Successive inactivation of the force-generating units of sodium-driven bacterial flagellar motors by a photoreactive amiloride analog.

AbstractThe dynamic assembly of the cell wall is key to the maintenance of cell shape during bacterial growth. Here, we present a method for the analysis of Escherichia coli cell wall growth at high spatial and temporal resolution, which is achieved by tracing the movement of fluorescently labeled cell wall-anchored flagellar motors. Using this method, we clearly identify the active and inert zones of cell wall growth during bacterial elongation. Within the active zone, the insertion of newly synthesized peptidoglycan occurs homogeneously in the axial direction without twisting of the cell body. Based on the measured parameters, we formulate a Bernoulli shift map model to predict the partitioning of cell wall-anchored proteins following cell division.

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Na+-driven flagellar motors of an alkalophilic Bacillus strain YN-1.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)44495-4 ◽

1983 ◽

Vol 258 (17) ◽

pp. 10577-10581

Author(s):

N Hirota ◽

Y Imae

Keyword(s):

Bacillus Strain ◽

Flagellar Motors ◽

Alkalophilic Bacillus

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2C1700 The dynamics of proton-driven flagellar motors of Salmonella at low speed conditions

Seibutsu Butsuri ◽

10.2142/biophys.42.s107_1 ◽

2002 ◽

Vol 42 (supplement2) ◽

pp. S107

Author(s):

T. Minamino ◽

N. Kami-ike ◽

K. Oosawa ◽

K. Namba

Keyword(s):

Low Speed ◽

Flagellar Motors

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The presence and absence of periplasmic rings in bacterial flagellar motors correlates with stator type

eLife ◽

10.7554/elife.43487 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 17

Author(s):

Mohammed Kaplan ◽

Debnath Ghosal ◽

Poorna Subramanian ◽

Catherine M Oikonomou ◽

Andreas Kjaer ◽

...

Keyword(s):

Legionella Pneumophila ◽

Pathogenic Bacteria ◽

Bioinformatics Analysis ◽

Cell Envelope ◽

Shewanella Oneidensis ◽

Flagellar Motor ◽

Flagellar Motors ◽

A Cell ◽

Animal Hosts

The bacterial flagellar motor, a cell-envelope-embedded macromolecular machine that functions as a cellular propeller, exhibits significant structural variability between species. Different torque-generating stator modules allow motors to operate in different pH, salt or viscosity levels. How such diversity evolved is unknown. Here, we use electron cryo-tomography to determine the in situ macromolecular structures of three Gammaproteobacteria motors: Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the motor’s stator system and its structural elaboration. Motors with a single H+-driven stator have only the core periplasmic P- and L-rings; those with dual H+-driven stators have an elaborated P-ring; and motors with Na+ or Na+/H+-driven stators have both their P- and L-rings embellished. Our results suggest an evolution of structural elaboration that may have enabled pathogenic bacteria to colonize higher-viscosity environments in animal hosts.

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Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

10.1101/367110 ◽

2018 ◽

Author(s):

Clémence Roggo ◽

Jan Roelof van der Meer

Keyword(s):

Escherichia Coli ◽

Protein Interactions ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Bacterial Chemotaxis ◽

Ph Sensitive ◽

Flagellar Motors ◽

Receptor Interactions ◽

Split Egfp ◽

Green Fluorescent

ABSTRACTChemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically activeEscherichia colicells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically activeE. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.IMPORTANCEBacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in theEscherichia colichemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.

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Biphasic chemotaxis ofEscherichia colito the microbiota metabolite indole

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916974117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 6114-6120 ◽

Cited By ~ 4

Author(s):

Jingyun Yang ◽

Ravi Chawla ◽

Kathy Y. Rhee ◽

Rachit Gupta ◽

Michael D. Manson ◽

...

Keyword(s):

Escherichia Coli ◽

Microbial Communities ◽

Bacterial Chemotaxis ◽

Dynamic Responses ◽

Time Dependent ◽

Flagellar Motor ◽

Wild Type ◽

Gi Tract ◽

Flagellar Motors

Bacterial chemotaxis to prominent microbiota metabolites such as indole is important in the formation of microbial communities in the gastrointestinal (GI) tract. However, the basis of chemotaxis to indole is poorly understood. Here, we exposedEscherichia colito a range of indole concentrations and measured the dynamic responses of individual flagellar motors to determine the chemotaxis response. Below 1 mM indole, a repellent-only response was observed. At 1 mM indole and higher, a time-dependent inversion from a repellent to an attractant response was observed. The repellent and attractant responses were mediated by the Tsr and Tar chemoreceptors, respectively. Also, the flagellar motor itself mediated a repellent response independent of the receptors. Chemotaxis assays revealed that receptor-mediated adaptation to indole caused a bipartite response—wild-type cells were attracted to regions of high indole concentration if they had previously adapted to indole but were otherwise repelled. We propose that indole spatially segregates cells based on their state of adaptation to repel invaders while recruiting beneficial resident bacteria to growing microbial communities within the GI tract.

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Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes

mBio ◽

10.1128/mbio.02286-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 4

Author(s):

Louie D. Henderson ◽

Teige R. S. Matthews-Palmer ◽

Connor J. Gulbronson ◽

Deborah A. Ribardo ◽

Morgan Beeby ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Content Type ◽

Cellular Processes ◽

Spatial Regulation ◽

Flagellar Motors ◽

Core Components ◽

Flagellar Assembly ◽

And Function ◽

Conserved Core ◽

Flagellar Biogenesis

ABSTRACT Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species. IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni. Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.

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