Limits...
Molecular architecture of the bacterial flagellar motor in cells.

Zhao X, Norris SJ, Liu J - Biochemistry (2014)

Bottom Line: Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior.Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor-stator complex and secretion apparatus.This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor-stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston , Houston, Texas 77030, United States.

ABSTRACT
The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellum, providing unparalleled insights into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor-stator complex and secretion apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Because the resolution that can be achieved by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small-diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional insights into the in situ structure of the flagellar motor and other cellular components. This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor-stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components.

Show MeSH

Related in: MedlinePlus

Sequentialflagellar assembly process revealed in B. burgdorferi.50 In the pre-T3S assembly state, mostflagellar motor components except for the flagellar rod have beenassembled, which includes the MS ring, the C ring, the stators, theexport apparatus, and the unique periplasmic structure collar. Thesecretion channel in the MS ring is closed (first panel). The exportationof rod substrates opens the channel, and proximal rod substrates [FliE,FlgB, FlgC, and FlhO (FlgF homologue)] cooperatively assemble intoa stable proximal rod structure (second panel). The distal rod proteinFlgG adds onto the proximal rod and polymerizes until it reaches adetermined length. With the completion of rod assembly, the hook capis exported and the P ring is assembled around the rod (third panel).The hook assembly is promoted by a hook cap.83 With the completion of hook assembly, the hook–filament junctionand filament cap are exported (fourth panel). The filament assemblyis promoted by the filament cap (fifth panel).82
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4221660&req=5

fig5: Sequentialflagellar assembly process revealed in B. burgdorferi.50 In the pre-T3S assembly state, mostflagellar motor components except for the flagellar rod have beenassembled, which includes the MS ring, the C ring, the stators, theexport apparatus, and the unique periplasmic structure collar. Thesecretion channel in the MS ring is closed (first panel). The exportationof rod substrates opens the channel, and proximal rod substrates [FliE,FlgB, FlgC, and FlhO (FlgF homologue)] cooperatively assemble intoa stable proximal rod structure (second panel). The distal rod proteinFlgG adds onto the proximal rod and polymerizes until it reaches adetermined length. With the completion of rod assembly, the hook capis exported and the P ring is assembled around the rod (third panel).The hook assembly is promoted by a hook cap.83 With the completion of hook assembly, the hook–filament junctionand filament cap are exported (fourth panel). The filament assemblyis promoted by the filament cap (fifth panel).82

Mentions: Flagellar assembly is a finelyorchestrated biochemical processinvolving both highly regulated motility gene expression and orderedprotein assembly.30,80,81 The morphogenetic pathway for flagellar synthesis has been well-establishedin S. enterica.82−84 Recently, the combinationof cryo-ET and genetic analysis in B. burgdorferi has permitted determination of the location of specific flagellarproteins48,49,52 and the visualizationof the process of flagellar assembly in cells. As an example, keyintermediates in the flagellar assembly of B. burgdorferi can be genetically trapped by systematically targeting individualflagellar genes (encoding the rod, hook, and filament proteins)50 (Figure 5). Interestingly,the MS ring channel appeared to be closed in a ΔfliE mutant, and no rod-associated density was visualized; thus, assemblyof the rod in B. burgdorferi is FliE-dependent, consistentwith studies conducted with S. enterica.71 Cryo-ET analysis of each rod mutant (ΔfliE, ΔflgB, ΔflgC, ΔflhO, and ΔflgG)permitted assessment of the contribution of each rod protein to rodassembly.50 Similarly, analysis of hookmutant ΔflgE revealed a structure thought torepresent a hook cap attached to the end of distal rod. Examinationof a flaB filament deletion mutant also exhibiteda filament cap structure, which is likely related to the cap proteinFliD. In this study, high-throughput cryo-ET procedures permittedthe comparative analysis of seven flagellar mutants and more than20000 gigabytes of data and thereby provide a large set of 3D flagellarstructures, which may represent key intermediates during flagellarassembly (Figure 5).50


Molecular architecture of the bacterial flagellar motor in cells.

