3.1 Selenate tolerance of four different strains
H. camelliae WT00C was an endophytic bacterium isolated from tea plant [26], and it was able to grow in 400 mM selenate as shown in Fig. 2A (a). Clearly, the lag phase of this bacterium was prolonged with the increase of selenate concentrations. For instance, its lag phases in 50, 100, 200 and 400 mM selenate were 3, 6, 12 and 60 h respectively. When selenate concentration was ≥ 500 mM, this bacterium did not grow. To improve the selenate tolerance, H camelliae WT00C was exposed to 200 mM selenate for multiple rounds, and each round was 24 h, After 4 rounds of exposures to 200 mM selenate, the lag phases of the strain named as CT00C reduced to 2 and 3 h in 200 and 400 mM selenate (see Fig. 2B (a)). Figure 2B (a) also showed that the strain CT00C was able to grow in 600 and 800 mM selenate although its growth required relatively long time. After 6 rounds of exposures, the bacterium named as NCT00C further shortened its lag phase. Different from the original strain WT00C, the growth of the strain NCT00C in the range of 0-400 mM selenate did not show significant difference (see Fig. 2C(a)). Moreover, the lag phases of the strain NCT00C were only 2 and 8 h when it grew in 600 and 800 mM selenate. Shortening lag phase and growing in > 500 mM selenate illustrated that the selenate tolerance of bacterial cells was greatly improved via 4–6 rounds of exposures. Figure 2C (a) also showed that the strain NCT00C grew much better than the strain CT00C under the conditions of 600 and 800 mM selenate. The results from growth-curve comparison indicated that the selenate tolerance of the strain NCT00C was the strongest among four strains. We also performed 8 rounds of exposures, and found that the strain NT00C was able to grow in 200 mM selenate but it did not grow when selenate concentration was ≥ 400 mM (see Fig. 2D (a)). Its lag phases in 50, 100 and 200 mM selenate were 1, 2 and 4 h. Although its lag phases were shorter than those of the strain WT00C in ≤ 200 mM selenate, the strain NT00C was unable to grow when selenate concentrations were ≥ 400 mM. Obviously, too many exposures to high concentration of selenate were detrimental to the selenate tolerance of bacterial cells.
The growth status of four strains in different concentrations of selenite was also examined. The results (see the supplementary data 2) showed that H. camelliae WT00C only grew in ≤ 10 mM selenite, and its lag phases in 0.5, 1, 5 and 10 mM selenite were 10, 20, 30 and 60 h respectively. Compared to the strain WT00C, the strains NCT00C and CT00C not only markedly shortened lag phases but also grew in 40 mM selenite. After 8 rounds of exposures, the strain NT00C displayed good growth status in 0–10 mM selenite but it did not grow when selenite concentration was > 20 mM. Clearly, the strains NCT00C and CT00C also displayed strong selenite tolerance, which agreed with those results obtained from bacterial growth status in selenate.
3.2 Red elemental selenium (Se0) formation of four different strains
When bacterial cells grew in the medium containing selenate or selenite, red color formation in the culture indicated the generation of red elemental selenium (Se0) [29, 33, 34]. As shown in Fig. 2A (b), the cultures of H camelliae WT00C became red in 50─400 mM selenate. When 600 and 800 mM selenate were used, the bacterial cultures did not show any color change. Different from H camelliae WT00C, the strains CT00C and NCT00C changed the color red in 50–800 mM selenate (see Fig. 2B (b) and Fig. 2C (b)). Unlike the strain CT00C and NCT00C, the strain NT00C only changed the color of cultures containing 50–200 mM selenate. When selenate concentration was over 200 mM, the culture color did not change anymore (see Fig. 2D (b)). Table 2 also showed the time of red-color appearance in the cultures containing different selenate concentrations. The strain NCT00C turned the culture red more rapidly than the strain CT00C, NT00C and WT00C. The red color appearance in the culture containing 200 mM selenate was 16 h for WT00C and 5 h for the strain CT00C, 4 h for the strain NCT00C and 10 h for the strain NT00C. In 400 mM selenate, red color appearance was 66 h for the strain WT00C, 7 h for CT00C and 4 h for NCT00C, respectively. These results indicated that the strain NCT00C growing in high concentrations of selenate was able to effectively catalyze selenate reduction to form red elemental selenium (Se0) in a relatively short time.
