Bacterial Chemotaxis Michael Eisenbach, The Weizmann Institute of Science, Israel Bacteria can move by a variety of means, the most com- mon one by rotating their flagella. This movement is often directed towards favourable chemicals (chemoat- tractants) or away from unfavourable chemicals (che- morepellents), a process termed chemotaxis. This modulation of swimming direction is the outcome of controlled changes in the direction of flagellar rotation. Therefore, mechanistically, the essence of bacterial chemotaxis is to control the direction of flagellar rota- tion. This control is done by a sophisticated signal trans- duction system, involving a small protein, CheY, which shuttles back and forth between the receptor complexes clustered at the pole of the cell and the flagellar motor complexes around the cell. These interactions are modu- lated by phosphorylation and acetylation. The excitatory signalling process involves amplification. The adaptation signalling involves methylation of the receptors. Even though bacterial chemotaxis is considered the best understood signalling system at the molecular level, many major questions are still waiting to be resolved. Introduction The phenomenon of bacterial chemotaxis was independ- ently discovered by Theodor Wilhelm Engelmann and Wilhelm Pfeffer in the 1880s. Thorough investigation of the phenomenon started in the 1960s with the quantitative, genetic and biochemical studies of Adler (1966). What is chemotaxis? Today, this term is used to denote cell move- ment towards or away from a chemical source, defined as positive or negative chemotaxis, respectively. The chemical is defined as a chemoattractant or chemorepellent, respectively. According to the common, broad definition of chemotaxis, any cell motion that is affected by a chemical gradient in a way that results in net propagation, up a chemoattractant gradient or down a chemorepellent gra- dient, is defined as chemotaxis. This definition includes three narrower definitions that were used in the past to distinguish between different behavioural mechanisms by which cells can approach chemoattractants and avoid chemorepellents: topotaxis (a change in the direction of movement resulting from active alignment of the cell’s axis according to a chemical gradient); a phobic response (a decreased linear velocity in response to a chemical stimulus followed by a change of direction); and klinokinesis (a change in the frequency of spontaneous directional chan- ges in response to a chemical stimulus). Bacterial populations may encounter a large spectrum of environments during their life cycles. Owing to their small size and relative simplicity, their ability to adjust the environment to their needs is very limited. Instead, they apparently adopted a strategy of moving from one envir- onment to another. Chemotaxis as well as other types of taxis (e.g. thermotaxis and phototaxis; see below) thus enable bacteria to approach (and remain in) beneficial environments and escape from hostile ones. (This phe- nomenon, taxis, is prevalent not only in bacteria but also in many other cell types and unicellular organisms that are capable of movement.) Chemotaxis also serves as a means of cell-to-cell communication and cell recruitment under stress conditions. It is therefore not surprising that a very large number of bacterial species are motile and chemo- tactic. As a matter of fact, most rod-shaped bacteria are motile, independent of their classification (e.g. Gram- positive or Gram-negative, aerobes or anaerobes, spore- formers or not). In contrast, most round bacteria, cocci, are nonmotile. See also: Bacterial Cells; Bacterial Ecology; Bacterial Taxis; Phototaxis: Microbial As summarised below, bacterial species vary from each other in their modes of motility, their strategies of response to external stimuli and the stimuli to which they are sensitive. Introductory article Article Contents . Introduction . Varieties of Bacterial Motility . Bacterial Flagella . The Flagellar Motor . Link Between Flagellar Rotation and the Bacterial Swimming Behaviour . Genes Controlling Chemotaxis . Signal Transduction Pathways of Chemotaxis Online posting date: 15 th December 2011 eLS subject area: Cell Biology How to cite: Eisenbach, Michael (December 2011) Bacterial Chemotaxis. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0001251.pub3 eLS & 2011, John Wiley & Sons, Ltd. www.els.net 1
Transcript
Bacterial ChemotaxisMichael Eisenbach, The Weizmann Institute of Science, Israel
Bacteria can move by a variety of means, the most com-
mon one by rotating their flagella. This movement is
often directed towards favourable chemicals (chemoat-
tractants) or away from unfavourable chemicals (che-
morepellents), a process termed chemotaxis. This
modulation of swimming direction is the outcome of
controlled changes in the direction of flagellar rotation.
Therefore, mechanistically, the essence of bacterial
chemotaxis is to control the direction of flagellar rota-
tion. This control is done by a sophisticated signal trans-
duction system, involving a small protein, CheY, which
shuttles back and forth between the receptor complexes
clustered at the pole of the cell and the flagellar motor
complexes around the cell. These interactions are modu-
lated by phosphorylation and acetylation. The excitatory
signalling process involves amplification. The adaptation
signalling involves methylation of the receptors. Even
though bacterial chemotaxis is considered the best
understoodsignalling system at the molecular level, many
major questions are still waiting to be resolved.
