| Popis: |
Transmembrane chemoreceptors of Escherichia coli bind periplasmic ligands and transduce the signal to the flagella motors, thereby adjusting the swimming behaviour of the cell according to the chemical nature of the ligand. Cell movement, directed either towards nutrients or away from toxic compounds, is known as chemotaxis. An important property of the chemotaxis signalling pathway essential for navigation in complex gradients of nutrients is adaptation, mediated by methylation of specific glutamate residues in the chemoreceptors cytoplasmic domain. The aspartate chemoreceptor Tar possesses four such sites, but it is still unclear why several sites of methylation are needed and if a certain hierarchy among these sites exists. In this study, we systematically and quantitatively characterized the efficiency of chemotaxis and the precision of adaptation for cells expressing Tar mutated at one or more modification sites as the only chemoreceptor. Therefore, we constructed Tar chemoreceptors with all possible combinations of alanine substitutions at the methylation sites to specifically render them non-methylatable. These Tar mutants were then tested for their ability to mediate chemotaxis on soft agar plates. Furthermore, adaptation kinetics of Tar mutants were analyzed by in vivo FRET microscopy and wild-type Tar was investigated by mass spectrometrical analysis, which allows to follow the order and kinetics of methylation at individual modification sites during the adaptation process. We found that the receptor methylation rate following addition of attractant differs for the individual methylation sites with methylation site 2 being fastest, followed by sites 1 and 3, and site 4 having the slowest rate of methylation. Demethylation upon removal of attractant occurs first at methylation site 3, followed by sites 2 and 1. Furthermore, we discovered that specific methylation sites are responsible for different features of chemotaxis and adaptation. Methylation site 1 mainly contributes to the adaptation precision and the methylation rate, whereas methylation site 2 is important for the methylation rate as well as for the demethylation rate. Methylation site 3 is responsible for the chemotaxis and the demethylation rate and methylation site 4 mainly contributes to the methylation rate. In summary, the results of the present study provide new insights into the molecular details of the adaptation process in E. coli chemotaxis and the subtle interplay of individual methylation sites in the regulation of chemotactic behavior. |