Zhao X, Norris SJ, Liu J - Biochemistry (2014)

Sequentialflagellar assembly process revealed in B. burgdorferi.50 In the pre-T3S assembly state, mostflagellar motor components except for the flagellar rod have beenassembled, which includes the MS ring, the C ring, the stators, theexport apparatus, and the unique periplasmic structure collar. Thesecretion channel in the MS ring is closed (first panel). The exportationof rod substrates opens the channel, and proximal rod substrates [FliE,FlgB, FlgC, and FlhO (FlgF homologue)] cooperatively assemble intoa stable proximal rod structure (second panel). The distal rod proteinFlgG adds onto the proximal rod and polymerizes until it reaches adetermined length. With the completion of rod assembly, the hook capis exported and the P ring is assembled around the rod (third panel).The hook assembly is promoted by a hook cap.83 With the completion of hook assembly, the hook–filament junctionand filament cap are exported (fourth panel). The filament assemblyis promoted by the filament cap (fifth panel).82
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221660&req=5

fig5: Sequentialflagellar assembly process revealed in B. burgdorferi.50 In the pre-T3S assembly state, mostflagellar motor components except for the flagellar rod have beenassembled, which includes the MS ring, the C ring, the stators, theexport apparatus, and the unique periplasmic structure collar. Thesecretion channel in the MS ring is closed (first panel). The exportationof rod substrates opens the channel, and proximal rod substrates [FliE,FlgB, FlgC, and FlhO (FlgF homologue)] cooperatively assemble intoa stable proximal rod structure (second panel). The distal rod proteinFlgG adds onto the proximal rod and polymerizes until it reaches adetermined length. With the completion of rod assembly, the hook capis exported and the P ring is assembled around the rod (third panel).The hook assembly is promoted by a hook cap.83 With the completion of hook assembly, the hook–filament junctionand filament cap are exported (fourth panel). The filament assemblyis promoted by the filament cap (fifth panel).82
Mentions: Flagellar assembly is a finelyorchestrated biochemical processinvolving both highly regulated motility gene expression and orderedprotein assembly.30,80,81 The morphogenetic pathway for flagellar synthesis has been well-establishedin S. enterica.82−84 Recently, the combinationof cryo-ET and genetic analysis in B. burgdorferi has permitted determination of the location of specific flagellarproteins48,49,52 and the visualizationof the process of flagellar assembly in cells. As an example, keyintermediates in the flagellar assembly of B. burgdorferi can be genetically trapped by systematically targeting individualflagellar genes (encoding the rod, hook, and filament proteins)50 (Figure 5). Interestingly,the MS ring channel appeared to be closed in a ΔfliE mutant, and no rod-associated density was visualized; thus, assemblyof the rod in B. burgdorferi is FliE-dependent, consistentwith studies conducted with S. enterica.71 Cryo-ET analysis of each rod mutant (ΔfliE, ΔflgB, ΔflgC, ΔflhO, and ΔflgG)permitted assessment of the contribution of each rod protein to rodassembly.50 Similarly, analysis of hookmutant ΔflgE revealed a structure thought torepresent a hook cap attached to the end of distal rod. Examinationof a flaB filament deletion mutant also exhibiteda filament cap structure, which is likely related to the cap proteinFliD. In this study, high-throughput cryo-ET procedures permittedthe comparative analysis of seven flagellar mutants and more than20000 gigabytes of data and thereby provide a large set of 3D flagellarstructures, which may represent key intermediates during flagellarassembly (Figure 5).50

Bottom Line: Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior.Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor-stator complex and secretion apparatus.This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor-stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston , Houston, Texas 77030, United States.

ABSTRACT
The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellum, providing unparalleled insights into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor-stator complex and secretion apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Because the resolution that can be achieved by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small-diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional insights into the in situ structure of the flagellar motor and other cellular components. This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor-stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components.

Show MeSH
Related in: MedlinePlus