| Abstract | In this study, the effects of growth conditions on archaellation in Methanococcus maripaludis were examined. Cells were grown in a variety of media including complex, minimal and with formate as the electron donor, with different nitrogen sources, varied salinities, and at a variety of growth temperatures. Of the conditions tested, western blot results showed that major archaellin FlaB2 levels only varied detectably as a result of growth temperature. While the amount of FlaB2 was similar for cells grown below 35°C, protein levels decreased at 38°C and were barely detectable at 42°C. Quantitative reverse transcription PCR experiments demonstrated that the flaB2 transcript levels were almost undetectable at 42°C. Electron microscopy confirmed that the FlaB2 levels detected by western blots corresponded to the state of archaellation, with cells grown at 42°C being mostly nonarchaellated. Unexpectedly, a lower apparent molecular mass for FlaB2 was observed in western blots of cells grown at temperatures beyond 38°C suggestive of a truncation in the attached N-linked tetrasaccharide at higher growth temperatures. Mass spectrometry analysis of archaella isolated from cells grown at 40°C confirmed that FlaB2 was now decorated with a trisaccharide in which the third sugar was also lacking the attached threonine and acetamidino modifications found in the wildtype glycan. |
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