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BOM-12: 4.13 Flagella and Motility p. 96 4.15 Cell Motion as a Behavioral Response: Microbial Taxes p. 102 9.7 Regulation of Chemotaxis p. 235 16.16.1 Motility of Spirochetes p. 477 Text Review Questions: Chap. 4: #16, 18 Chap. 9: #5 4.13 Flagella and Motility Motion, or movement, may not be true MOTILITY: Convection Brownian Motion Adhesion to moving objects Buoyancy: Gas Vesicles Ameboid Motility does not ocurr in Bacteria or Archaea Bacterial Flagella Bacterial “flagella” are mis-named because they are rigid; not a flexible, whip-like structure as are eucaryotic flagella. Filament: Up to 15 um length 20 nm diameter 3 nm channel Polar Lophotrichous Perithrichous Sheathed (endoflaglla) vs Unsheathed

Vibrio sp. unsheathed peritrichous flagella and sheathed polar flagellum

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Flagellar Structure Flagella is assembled from 40-50 different proteins. Flagellar baseplate is structurally complex and similar in all flagellated bacteria except for absence of L ring in Gram-positive species. Adherents of "Intelligent Design" use the bacterial flagellum as an example of "irreducible complexity".The flagellar "filament" is composed of many copies of a single protein, "flagellin". Flagellin Protein Subunit of Salmonella is a protein of 494 amino acids

3D Structure of Flagellin

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Flagellar Movement The baseplate of the bacterial flagellum is the only 'wheel' in biology, it is a 'motor' that converts energy stored in the electrochemical proton gradient into mechanical work (rotation). Flagellar Synthesis Flagellar filaments are self-assembled at distal end from single protein monomers which diffuse through hollow core. Structure (wavelength, amplitude sense) and mechanical properties of filament are determined by flagellin sequence and chemical environment during self-assembly. Cell Speed Flagellar swimming is a luxury, there is a high energy cost in terms of drain on proton gradient. Approx. 1,000 H+ per revolution. Swimming is suppressed when cells are in poor nutritional status. Water is a viscous medium to a particle as small as a bacterial cell; when flagellar motor stops, cell stops immediately-no coasting. Rotation velocity = 100 rpm approx. Swimming velocity = 25-100 um/sec approx. Energy cost = 1,000 protons/revolution/flagellum

4.15 Cell Motion as a Behavioral Response: Chemotaxis and Phototaxis Chemotaxis Temporal Gradient vs Spatial Gradient More than 60 genes are required for flagellar swimming and chemotaxis in E. coli. Approximately 40 dozen of these are for structural components of the flagellum and baseplate. The rest are involved with chemotactic behavior. Tactic responses in E. coli are accomplished by a biased random walk composed of alternating episodes of "running" and "tumbling". The random walk is biased by decreasing the probability of twiddling when the direction of swimming is favorable.

I S O T R O P I C C H E M O A T T R A C T A N T C O N C E N T R A T I O N

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The transition between running and twiddling is caused by change in direction of flagellar rotation.

d

Morphology of flagella during running (a and b) and twiddling (c and d). a and c are darkfield light photomicrographs. b and d are diagrammatic representations. In b, motors are running CCW and flagellar filaments are CCW throughout their length; therefore filaments form a bundle. In d reversal of motors to CW rotation induces a transient heteromorphic state in which the filaments are CW proximally and CCW diatally; this disrupts bundle.

ccw cw

ccw

In an isotropic environment, rotation is already CCW biased; therefore run times are longer than twiddle times.

1 SECOND RUNNING CCW rotation; flagellar filaments in CCW state form bundle at

pole of cell 0.1 SECOND TUMBLING CW rotation initiates transition from CW state of filament

beginning at the base of the filament. In this transient heteromorphous state the filament bundle is dispersed

RUN TUMBLE Average Duration 1 to several seconds Fraction of second Motor Direction (from outside) CCW CW Helical Conformation of Filament LH LH -> RH Filament Interaction Bundle Independent

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Measuring Chemotaxis Phototaxis Other Taxes 9.7 Regulation of Chemotaxis

Step One: Response to Signal Methyl-accepting chemotaxis proteins (MCP's) in E. coli TSR serine/temperature/pH/hydrophobic amino acids TAR aspartate / maltose (+malE) / heavy metals TRG ribose (+rbsB) / galactose (+mglB) TAP dipeptides (+dpp)

All are transmembrane signal transducing proteins. All are constitutive. Periplasmic domain of MCP binds attractants/repellents directly, or complexed with PBP’s. Cytoplasmic domain regulates autophosphorylation of CheA in response to ligand (chemoattractant) binding, subject to modulation by methylation of cytoplasmic domain.

