Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

Abstract

MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.The MotA 4 MotB 2 complex of Escherichia coli is the stator of the rotary flagellar motor (5) and the H ϩ channel that couples the proton motive force to motility (1,9,(11)(12)(13)(14)23). At least 11 complexes can be accommodated per motor (17). MotA spans the cytoplasmic membrane four times and has short periplasmic loops between transmembrane segment 1 (TM1) and TM2 and between TM3 and TM4 (3, 21). Large cytoplasmic domains occur between TM2 and TM3 and following TM4. MotB has a cytoplasmic N terminus of 25 residues, a single TM domain, and a periplasmic domain with a conserved peptidoglycan-binding motif (2, 18) that anchors the Mot complex to the cell wall (4, 10).We asked whether electrostatic interactions between negatively charged residues near the C terminus of MotA and positively charged residues near the N terminus of MotB facilitate formation of the MotA 4 MotB 2 complex for three reasons. (i) MotB is unstable in the absence of MotA (20), so coinsertion into the membrane and rapid complex assembly might be advantageous. (ii) Translation of motA and motB is coupled (20). Thus, the initial proximity of the C terminus of MotB and the N terminus of MotB would facilitate their early interaction. (iii) Clusters of charged residues in MotA and MotB are conserved.

Analysis of the Borrelia burgdorferi Cyclic-di-GMP-Binding Protein PlzA Reveals a Role in Motility and Virulence

“…We speculate that PlzA or a PlzA-regulated factor(s) may modulate the function of a motor protein, perhaps by modulating the MotB proton channel. A recent study with E. coli reported that amino acid residues K53 to T64 of MotB act as a plug to prevent proton flow before the MotA-MotB complex associates with the flagellar structure (31,32). Because in plzA cells the swarming motility is attenuated but the swimming pattern was not altered, it appears likely that PlzA acts as a positive regulator in B. burgdorferi motility or chemotaxis.…”

The Flagellar Protein FliL Is Essential for Swimming in Rhodobacter sphaeroides

“…MotB from R. sphaeroides has 366 residues, and remarkable differences were detected when it was compared to the E. coli MotB sequence (29). In fact, from the alignment of the MotB sequences of these bacteria, it can be observed that the similarity is restricted mainly to the C-terminal region while the region proposed as the MotB channel plug is not conserved in R. sphaeroides.…”

Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation

“…The occurrence of the motB(L149V) and motB(N278K) suppressor mutations in the periplasmic region of MotB of the pseudorevertants, which are located rather away from the proton pathway, implies that these mutations restore the motor function by acting indirectly rather than by altering the protonconducting pathway directly. As proper positioning of the MotA/B complex relative to the rotor would require a defined conformation of the C-terminal periplasmic domain of MotB (17)(18)(19), the reduced energy-coupling efficiency caused by the D33E mutation may be restored by modifications in the geometrical coupling of the rotor-stator interaction.…”