Western Blot analysis of dodecin was performed for the determination of the dodecin expression level during H. salinarum growth. Aliquots of cell lysates normalized to the internal cell volume were subjected to SDS-PAGE. After transfer of proteins by Electro- blotting, PVDF membranes were reacted in a standard Western-blotting procedure with polyclonal chicken anti-dodecin antibodies directed as primary antibodies. Dodecin was identified using chicken anti-dodecin as primary and horseradish peroxidase labelled anti- chicken as secondary antibodies. Blots were exposed by chemiluminescence reaction and for the investigation of the dodecin expression level during a H. salinarum growth curve evaluated and compared by eye.
As lumichrome was considered as a potential stimulus for the dodecin expression, the development of dodecin expression was also monitored in cells grown in media supplemented with 40 µM of lumichrome. As shown in Figure 38, at this concentration lumichrome strongly affected cell growth in light independent of the presence (R1) or absence (R1∆3073) of dodecin.
Figure 38. Growth Curves of H. salinarum
Growth of the H. salinarum wild type strain R1 in the dark (R1_d, black) and light (R1_l, red) as well as of the deletion strain R1∆3073 in the dark (R1∆3073_d, gray) and light (R1∆3073_l, orange). Curves in A illustrate cell growth in standard growth medium for H. salinarum; curves in B growth in medium supplemented with lumichrome to 40 µM. Uniform cell growth could be observed except for the combination of light and lumichrome (at 40 µM) which led to a depression of growth in the stationary phase (irrespective of the presence of dodecin).
Growth curves summarized in Figure 38 clearly indicate the dodecin gene product not to be required for general viability. Western Blot analysis of the respective cultures, shown in
0 5 10 15 20 0 40 80 120 R1_d R1_l R1del3073_d R1del3073_l Kl et t time [d]
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0 5 10 15 20 0 40 80 120 R1_d R1_l R1del3073_d R1del3073_l Kl et t time [d]Figure 39, revealed that dodecin is regulated during H. salinarum growth. In general, dodecin expression was found to be induced upon transition into the logarithmic growth phase. Figure 39.A and 39.B illustrate the dodecin expression level in wildtype cells grown in light and dark, respectively. While in Figure 39.B the staining intensities of dodecin bands vary throughout the experiment, weak intensities at day 12 and 13 in Figure 39.A imply down- regulation of dodecin in the late stationary phase, when cells are grown in light.
A different pattern of the dodecin expression was observed when the growth medium was supplemented with lumichrome (40 µM). Figure 39.C indicate a fast induction of dodecin expression in cells grown in light and lumichrome, followed by a fast down-regulation of dodecin starting at day 5. According to growth curves in Figure 38, it should be taken into account that the reduction in staining intensities might be supported by an extensive decay of viable cells which is not correspondingly followed by the optical density and which could lead to an overestimation of dodecin down-regulation. In cells grown in presence of lumichrome in the dark, dodecin expression is retarded (Figure 39.D). This is in strong contrast to the dodecin regulation in cells grown in light and lumichrome (Figure 39.C).
Figure 39. Western Blot Analysis of the Dodecin Expression Level
Western Blot Analysis was performed with H. salinarum wildtype cells grown in light (A) and in the dark (B) as well as in light and in dark in presence of 40 µM lumichrome (C,D). The dodecin expression is generally found to be induced at day two or three irrespective of the conditions tested. Variations in staining intensities of blot B might rather reflect experimental errors than a variation in the dodecin expression level.
1 2 3 4 5 8 12 13 day 1 2 3 4 5 8 12 13 day 1 2 3 4 5 8 12 13 day 1 2 3 4 5 8 12 13 day
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By Western Blot analysis the dodecin regulation in cultures of H. salinarum was qualitatively determined. No effort was spent into the determination of the dodecin copy numbers and blots do not allow comparing dodecin concentrations among the respective conditions. However, in a single experiment the dodecin cellular concentration in the cytosol of H. salinarum grown in the dark to the stationary phase was analyzed, as shown in Figure 40. Heterologously expressed dodecin at different concentrations was used as a standard. Staining intensities were integrated with imageG (www.ncbi.nlm.nih.gov). The dodecin molarity in H. salinarum wildtype cells R1 was calculated to 9.2 µM which corresponds to a dodecin copy number of 8000 at the respective cellular state (9.2 10-6 M x 1.47 10-15 l (volume of a single cell) x 6.022 1023 (NL, Loschmidt number)).
Figure 40. Quantitative Western Blot Analysis of Dodecin
(A) Cell lysate of a H. salinarum R1 wildtype culture grown to stationary phase in the light was loaded at two concentrations onto a SDS PAGE gel (X and X2) next to heterologously expressed and refolded apododecin at defined concentrations (A: 10 µM, B: 15 µM, C: 20 µM and D: 25 µM). (B) Determination of the dodecin molar cellular concentration to 9.2 µM by regression analysis of the reference staining intensities (bands A, B, C and D, Figure 3.A) and extrapolation to the staining intensity of band X (red dot). Differences in the run distances (X, X2 vs. A, B, C, D) reflect the higher mass of the heterologously expressed dodecin compared to wildtype dodecin due to N-terminally attached His-affinity tag (additional 8 amino acids).