Introduction
The phenomenon of bacterial chemotaxis was independ-ently discovered by Theodor Wilhelm Engelmann andWilhelmPfeffer in the 1880s. Thorough investigation of thephenomenon started in the 1960s with the quantitative,genetic and biochemical studies of Adler (1966). What ischemotaxis? Today, this term is used to denote cell move-ment towards or away from a chemical source, defined aspositive or negative chemotaxis, respectively. The chemical
is defined as a chemoattractant or chemorepellent,respectively.According to the common, broaddefinition ofchemotaxis, any cell motion that is affected by a chemicalgradient in a way that results in net propagation, up achemoattractant gradient or down a chemorepellent gra-dient, is defined as chemotaxis. This definition includesthree narrower definitions that were used in the past todistinguish between different behavioural mechanisms bywhich cells can approach chemoattractants and avoidchemorepellents: topotaxis (a change in the direction ofmovement resulting from active alignment of the cell’s axisaccording to a chemical gradient); a phobic response (adecreased linear velocity in response to a chemical stimulusfollowed by a change of direction); and klinokinesis (achange in the frequency of spontaneous directional chan-ges in response to a chemical stimulus).Bacterial populationsmay encounter a large spectrumof
environments during their life cycles. Owing to their smallsize and relative simplicity, their ability to adjust theenvironment to their needs is very limited. Instead, theyapparently adopted a strategy of moving from one envir-onment to another. Chemotaxis as well as other types oftaxis (e.g. thermotaxis and phototaxis; see below) thusenable bacteria to approach (and remain in) beneficialenvironments and escape from hostile ones. (This phe-nomenon, taxis, is prevalent not only in bacteria but also inmany other cell types and unicellular organisms that arecapable of movement.) Chemotaxis also serves as a meansof cell-to-cell communication and cell recruitment understress conditions. It is therefore not surprising that a verylarge number of bacterial species are motile and chemo-tactic. As a matter of fact, most rod-shaped bacteria aremotile, independent of their classification (e.g. Gram-positive or Gram-negative, aerobes or anaerobes, spore-formers or not). In contrast,most roundbacteria, cocci, arenonmotile. See also: Bacterial Cells; Bacterial Ecology;Bacterial Taxis; Phototaxis: MicrobialAs summarised below, bacterial species vary from each
other in their modes ofmotility, their strategies of responseto external stimuli and the stimuli to which they aresensitive.
Introductory article
Article Contents
. Introduction
. Varieties of Bacterial Motility
. Bacterial Flagella
. The Flagellar Motor
. Link Between Flagellar Rotation and the Bacterial
Swimming Behaviour
. Genes Controlling Chemotaxis
. Signal Transduction Pathways of Chemotaxis
Online posting date: 15th December 2011
eLS subject area: Cell Biology
How to cite:Eisenbach, Michael (December 2011) Bacterial Chemotaxis. In: eLS.John Wiley & Sons, Ltd: Chichester.
The most common strategy for motility is movement dri-ven by flagellar rotation. By means of their rotation, theflagella – external organelles that serve as ‘propellers’ –exert thrust that drives the bacteria relatively quickly, up to30 body lengths per second (20–60 mms21). As shown inTable 1, there are various types of flagellar motility, whichdepend on the location and number of flagella as well ason the species. In some species (e.g. Pseudomonas spp.,Spirillum spp., Chromatium spp. and Halobacteria), thecells swim forward and backward, and reorientationappears to be passive byBrownianmotion. In other species(e.g. Escherichia coli, Salmonella enterica serovar Typhi-murium (earlier called Salmonella typhimurium; here it willbe called in short ‘Salmonella’), Sinorhizobium meliloti,Rhodobacter sphaeroides and Agrobacterium tumefaciens),the cells move in a rather straight line and, occasionally,actively reorient themselves. It should be noted that com-binations of the varieties mentioned in Table 1 are alsopossible. For example,Vibrio alginolyticus cells each have asheathed flagellum at one pole that pushes the cells forwardor pulls backward.When on the surface of a solid medium,the cells produce lateral flagella in addition to the polarones. The lateral flagella enable swarming. See also:
Archaeal Flagella; Bacterial Flagella; Bacterial Flagella:Flagellar Motor
Swarming
Flagella are not only swimming tools but they also serve forswarming (Table 1). Swarming is an organised surfacemotility of cells in a colony, which depends on massiveflagellation and cell-to-cell communication (Harshey,2003). This organised surface translocation has beendemonstrated in both Gram-negative (primarily) andGram-positive species. Even bacteria such as E. coli andSalmonella with well-characterised swimming motility,when on a hard surface, are able to differentiate into fila-mentous (up to 50mm long), multinucleated, hyper-flagellated cells that translocate together as a colony on thesurface. Similar to swarming bees, the differentiated bac-teria in the colonyare organised in such away that the outerlayer of the colony moves like a swirl and expands out-wardly, and the evacuated space inside the colony is filledwith newly grown bacteria. The result is fast colonyexpansion (up to � 3 mms21 or 1 cmh21). Quorum sensingappears to be essential for swarming. Of themotilitymodesdescribed in this section, swarming is the only one forwhichthere is no evidence for being involved in chemotaxis.See also: Quorum Sensing
Gliding
Other strategies of motility, which do not depend on fla-gella, have also been recognised. Of these, the most
abundant one is gliding motility. Gliding motility is themovement of an organism on a solid surface with no visibleexternal organelles for themovement and no shape change.Gliding bacteria can be divided into two classes accordingto their speed and possibly their motility mechanism: slowand fast gliders.
