Distinct Roles of Highly Conserved Charged Residues at the MotA-FliG Interface in Bacterial Flagellar Motor Rotation

Morimoto, Yusuke V.; Nakamura, Shuichi; Hiraoka, Koichi D.; Namba, Keiichi; Minamino, Tetsuo

doi:10.1128/jb.01971-12

Cited by 81 publications

(107 citation statements)

References 47 publications

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“…Considering the torque produced by N stators as Nτ 1 = γ2πω N (where τ 1 is the torque produced by a single stator, γ the drag coefficient of the bead, and ω N the measured speed (in Hz) of the motor with N stators), all the strains tested are driven by N ~ 8-10 stators, in line with previous measurements at high load 15,44 . This result supports previous works focused on quantifying steady-state stator stoichiometry by fluorescent stators 15,[18][19][20][21]28,29,[45][46][47] .…”

Section: Resultssupporting

confidence: 81%

“…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 .…”

mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

Heo

Nord

Chamousset

et al. 2018

Biophysical Journal

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Fluorescent 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, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation-new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Fusion of a fluorescent protein to a protein of interest is an invaluable tool for the study of protein function in a broad, and still expanding range of in vivo and in vitro systems. However, it is widely recognized that the presence of fluorescent proteins can alter functional properties of the native protein [1][2][3][4][5] . Careful assessment of these side effects and strategies to minimize them have therefore been critical to the success of fluorescent protein fusions (FPFs) [6][7][8][9] . The continuing development of FPF-based approaches to explore novel phenomena also calls for new insight into the associated side effects and methods to alleviate them [10][11][12] . 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. Such perturbations limit the depth to which the internal dynamics and function of the BFM, and protein complexes in general, can be probed with FPFs. Linker peptides inserted at the fusion point have been instrumental in minimizing non-native behaviour of FPFs in many biological systems [8][9][10][11] , but their use in the study of protein-complex dynamics is limited in the BFM has thus far been limited.The BFM is located at the base of each flagellum in the membrane of many motile bacteria (Fig. 1A). The torque gene...

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Section: Resultssupporting

confidence: 81%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

Heo

Nord

Chamousset

et al. 2018

Biophysical Journal

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“…Desafortunadamente, no se conoce que proporción del genoma falta y cabe la posibilidad que el gen MotB se encuentre entre lo faltante. Aunque la ausencia de MotB no afecta el ensamble del flagelo, es importante precisar la existencia del gen MotB porque en su ausencia afecta la fuerza del protón motriz al inhibirse en el canal de iones resultando un flagelo sin movilidad, afectando esto a la motilidad y adhesión de la bacteria (Morimoto et al 2013).…”

Section: El Análisis De Los 5 Fragmentos Del Genoma De Candidatusunclassified

“…Esto se debe a que Arg90 y Glu98 de MotA, presentan interacciones electroestáticas con los aminoácidos de la proteína FliG en la cadena C-terminal. El Aspartato en la posición 289 (Asp289) y Arginina en la posición 281 (Arg281) del FliG, son aminoácidos que interactúan con Arg90 y Glu98 de MotA, respectivamente (Morimoto et al 2013). La presencia de estos aminoácidos en las secuencias deducidas para MotA y FliG de Candidatus Hepatobacter penaei es de importancia en el sistema flagelar para la motilidad de la bacteria, por ser uno de los factores de virulencia más usados en la invasión al hospedero.…”

Section: El Análisis De Los 5 Fragmentos Del Genoma De Candidatusunclassified

Bioinformatic analysis of the flagellar system of the rickettsia-like alphaproteobacteria Candidatus Hepatobacter penaei

Leyva

Martínez‐Porchas

Vargas‐Albores

et al. 2019

Rev. biol. mar. oceanogr.

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La presencia de sistemas flagelares es común en muchas bacterias de vida libre, sin embargo, estos han sido considerados ausentes en bacterias del orden Rickettsiales. Candidatus Hepatobacter penaei es una bacteria Rickettsial de origen marino descrita como un microorganismo con motilidad, propulsada por un sistema flagelar, el cual también facilita la infección. Siendo esta bacteria uno de los principales riesgos de infección en granjas camaronícolas. El objetivo de este trabajo fue determinar la presencia de genes que codifiquen proteínas del sistema flagelar de la bacteria Candidatus Hepatobacter penaei. Para el análisis se utilizó la secuencia genómica la base de datos del GenBank con número de acceso NZ_JQAJ00000000.1, una vez detectados los genes, se dedujeron las secuencias de aminoácidos. Las secuencias de aminoácidos del sistema flagelar se identificaron mediante BlastP, por medio de un alineamiento múltiple de dominios conservados. Posteriormente, se realizó un modelado de la proteína FlhA y se comparó con la proteína reportada para Oceanibaculum indicum para la conformación hojas  y  hélices. Los resultados mostraron la presencia de algunas secuencias de aminoácidos relacionadas con los genes del sistema flagelar de la bacteria como MotA, FliG, FliN, FliL FlhA, FlhB, FliQ, FliR y FliF. Finalmente, estos resultados sugieren que Candidatus Hepatobacter penaei es una bacteria tipo Rickettsia que posee un sistema flagelar, el cual posiblemente le confiere motilidad y forma parte del proceso de infección en hospederos como crustáceos marinos.

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Functional chimeras of flagellar stator proteins between E. coli MotB and Vibrio PomB at the periplasmic region in Vibrio or E. coli

et al. 2015

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The bacterial flagellar motor has a stator and a rotor. The stator is composed of two membrane proteins, MotA and MotB in Escherichia coli and PomA and PomB in Vibrio alginolyticus. The Vibrio motor has a unique structure, the T ring, which is composed of MotX and MotY. Based on the structural information of PomB and MotB, we constructed three chimeric proteins between PomB and MotB, named PotB91, PotB129, and PotB138, with various chimeric junctions. When those chimeric proteins were produced with PomA in a ΔmotAB strain of E. coli or in ΔpomAB and ΔpomAB ΔmotX strains of Vibrio, all chimeras were functional in E. coli or Vibrio, either with or without the T ring, although the motilities were very weak in E. coli. Furthermore, we could isolate some suppressors in E. coli and identified the mutation sites on PomA or the chimeric B subunit. The weak function of chimeric PotBs in E. coli is derived mainly from the defect in the rotational switching of the flagellar motor. In addition, comparing the motilities of chimera strains in ΔpomAB, PotB138 had the highest motility. The difference between the origin of the α1 and α2 helices, E. coli MotB or Vibro PomB, seems to be important for motility in E. coli and especially in Vibrio.

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Distinct Roles of Highly Conserved Charged Residues at the MotA-FliG Interface in Bacterial Flagellar Motor Rotation

Cited by 81 publications

References 47 publications

Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

Bioinformatic analysis of the flagellar system of the rickettsia-like alphaproteobacteria Candidatus Hepatobacter penaei

Functional chimeras of flagellar stator proteins between E. coli MotB and Vibrio PomB at the periplasmic region in Vibrio or E. coli

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