Table 2
The time of beginning to show red color in bacterial cultures. Four bacterial strains were respectively incubated at 37°C in the LB medium containing 0-800 mM selenate for different times.
Selenate (mM)
|
0
|
50
|
100
|
200
|
400
|
800
|
WT00C
|
-
|
8 ± 0.5 h
|
12 ± 1.0 h
|
16 ± 1.5 h
|
66 ± 1.5 h
|
-
|
CT00C
|
-
|
5 ± 0.5 h
|
5 ± 0.5 h
|
5 ± 0.4 h
|
7 ± 0.5 h
|
50 ± 2.5 h
|
NCT00C
|
-
|
4 ± 0.5 h
|
4 ± 0.5 h
|
4 ± 0.2 h
|
4 ± 0.2 h
|
14 ± 1.2 h
|
NT00C
|
-
|
6 ± 0.5 h
|
7 ± 0.5 h
|
10 ± 1.0 h
|
-
|
-
|
When bacterial cells grew in different concentrations of selenite, H camelliae WT00C only turned the culture red when selenite concentration was ≤ 10 mM. Different from the strain WT00C, the strains CT00C and NCT00C were able to change the color of the culture containing 40 mM selenite. However, the strain NT00C only changed the culture color when selenite concentration was ≤ 20 mM (see the supplementary data 2). Among four strains, the strains, CT00C and NCT00C were the best candidates for effectively catalyzing selenite reduction to form red elemental selenium (Se0).
3.3 Productivities of red elemental selenium and selenoproteins
Productivities of red elemental selenium (Se0) in four different strains were examined by growing bacterial cells in LB broth containing 100 mM selenate to logarithmic phase. Figure 3 showed that selenium contents ( µg/ml) of the strains CT00C and NCT00C were 36% more than that of the strain WT00C (see Fig. 3(a)), and selenium conversion efficiencies ( µg/108 cells) were also 30% higher than that of the strain WT00C (see Fig. 3(b)). High selenium conversion efficiencies indicated that the capability of the strains CT00C and NCT00C to generate red elemental selenium via selenate reduction were more effectively than the strain WT00C.
We also used the strains NCT00C and WT00C-se [34] (two rounds of forced evolution) to produce red elemental selenium via fermentation in LB broth containing 200 mM selenate. The yields of pure elemental selenium gave 1028 mg/L for the strain NCT00C and 560 mg/L for the strainWT00C-se. High yield of red elemental selenium demonstrated that the strains NCT00C indeed was a better candidate for effectively catalyzing selenate reduction to produce red elemental selenium (Se0). Figure 4 showed fermentation in 5L fermantor, Se-nanospheres observed by TEM in bacterial culture and the red elemental selenium (Se0) purified from the fermentation culture.
Productivities of selenoproteins synthesized in four different strains were also examined by growing bacterial cells in LB broth containing 50 mM selenate to the stationary phase. Selenoproteins were estimated by measuring selenium contents in bacterial proteins (µg/mg). As showed in Fig. 3c, selenium contents in bacterial proteins of the strains CT00C and NCT00C were 40% more than that of the strain WT00C, Thus, the strains CT00C and NCT00C integrated selenium into proteins to form selenoproteins more effectively as compared to the strain WT00C.
