of 13 Herbaspirillum species. RESULTS: Their colonies were round, opaque, central uplift and regular edge with a milky white color. Their cells were Gram-negative, rod-shaped with the size of (0.5-0.7) mm x (1.4-1.8) mm and flagellers, but without spore. Both isolates produced indole-3-acetic acid (IAA) (18.7 mg/L for WT00C and 24.9 mg/L for WT00F), ammonia and siderophores, but no nitrogen-fixing activity. The 16S rDNA had sequences similarities of 99.7% each other and 99% with 13 Herbaspirillum species. Two isolates used carbon source as described in the genus Herbaspirillum, except for propionate salt. The neighbor-joining tree built using the 16S rDNA showed that two isolates formed an independent group, which kept certain genetic distance from the 13 Herbaspirillum species. Their physiological and biochemical characteristics and genotypes were different from those for 13 Herbaspirillum species. CONCLUSION: Two isolates WT00C and WT00F were classified as novel members in the genus Herbaspirillum.

Bioleaching characteristics, influencing factors of Cu solubilization and survival of Herbaspirillum sp. GW103 in Cu contaminated mine soil.

This study was aimed at assess the potential of diazotrophic bacteria, Herbaspirillum sp. GW103, for bioleaching of Cu in mine soil. The strain exhibited resistance to As (550mgL(-1)), Cu (350mgL(-1)), Zn (300mgL(-1)) and Pb (200mgL(-1)). The copper resistance was further confirmed by locating copA and copB genes. The survival of the isolate GW103 during bioleaching was analyzed using green fluorescent protein tagged GW103. Response surface methodology based Box-Behnken design was used to optimize the physical and chemical conditions for Cu bioleaching. Five significant variables (temperature, incubation time, CaCO3, coconut oil cake (COC), agitation rate) were selected for the optimization. Second-order polynomials were established to identify the relationship between Cu bioleaching and variables. The optimal conditions for maximum Cu bioleaching (66%) were 30°C, 60h of incubation with 1.75% of CaCO3 and 3% COC at 140rpm. The results of Cu sequential extraction studies indicated that the isolate GW103 leached Cu from ion-exchangeable, reducible, strong organic and residual fractions. Obtained results point out that the isolate GW103 could be used for bioleaching of Cu from mine soils.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.