FIGURE 3:

Investigation of the role of SltA32kDa forms. (A) Conditional expression of SltA chimeras driven by the thiamine-repressible thiA^p promoter. Addition of 100 μM thiamine to AMM prevents expression of thiA^p. When SltA fusions were expressed through thiA^p as sole source of this transcription factor, the presence of thiamine led to a null sltA phenotype for sensitivity to high concentrations of sodium or lithium and to alkalinity but a nearly WT phenotype in the absence of vitamin B1. (B) Western blots detect the three forms of SltA in strain MAD4294 (thiA^p::sltA::ha₃) in the absence of thiamine. Two bands having almost identical mobilities to the SltA32kDa bands are detectable when SltA^400–698-HA₃ is expressed. The absence of SltB has no effect on the presence or mobility of SltA^400–698-HA₃ bands. (C) Dephosphorylation assay of extracts from strain MAD4661 expressing the SltA^400–698-HA₃ fusion. Treatment with λ-protein phosphatase (λPP) results in loss of the low-mobility band and increases the intensity of the high-mobility band for the SltA^400–698-HA₃ fusion, demonstrating phosphorylation of this chimera (indicated on the left); c, contains total crude protein extracts obtained using the alkaline lysis procedure, resulting in a higher level of detectable SltA^400–698-HA₃ forms. (D) Western blots showing the presence of the 100-kDa truncated SltB-GFP form when the SltA^400–698-HA₃ fusion was expressed. Actin was used as loading control. The SltA-HA₃ forms in those protein extracts are shown below.