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.

Red elemental selenium (Se0 ) improves the immunoactivities of EPC cells, crucian carp and zebrafish against spring viraemia of carp virus.

Selenium, as an essential trace element, interferes through selenoproteins in many physiological processes of plants and mammals. Its antiviral activity has recently attracted much attention because selenium improves the antiviral capacity of animal cells against a few viruses relevant to human diseases. In this study, the red elemental selenium was purified from the fermentative culture of Herbaspirillum camelliae WT00C and then used to culture epithelioma papulosum cyprinid (EPC) cells or feed crucian carp and zebrafish. Finally, its antiviral effects were investigated at the cell level and living fishes after spring viraemia of carp virus infection. At the cell level, 5, 10 and 20 µg ml-1 red elemental selenium significantly induced the expression of interferon (IFN) and ISG15 genes in EPC cells. The viral TCID50 (50% tissue culture infective dose) values in the EPC cells incubated with 5, 10 and 20 µg ml-1 red elemental selenium were significantly less than those of the control. More expression of IFN and ISG15 genes and less TCID50 values indicate that red elemental selenium indeed improves the antiviral capability of EPC cells. In the crucian carp fed with the food containing 5 and 10 µg g-1 red elemental selenium, IFN expressions showed 13- and 39-fold increases at the 16th day of post-injection, and its expression was dependent on selenium concentrations. Meanwhile, no fish death occurred in all the experimental groups. In the zebrafish fed with the red worm containing 5 µg g-1 red elemental selenium, IFN and Mx expressions and survival rate were significantly higher than those of the control. The results of this study show that red elemental selenium indeed improves the antiviral activity of fish. The antiviral effects of selenium mainly come from its immune regulation through its incorporation into selenoproteins. The optimum level of selenium contributes to improving fish immunity, whereas excess selenium causes excessive immune and inflammatory responses.

Herbaspirillum camelliae sp. nov., a novel endophytic bacterium isolated from Camellia sinensis L.

Bacterial strain WT00CT is an endophytic bacterium that was isolated from the tea plant (Camellia sinensis L.). The phylogenetic analysis of 16S rRNA genes demonstrated that strain WT00CT was a member of the genus Herbaspirillum. This strain is microaerobic, gram-negative and non-pigmented, and its cells are rod shaped, with a polar flagellum. It grew optimally at 34-37 °C, pH 5.0-8.0 and 0-1.5% NaCl (w/v). The G + C content of its genomic DNA was 62.36 mol%. C16:0, iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0 were major fatty acids. The strain WT00CT contained six polar lipids, namely DPG (diphosphatidylglycerol), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), PC (phosphatidylcholine), GL (glycolipid) and APL (aminophospholipids), and its respiratory quinone was Q8. The strain WT00CT had a genome size of 6.08 Mb with a total ORF of 5,537, in which one gene cluster (36 genes) encoding a type IV secretion system was absent in other members of the Herbaspirillum genus. ANI values of genomic comparison between the strain WT00CT and other Herbaspirillum species were 75-96%. Based on the phylogenetic, chemotaxonomic and phenotypic data presented here, the strain WT00CT represents a novel species in the Herbaspirillum genus, for which the name Herbaspirillum camelliae sp. nov. is proposed. The type strain of H. camelliae sp. nov. is WT00CT (AB 2018017 T and KCTC 62527 T).

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.