Perspectives on Bacterial Flagellar Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

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Impact of fluorescent protein fusions on the bacterial flagellar motor

10.1101/152595 ◽

2017 ◽

Cited By ~ 1

Author(s):

M Heo ◽

AL Nord ◽

D Chamousset ◽

E van Rijn ◽

HJE Beaumont ◽

...

Keyword(s):

Side Effects ◽

Protein Function ◽

Fusion Proteins ◽

Fluorescent Proteins ◽

Protein Complexes ◽

Switching Frequency ◽

Flagellar Motor ◽

Internal Dynamics ◽

Biologically Relevant ◽

Bacterial Flagellar Motor

AbstractFluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects. Such insight, however, is still lacking for many applications of fluorescent fusions. This is particularly relevant in the study of the internal dynamics of motor protein complexes, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to thestatorof the bacterial flagellar motor (BFM) complex have previously been used to successfully unveil the internal subunit dynamics of the motor. Here we report the effects of three different fluorescent proteins fused to the stator, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry in the complex remained unaffected. MotB fusions decreased the rotation-direction switching frequency of single motors and induced a novel BFM behavior: a bias-dependent asymmetry in the speed attained in the two rotation directions. All these effects could be mitigated by the insertion of a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions on BFM dynamics and their alleviation—new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Author summaryMuch of what is known about the biology of proteins was discovered by fusing them to fluorescent proteins that allow detection of their location. But the label comes at a cost: the presence of the tag can alter the behavior of the protein of interest in unforeseen, yet biologically relevant ways. These side effects limit the depth to which fluorescent proteins can be used to probe protein function. One of the systems that has been successfully studied with fluorescent fusions for which these effects have not been addressed are dynamic protein complexes that carry out mechanical work. We examined how fluorescent proteins fused to a component of the bacterial flagellar motor complex impacts its function. Our findings show that the fusion proteins altered biologically relevant dynamical properties of the motor, including induction of a novel mechanical behavior, and demonstrate an approach to alleviate this. These results advance our ability to dissect the bacterial flagellar motor, and the internal dynamics of protein complexes in general, with fluorescent fusion proteins while causing minimal perturbation.

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Spontaneous adaptation of ion selectivity in a bacterial flagellar motor

10.1101/2021.01.26.427765 ◽

2021 ◽

Author(s):

Pietro Ridone ◽

Tsubasa Ishida ◽

Yoshiyuki Sowa ◽

Matthew A. B. Baker

Keyword(s):

Genome Engineering ◽

Environmental Changes ◽

Ion Selectivity ◽

Bacterial Species ◽

Selective Advantage ◽

Flagellar Motor ◽

Novel Mutations ◽

Power Sources ◽

Motor Complex ◽

Bacterial Flagellar Motor

ABSTRACTMotility provides a selective advantage to many bacterial species and is often achieved by rotation of flagella that propel the cell towards more favourable conditions. In most species, the rotation of the flagellum, driven by the Bacterial Flagellar Motor (BFM), is powered by H+ or Na+ ion transit through the torque-generating stator subunits of the motor complex. The ionic requirements for motility appear to have adapted to environmental changes throughout history but the molecular basis of this adaptation, and the constraints which govern the evolution of the stator proteins are unknown. Here we use CRISPR-mediated genome engineering to replace the native H+-powered stator genes of Escherichia coli with a compatible sodium-powered stator set from Vibrio alginolyticus and subsequently direct the evolution of the stators to revert to H+-powered motility. Evidence from whole genome sequencing indicates both flagellar- and non-flagellar-associated genes that are involved in longer-term adaptation to new power sources. Overall, transplanted Na+-powered stator genes can spontaneously incorporate novel mutations that allow H+-motility when environmental Na+ is lacking.

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