RESUMEN
The bacterial flagellar motor is a complex macromolecular machine whose function and self-assembly present a fascinating puzzle for structural biologists. Here, we report that in diverse bacterial species, cell lysis leads to loss of the cytoplasmic switch complex and associated ATPase before other components of the motor. This loss may be prevented by the formation of a cytoplasmic vesicle around the complex. These observations suggest a relatively loose association of the switch complex with the rest of the flagellar machinery. IMPORTANCE We show in eight different bacterial species (belonging to different phyla) that the flagellar motor loses its cytoplasmic switch complex upon cell lysis, while the rest of the flagellum remains attached to the cell body. This suggests an evolutionary conserved weak interaction between the switch complex and the rest of the flagellum which is important to understand how the motor evolved. In addition, this information is crucial for mimicking such nanomachines in the laboratory.
Asunto(s)
Bacterias/metabolismo , Flagelos/fisiología , Bacterias/química , Bacterias/clasificación , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/química , Conformación ProteicaRESUMEN
The cytoplasmic C ring of the bacterial flagellum is known as the switch complex. It binds the response regulator phospho-CheY to control the direction of flagellar rotation. The C ring of enteric bacteria is well characterized. However, no Gram-positive switch complex had been modeled. Ward et al. (E. Ward, E. A. Kim, J. Panushka, T. Botelho, et al., J Bacteriol 201:e00626-18, 2019, https://doi.org/10.1128/JB.00626-18) propose a structure for the Bacillus subtilis switch complex based on extensive biochemical studies. The work demonstrates that a similar architecture can accommodate different proteins and a reversed signaling logic.
Asunto(s)
Bacillus subtilis , Flagelos , Proteínas Bacterianas , Rotación , Transducción de SeñalRESUMEN
Flagellar rotation regulates the phenomenon of chemotaxis in bacteria. The interaction between the stator unit and the rotor unit of the flagellar motors is responsible for switching the direction of bacterial flagellar rotation. However, the molecular interaction mechanism between the stator (MotA/MotB) and the rotor (FliG/FliM/FliN) proteins for the flagellar rotational direction switching was not very clear. To address this, the asymmetry in the copies of FliG, FliM, and FliN molecules was resolved by reconstructing the switch complex using a modeled rotor unit that fulfills the experimentally available geometric constraints. The diameter of our assembled switch complex supported the existing literature. Experimental evidence and the conformational spread model validates our constructed switch complex. Subsequently, normal mode analysis (NMA) on these constructed protomer units revealed that the most fluctuating molecule in the rotor unit is FliG, which interacts with the bacterial stator through its C-terminal domain. NMA also facilitates our understanding of the reorientation mechanism of FliG between the two states of its flagellar rotation, i.e., counter-clockwise to clockwise and vice versa. Our observations regarding speed regulation, the gap between rotor and stator, and the flagellar switching due to the activity of cytoplasmic proteins, indicate that the bacterial flagellar motor uses the same mechanism as that of an electric motor. Graphical abstract Molecular mechanism of the bacterial flagellar switch.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Rotación , Thermotoga maritima/metabolismo , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Bacterial flagella are rotary nanomachines that contribute to bacterial fitness in many settings, including host colonization. The flagellar motor relies on the multiprotein flagellar motor-switch complex to govern flagellum formation and rotational direction. Different bacteria exhibit great diversity in their flagellar motors. One such variation is exemplified by the motor-switch apparatus of the gastric pathogen Helicobacter pylori, which carries an extra switch protein, FliY, along with the more typical FliG, FliM, and FliN proteins. All switch proteins are needed for normal flagellation and motility in H. pylori, but the molecular mechanism of their assembly is unknown. To fill this gap, we examined the interactions among these proteins. We found that the C-terminal SpoA domain of FliY (FliYC) is critical to flagellation and forms heterodimeric complexes with the FliN and FliM SpoA domains, which are ß-sheet domains of type III secretion system proteins. Surprisingly, unlike in other flagellar switch systems, neither FliY nor FliN self-associated. The crystal structure of the FliYC-FliNC complex revealed a saddle-shaped structure homologous to the FliN-FliN dimer of Thermotoga maritima, consistent with a FliY-FliN heterodimer forming the functional unit. Analysis of the FliYC-FliNC interface indicated that oppositely charged residues specific to each protein drive heterodimer formation. Moreover, both FliYC-FliMC and FliYC-FliNC associated with the flagellar regulatory protein FliH, explaining their important roles in flagellation. We conclude that H. pylori uses a FliY-FliN heterodimer instead of a homodimer and creates a switch complex with SpoA domains derived from three distinct proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/química , Helicobacter pylori/química , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Sistemas de Secreción Tipo III/química , Cristalografía por Rayos X , Flagelos/ultraestructura , Proteínas de la Membrana , Complejos Multiproteicos/química , Dominios ProteicosRESUMEN
The bacterial flagellar motor is capable of adapting to changes in the concentrations of extracellular chemical stimuli by changing the composition of the switch complex of the flagellar motor. Such remodeling-based adaptation complements the receptor-mediated adaptation in the chemotaxis network to help maintain high sensitivity in the response of the motor to phospho-CheY concentrations, despite cell-to-cell variability in the abundances of chemotaxis proteins. In this chapter, a modeling approach is described that explains the mechanisms of switch-remodeling and motor-mediated adaptation. The approach is based on observations of structural differences, associated with the direction of motor rotation, that modulate the strength of FliM/FliN binding within the switch. By modulating the number of CheY-P-binding sites within the motor, remodeling maximizes sensitivity over a range of signal levels.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/fisiología , Proteínas Quimiotácticas Aceptoras de Metilo/química , Adaptación Fisiológica , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/química , Sitios de Unión , Quimiotaxis , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Modelos Teóricos , Unión ProteicaRESUMEN
The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliFC) bound to the N terminal domain of the switch complex protein FliG (FliGN) reveals that FliGN folds around FliFC to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The interface is consistent with solution-state nuclear magnetic resonance, small-angle X-ray scattering, in vivo interaction studies, and cellular motility assays. Co-folding with FliFC induces substantial conformational changes in FliGN and suggests that FliF and FliG have the same stoichiometry within the rotor. Modeling the FliFC:FliGN complex into cryo-electron microscopy rotor density updates the architecture of the middle and upper switch complex and shows how domain shuffling of a conserved interaction module anchors the cytoplasmic rotor to the membrane.
Asunto(s)
Proteínas Bacterianas/química , Membrana Celular/química , Flagelos/química , Proteínas de la Membrana/química , Thermotoga maritima/química , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Fenómenos Biomecánicos , Membrana Celular/ultraestructura , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/ultraestructura , Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Thermotoga maritima/ultraestructuraRESUMEN
Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (ΔPSA) into FliG from Vibrio alginolyticus, whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mutant could not produce flagellar filaments in a fliG null strain but the FliG(ΔPSA) protein could localize at the cell pole as does the wild-type protein. Unexpectedly, when this mutant was expressed in a wild-type strain, cells formed flagella efficiently but the motor could not rotate. We propose that this different phenotype in Vibrio and Salmonella might be due to distinct interactions between FliG mutant and FliM in the C ring between the bacterial species.