A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli

Marykwas, Donna L.; Berg, Howard C.

doi:10.1128/jb.178.5.1289-1294.1996

Cited by 49 publications

(61 citation statements)

References 17 publications

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“…Also, our knowledge on the topology of membrane-associated VirB proteins, such as VirB8 and VirB10, may allow the construction of fusions to analyze interactions with their periplasmic domains. This strategy may greatly accelerate detailed mapping as well as mutational analysis of interacting domains (31).…”

Section: Discussionmentioning

confidence: 99%

The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens

1997

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Section: Discussionmentioning

confidence: 99%

The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens

1997

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“…Yeast can recombine linearized plasmid DNA fragments with linear restriction fragments, provided that a region of homology exists between them and that there is a nutritional advantage (Kunes et al, 1985). Numerous screens utilizing gap repair have previously been reported for many different genes within yeast (Muhlrad et al, 1992;Ishioka et al, 1995;Marykwas and Berg, 1996;Kostrub et al, 1998;Goddard et al, 2005;Ladds et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non‐lethal ricin A chain variants

Allen

Byron

Lord

et al. 2005

Yeast

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Knowledge of the uptake, membrane translocation, refolding and ribosome interaction of the ribosome-inactivating toxin ricin is incomplete at the present time. Ricin A chain (RTA) is the catalytic subunit of holotoxin and is also of particular interest as a vaccine candidate. For many studies into the uptake and immunological applications of ricin, it is essential to have inactive variants. Here, following error-prone polymerase chain reaction of the RTA open reading frame, we have used a modified gap-repair protocol in Saccharomyces cerevisiae to show that it is possible to rapidly generate a panel of inactive RTA mutants. Since yeast cells have ribosomes that are highly sensitive to RTA, we utilized a genetic selection based on the viability of transformants. This enabled the recovery of a number of mutations, some not previously identified, which permitted production of full-length but non-toxic RTA proteins. Such disarmed toxins may have utility as tools to study the cytosolic entry and action of RTA, and as potential vaccine candidates.

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“…The default direction of the motor is CCW, and binding of CheY-phosphate (2) or a mutationally activated form of CheY (37) to FliM increases the probability of CW rotation. FliM also associates with FliG (31,32,33,48). Conformational changes in FliM upon binding CheY-phosphate are presumably transmitted to FliG, but the mechanism by which these proteins communicate is uncertain.…”

mentioning

confidence: 99%

Rusty, Jammed, and Well-Oiled Hinges: Mutations Affecting the Interdomain Region of FliG, a Rotor Element of the Escherichia coli Flagellar Motor

et al. 2004

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Escherichia coli swims by spinning its four to eight flagella (1, 38), which are distributed randomly around the surface of the cell (28). When the flagella turn counterclockwise (CCW), the left-handed helical flagellar filaments coalesce into a bundle that pushes the cell in a gently curved path known as a run. When one or more flagella rotate clockwise (CW), the bundle comes apart (29, 49) and the cells undergo sudden, random changes in direction that are called tumbles. Alternation of CCW and CW flagellar rotation produces a series of runs and tumbles that generates a three-dimensional random walk. Suppression of CW rotation by signals from chemoreceptors enables a cell to migrate up an attractant gradient or down a repellent gradient (41).Most of the ϳ40 flagellar proteins are involved in the assembly of the basal body, hook, and filament. Three proteins have been shown to be directly involved in torque generation: MotA, MotB, and FliG. Null mutations in motA and motB produce paralyzed flagella (3, 4). MotA and MotB are located in the cell membrane (36) surrounding the MS ring (2 2), and they form the transmembrane channel that delivers protons to the motor (3,4,5,42,51). The large C-terminal periplasmic domain of MotB has a putative peptidoglycan-binding domain that is believed to anchor the Mot proteins to the peptidoglycan cell wall (9,11,17,51).FliG is mounted on the cytoplasmic face of the MS ring (15), which is composed of the single polypeptide FliF (50). FliG also contacts the C ring, which contains FliM and FliN (16,21,34). FliG, FliM, and FliN form the switch-motor complex (52), which is required for torque generation, flagellar assembly, and switching the direction of flagellar rotation (19,39,53).FliG from E. coli (FliG Ec contains 330 residues and can be separated into two stable, overlapping domains (25). A fliG mutant with a stop codon at position 246 has paralyzed flagella, whereas a ⌬fliG strain is not flagellated. Certain charged residues in the carboxyl-terminal region of FliG are essential for motility (26) and interact electrostatically with residues of opposite charge (55) in the cytoplasmic loop between transmembrane helices 2 and 3 of MotA (54). The picture of FliG that has emerged is that the amino-terminal region of FliG is required for flagellar assembly, whereas the carboxyl-terminal domain of FliG is required for torque generation.The C-terminal 126 residues of E. coli FliG can be expressed as a stable, soluble cytoplasmic fragment (25). The crystal structure of the C-terminal motility domain of FliG from Thermotoga maritima (FliG Tm ) has been determined (27). A more recent publication describes the structure of a larger C-terminal region that includes the putative hinge between the domains (8).The FliM protein plays an essential role in switching between CCW and CW rotation (39,44,47). FliM interfaces with the chemotactic circuit via the response regulator CheY (7,33,48). The default direction of the motor is CCW, and binding of CheY-phosphate (2) or a mutationally activated fo...

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A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli

Cited by 49 publications

References 17 publications

The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens

The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens

Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non‐lethal ricin A chain variants

Rusty, Jammed, and Well-Oiled Hinges: Mutations Affecting the Interdomain Region of FliG, a Rotor Element of the Escherichia coli Flagellar Motor

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