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CheA A “sensor kinase”. cheA autophosphorylation using ATP substrate is regulated by MCP. This in turn is regulated by chemoattractant binding and by methylation of MCP. Chemoattractant binding decreases cheA phosphorylation rate. (Note that Fig. 8.27 misleadingly suggests the opposite.)

Methylation increases cheA phosphorylation rate. cheA-P phosphorylates both CheY (fast) and CheB (slow). Che W “Mediates” interaction of MCP with CheA Step Two: Controlling Flagellar Rotation  Tumbles are generated by inducing reversal of rotation from CCW to CW. CheY When phosphorylated, cheY-P interacts with flagellar motor via fli proteins in baseplate to to induce tumble. Note typo on p. 226 (fla for fli). CheZ Dephosphorylates cheY at constant rate. Step Three: Adaptation CheR Methylates cytoplasmic domain of MCP’s at constant rate. cheB When phosphorylated, cheB-P demethylates MCP’s. Note that the text does not identify a component for de-phosphorylation of cheB-P.

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Summary of Signal Transduction and Adaptation A molecular model of chemotaxis must account for both response and adaptation. Chemoattractant binding signals a suppression of motor reversal by a complex sensory transduction mechanism involving products of genes cheA, cheW, cheY and cheZ and fli. Mutaions in any of these genes produce a phenotype in which cells are motile but are non-chemotactic to all substances. The product of cheY interacts directly with the flagellar baseplate to promote ccw to cw reversal. Binding of chemoattractants to chemoreceptors reduces this interaction and therefore extends the length of runs. Adaptation to higher concentrations of chemoattractant is accomplished by increasing the methylation of the chemoreceptor on the cytoplasmic side of the membrane by decreasing the activity of the methylesterase (cheB). Thus, the chemosensor-chemoattractant binding equilibrium on the periplasmic side of the membrane is a function of the immediate attractant concentration. The methylation state of the chemosensor on the inside of the membrane indicates what the binding equilibrium was a short time in the past. This is the molecular basis of the MEMORY that allows the cell to sense a temporal change in attractant concentration. In a nutshell: Attractant Concentration LOW HIGH HIGH Attractant Binding to MCP - + + Methylation of MCP - - + Tumble Generation +

RESPONSE

-

ADAPTATION

+

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16.16.1 Motility of Spirochetes Spirochete motility is variant of flagellar motility designed to facilitate migration through semisolid media such as tissue. The flagella (“endoflagella”) are assembled between the plasma membrane and the outer membrane (i.e. in the periplasmic space). I believe that the “Outer Sheath” referred to in the text is equivalent to the Outer Membrane of other Gram-Negative Bacteria. Spirochete motility allows migration through semisolid environments (such as human tissue, in the case of Treponema palladium).

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STUDY QUESTIONS: 1. Draw a thumbnail sketch of a flagellar baseplate from a Gram-negative baterium. Show the

cytoplasmic membrane, peptidoglycan, outer membrane, the L ring, the P ring, the MS ring and the C ring. Also show the location of fli and mot proteins. Finally, indicate the path of protons (H+).

2. What would be the phenotype of an E. coli strain in which mutation had inactivated the

methyl-accepting chemotactic protein "Tar"? i.e. What characteristics would you use to distinguish this mutant from wild-type, and from other types of motility/chemotaxis mutants?

3. How do bacterial flagella differ from eukaryotic flagella? 4. What makes an E. coli switch from running to twiddling? 5. How does the mechanism of Spirochete motility differ from flagellar swimming? 6. Match each of the listed proteins (1-8) with the appropriate function/s (A-K). 1. CheA _____ 2. CheB _____ 3. CheR _____ 4. CheW _____ 5. CheY _____ 6. CheZ _____ 7. Fli _____ 8. MCP _____

A. binds attractants/repellents B. bound to MCP and CheA; book does not give a function C. dephosphorylates CheY D. methylates MCP at constant rate E. phosphorylated form interacts with flagellar baseplate (Fli) to generate tumbles F. phosphorylated form demethylates MCP G. phosphorylates CheB H. phosphorylates CheY I. regulates the phosphorylation of CheA J. sensor kinase; autophosphorylates using ATP as substrate K. switches flagellar baseplate rotation