Regulation des geschwindigkeitsvariablen Flagellenmotors von Sinorhizobium meliloti: Analyse von Regulator- und Mobilitätsproteinen
Final Report Abstract
The rigid, complex flagella of Sinorhizobium meliloti rotate solely in the CW sense without reversals, but they vary their rotary speed to direct changes in the swimming path. This type of motility control by a speed-variable motor has its molecular corollary in three novel motility proteins, MotC, MotD and MotE, present in addition to the ubiquitous MotA/MotB energizing proton channel. MotC and MotE are periplasmic proteins. MotC stabilizes MotB, and MotE is a specific periplasmic chaperone responsible for the proper folding and stability of MotC. After many fruitless attempts to identify the function of MotD, we could disprove its previously published classification as motor protein. At this point, we were able to reassign MotD as the hook-length regulator FliK. Up to now,ßiK was one of the few flagellar genes not annotated in the S. meliloti genome. Characteristic for its class, the 475-residue FliK contains a conserved, compactly folded Flg_hook domain in its carboxy-terminal region. Deletion ofßiK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutants all map in the cytoplasmic region of the transmembrane export protein FlhB, restoring assembly of a flagellar filament, and thus motility, in the presence of polyhooks. Structural properties of FliK are concurrent with its function as substrate specificity switch of the flagellar export apparatus from rod/hook-type to filament-type substrates. The activated chemotaxis response regulator CheY2-P acts as a "brake" at the cytoplasmic surface of the motor by binding to the C-ring protein FliM. We analyzed this interaction using two independent in-vitro approaches. Both studies revealed, that activated CheY2 has a stronger affinity to FliM than non-phosphorylated CheY2. We could quantify the binding affinities in the order CheY2 < CheY2D14K (a constitutively active, non-phosphorylated mutant) < CheY2-P = CheY2-BeF3 (a stable CheY2-P analogue). According to behavioral assays of single site mutants, amino acid residues Asn105, Asn106 and Val107 of CheY2 are part of a signalling surface that form a perfect steric match with an yet unidentified FliM binding surface. Our future studies will focus on the role of the periplasmic MoC for speed regulation, as well as on the quantification of CheY2-FHM interaction in vivo using Fluorescence Energy Transfer and in vitro using surface plasmon resonance analysis.
Publications
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(2004) The novel protein MotE serves as chaperone for the periplasmic motor protein MotC in Sinorhizobium meliloti. Molecular Microbiology 52, 701-712
E. Eggenhofer, Haslbeck M., Scharf B.
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(2005) Control of speed modulation (chemokinesis) in the unidirectional rotary motor of Sinorhizobium meliloti. Molecular Microbiology 56, 708-718
U. Attmannspacher, Scharf B., Schmitt R.
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(2006) MotD of Sinorhizobium meliloti and related ct-proteobacteria is the flagellar hook-length regulator and therefore reassigned as FliK. Journal of Bacteriology 188, 2144-2153
E. Eggenhofer, Rachel R., Haslbeck M., and Scharf B.
