Load‐sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor

Abstract

SummaryThe Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild-type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild-type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their 'unplugged' forms were only half of the conductivity of the wildtype channel. These results suggest that the D33E mutation induces a load-dependent inactivation of the MotA/B complex. We propose that the stator element is a load-sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co-ordinated proton translocation.

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

“…Multiplying the number of stator complexes by the radius of the contact point lever and by the estimated 7.3-pN force exerted per stator complex, we observe excellent correlation between our predicted and the observed torque magnitudes in all four groups (Fig. 6B): a predicted torque of 1,606 pN·nm for enteric bacteria vs. an observed torque of 1,260 pN·nm (14), with some estimates of 2,000 pN·nm (20); a predicted torque of 2,040 pN·nm for Vibrio vs. an observed torque of 2,200 pN·nm at low sodium concentrations, increasing with higher concentrations; a predicted torque of 3,288 pN·nm for e-proteobacteria vs. an observed torque of 3,600 pN·nm; and a predicted torque of 3,562 pN·nm for spirochetes vs. an observed torque of 4,000 pN·nm. Although this model is clearly a simplification of the process, and biophysical studies reveal that additional stator complexes provide incrementally smaller torque contributions (14), we believe the salient features of our model's predictions are compelling.…”

“…For example, C. crescentus motors have been measured to produce torques of 350 pN·nm (2). Estimates for the torque of the enteric motor ranges from ∼1,300 to ∼2,000 pN·nm (20,21). The e-proteobacterium H. pylori has been estimated to swim with torque of 3,600 pN·nm (22), and spirochetes are capable of swimming with 4,000 pN·nm of torque (21,23).…”

Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

“…After fusing the fluorescent proteins YPet, eGFP and Dendra2 directly to the N-terminus of MotB (FPs-MotB), as done in several studies with (e) 15,18,19,21,29,38 , we verified that the constructed strains were motile in soft agar at the population level (Fig. 1B).…”

“…One direction of research for which this holds is the study of the internal dynamics of proteins complexes 13,14 . Recently, several studies have made use of FPFs to study the internal subunit dynamics of the bacterial flagellar motor (BFM) [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] . Although FPFs were shown to affect BFM function by decreasing the chemotactic motility of cells and average speed of motors 15 , a quantitative characterization of the impact on the BFM mechanical behavior is lacking.…”

“…The stators dynamically turn over between two populations: one that is bound to the BFM and one that passively diffuses in the inner membrane 15,37 . These dynamics have been observed directly at the single-stator level using fluorescent fusions to the N-terminus of MotB 15,18,19,21,28,29 . More recently, similar fusions have been used to unveil that the number of stators recruited into the complex depend on the load of the BFM 18,19,29 .…”