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.

Degradation of epigallocatechin and epicatechin gallates by a novel tannase TanHcw from Herbaspirillum camelliae.

BACKGROUND: Herbaspirillum camelliae is a gram-negative endophyte isolated from the tea plant. Both strains WT00C and WT00F were found to hydrolyze epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) to release gallic acid (GA) and display tannase activity. However, no tannase gene was annotated in the genome of H. camelliae WT00C. RESULTS: The 39 kDa protein, annotated as the prolyl oligopeptidase in the NCBI database, was finally identified as a novel tannase. Its gene was cloned, and the enzyme was expressed in E. coli and purified to homogeneity. Moreover, enzymatic characterizations of this novel tannase named TanHcw were studied. TanHcw was a secretary enzyme with a Sec/SPI signal peptide of 48 amino acids at the N-terminus, and it catalyzed the degradation of tannin, methyl gallate (MG), epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG). The optimal temperature and pH of TanHcw activities were 30 °C, pH 6.0 for MG and 40 °C, pH 7.0 for both EGCG and ECG. Na+, K+ Mn2+ and Triton-X100, Tween80 increased the enzyme activity of TanHcw, whereas Zn2+, Mg2+, Hg2+, EMSO, EDTA and ß-mercaptoethanol inhibited enzyme activity. Km, kcat and kcat /Km of TanHcw were 0.30 mM, 37.84 s-1, 130.67 mM-1 s-1 for EGCG, 0.33 mM, 34.59 s-1, 105.01 mM-1 s-1 for ECG and 0.82 mM, 14.64 s-1, 18.17 mM-1 s-1 for MG, respectively. CONCLUSION: A novel tannase TanHcw from H. camelliae has been identified and characterized. The biological properties of TanHcw suggest that it plays a crucial role in the specific colonization of H. camelliae in tea plants. Discovery of the tannase TanHcw in this study gives us a reasonable explanation for the host specificity of H. camelliae. In addition, studying the characteristics of this enzyme offers the possibility of further defining its potential in industrial application.

Bacterial growth, morphology, and cell component changes in Herbaspirillum sp. WT00C exposed to high concentration of selenate.

Selenium (Se) is a nonmetallic element of the chalcogens. It is primarily available in natural environments as selenate and selenite oxoanions. Although selenate/selenite reduction in many microbes is widely studied at low concentrations (<50 mM), the effects of high selenate stress on bacterial growth, morphology, and cell components have not yet been studied. In this study, the response of Herbaspirillum sp. WT00C to selenate stress at high concentration is investigated by microbiological and scanning electron microscopy (SEM) techniques as well as proteomic analysis. Bacterial growth was seriously inhibited under high selenate concentrations and its growth-inhibitory phase was prolonged with the increase of selenate concentrations. More interestingly, this bacterium was able to recover its growth even if the selenate concentration was up to 400 mM. Its growth inhibition period shortened to 6 h when the bacterium growing in 200 mM selenate for 28 h was reinoculated to the Luria-Bertani medium containing 200 mM selenate. The high concentration of selenate also induces marked changes in the cell dimension and surface roughness, as revealed by SEM, along with compositional changes in the cell wall shown by proteomic analysis. The bacterial growth inhibition results from the marked downregulation of the α-subunit of DNA polymerase III and RNA helicase, whereas its growth recovery is related to its high antioxidative activities. More NADPH synthesis and the upregulation of thioredoxin reductase and GPx are beneficial for Herbaspirillum sp. WT00C to establish and maintain a balance between oxidant and antioxidant intracellular systems for defending selenate toxicity. This study is an important contribution to understanding why Herbaspirillum sp. WT00C survives in a high concentration of selenate and how the bacterial cells respond physiologically to selenate stress at high concentration.