3.4 Transcriptional levels of the genes involving in selenate metabolism and tolerance
Transcriptional levels of 19 genes involving in elemental selenium (Se0) and selenoprotein synthesis as well as selenate tolerance in four different strains were investigated by using qPCR. 6 genes for elemental selenium synthesis were cysD (sulfate adenylyltransferase (SAT), EC 2.7.7.4), gsdA (glucose-6-phosphate 1-dehydrogenase (G6PD), EC 1.1.1.49), icd (isocitrate dehydrogenase (IDH), EC 1.1.1.42), gshA (glutamylcysteine synthetase (GCL), EC 6.3.2.2), gshB (glutathione synthase (GSS), EC 6.3.2.3) and gsr (glutathione reductase (GR), EC 1.8.1.7), in which GCL and GSS are responsible for glutathione (GSH) formation. 4 genes for selenoprotein synthesis were trxR (thioredoxin reductase (TRX), EC 1.8.1.9), selD (selenide, water dikinase, EC 2.7.9.3), selA (L-seryl-tRNA(Ser) seleniumtransferase, EC 2.9.1.1) and metG (methionyl-tRNA synthetase (MARS), EC 6.1.1.10). Those genes were involved in selenate metabolism as showed in Fig. 1. 8 genes (mdh, ligK, pntA, iolA, ghrA, gluC, mmsB, argC) thought to be related with selenate tolerance [34] were also examined. In addition, the expression of lpxB gene encoding lipid-A-disaccharide synthase [EC 2.4.1.182] was incidentally tested. To keep the consistency of analytical conditions, all four strains were incubated at 37°C, 200 rpm in LB medium containing 100 mM selenate. As shown in Fig. 5, the transcriptional levels of 6 genes for elemental selenium synthesis in CT00C, NCT00C and NT00C strains displayed the increase of ≥ 10-fold as compared to the wild-type strain WT00C (see Fig. 5(a)). Although the expression of icd and gshA genes in the NT00C strain increased significantly, the expression of cysD gene responsible for the reduction of selenate to selenite was much less that those of the strains CT00C and NCT00C. In the pathway of selenoprotein synthesis, the transcriptional levels of trxB, selD, selA and metG genes in CT00C and NCT00C strains were about 10-fold more than those for the strain WT00C. The strain NT00C displayed the increase of trxB and selD gene expressions but its transcription of selA and metG genes did not show significant increase as compared to the strain WT00C (see Fig. 5(b)). Those results suggested that two strains CT00C and NCT00C held a strong capacity of synthesizing elemental selenium and selenoproteins.
Figure 5(c) showed the transcriptional levels of mdh, ligK, pntA, iolA, ghrA, gluC, mmsB and argC genes in four different strains. As compared to the strain WT00C, the strains CT00C and NCT00C significantly increased transcriptional levels of 8 genes (≥ 5-fold), whereas the strain NT00C only increased the transcription of 5 genes (mdh, pntA, iolA, ghrA, gluC). Although the expression of mdh, pntA and gluC genes was markedly increased in the strain NT00C, its mmsB and argC genes were poorly transcribed. The transcriptional levels of the mmsB and argC genes in the strain NT00C was even less than that of the strain WT00C. Clearly, the strains CT00C and NCT00C indeed held strong selenate tolerability. In addition, Fig. 5 (d) also showed the expression of lpxB gene involving in lipopolysaccharide (LPS) biosynthesis. The strains CT00C and NCT00C displayed 10-fold less expression, whereas the strain NT00C increased 7 folds as compared to the strain WT00C. Less transcription of lpxB gene suggested that the activity of lpxB gene was seriously inhibited by high concentration of selenate, which was in accord with the results observed in the previous study [34]. Nevertheless, the expression of lpxB gene was gradually increased along with the increase of exposure times in selenate as shown in Fig. 5 (d). After 8 rounds of exposure to high concentration of selenate, the lpxB gene in the strain NT00C was highly activated.
3.5 Morphological characteristics of bacterial cells
Cell morphologies of the strain WT00C growing in the LB medium and the strains CT00C, NCT00C and NT00C exposing to 200 mM selenate for 4, 6 and 8 rounds were observed by scanning electron microscopy. As shown in Fig. 6, bacterial sizes were varied at different growth stages. The cells of the strains WT00C, CT00C and NCT00C began to grow and divide at 2 h incubation, whereas the cells of the strain NT00C were in the inhibitory state. At the stationary phase (24 h), the cell sizes were 1.17 ± 0.04 µm×327 ± 6 nm for the stain WT00C growing in LB medium [34], 1.81 ± 0.06×1.02 ± 0.05 µm for the stain CT00C. 1.91 ± 0.05×1.03 ± 0.04 µm for the stain NCT00C and 2.10 ± 0.07×1.06 ± 0.08 µm for NT00C. Clearly, the size of bacterial cells became bigger as the number increase of exposures to high concentration of selenate. The size change mainly manifested in the increase of cell width although cell length was also increased. Three strains CT00C, NCT00C and NT00C in cell width were almost 3-fold of the strain WT00C. The strain NT00C had doubled in cell length as compared to the original strain. Those enlarged cells were incubated in LB medium for 24 h, their sizes did not return to the original size of the strain WT00C. We also observed the changes of cell surface roughness under 50,000 magnifications. Bacterial cells displayed surface changes from the rough to the smooth during multiple forced evolutions. After 8 rounds of forced evolution, the cell surface of the stain NT00C did not recover to the original roughness although its lpxB gene was markedly up-regulated. These results clearly demonstrated that bacterial cells underwent obviously physiological and morphological changes when H camelliae WT00C was exposed to high concentration of selenate.