Slow gliders
The most investigated class is the myxobacteria (e.g.Myxococcus xanthus) – Gram-negative bacteria that live insoil. They glide very slowly (1–20 mmmin21). The speed ofgliding within this range depends on the distance betweenthemoving cell and its nearest cell: the greater the distance,the lower the speed. Myxobacteria have two independentmotility systems, ‘adventurous’ and ‘social’, which aregenetically and functionally distinct (Mauriello et al.,2010). Each motility system is sensitive (although in a dif-ferent way) to the local cell density. The adventurousmotility is the motility of cells located more than a cell’slength from any neighbouring cell. It is effective mainly onrelatively hard and dry surfaces (such as 1.5% agar). Themechanism of this motility seems to involve proton-motiveforce-driven rotation of AgmU, a periplasmic A-motilityprotein that decorates a looped continuous helix, whichrotates clockwise as cells glide forward, reversing its rota-tion when cells reverse. It was proposed that these motorsrun along the looped helical track, driving rotation of thetrack; deformation of the cell surface by AgmU-associatedproteins creates pressure waves in the slime, pushing cellsforward (Nan et al., 2011). Social motility is movement ingroups involving continuous reorientation and reassocia-tion of the cells in the group. It is mostly effective on softerand wetter surfaces (such as .3% agar). Social motilityinvolves type IV pili, extruding from the cell pole. Theyadhere to a surface and then retract, pulling the cell in thedirection of the adhering pili. Social motility also involvesproduction of extracellular slime fibrils, thought tofunction as tactile antennae that transmit a signal back tothe cell indicating the proximity of another cell. See also:Bacterial Pili and Fimbriae
Fast gliders
The other class of gliding bacteria involves faster gliders(1–10 mms21) such as cyanobacteria and Cytophaga(McBride, 2001). (Some of the fast gliders, e.g. Deleyamarina, have, depending on the conditions, both flagellarand glidingmotility.) Although gliding organelles have notbeen found, latex beads, artificially attached to Cytophagacells, were seen to move back and forth at the speed ofcell gliding, or, instead, rotate. A carbohydrate-secretingorganelle was identified in cyanobacteria, Cytophaga andFlexibacter, suggesting that steady secretion of slimethrough this organelle may generate the thrust required forglidingmotility. The finding of an ordered array of parallelfibrils between the peptidoglycan layer and the outermembrane of cyanobacteria may provide another mech-anism for gliding motility. See also: Cyanobacteria
Table 1 Varieties of flagellar motility in bacteria
Flagellation Appearance Species for example Description of motility
A single flagellum at
(or near) one of the cell
poles
Pseudomonas spp. The flagellum – depending on its direction of
rotation – pushes or pulls the cell. Consequently, the
cell goes back and forth
A single flagellum
roughly in the middle
between the poles
Rhodobacter sphaeroides The flagellum either rotates clockwise or pauses.
Consequently the cell swims in a rather straight line
and occasionally stops for reorientation.
The bundle of flagella
at one of the poles
Chromatium okenii, some
cells of Halobacterium
salinarium
The bundle – depending on its direction of rotation
– pushes or pulls the cell. Consequently, the cell goes
back and forth
The bundle of flagella
at each of the two poles
Some cells of H. salinarium The bundles – depending on their direction of
rotation – push or pull the cell. Consequently, the
cell goes back and forth or stops
The bundle of flagella
at each of the two poles
in Spirillum spp.
Spirillum volutans Forward and backward swimming is carried out in
the same manner, only that the bundles flip over
when the cell reverses. The helical cell body rotates
in reaction to the rotation of the flagella and this
rotation produces the thrust for motility
5–10 Flagella
randomly distributed
around the cell
Escherichia coli, Salmonella
typhimurium, Bacillus
subtilis
Most of the time the flagella rotate
counterclockwise and the cell swims in a rather
straight line (a run). Intermittently, the flagella
rotate clockwise or pause, as a result of which the
cell undergoes a vigorous angularmotion (a tumble)
5–10 Flagella
randomly distributed
around the cell
Sinorhizobium meliloti Most of the time the flagella rotate
counterclockwise and the cell swims in a rather
straight line. Occasional changes in the speed of
flagellar rotation cause the cell to turn (without
tumbles)
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Table 1 Continued
Flagellation Appearance Species for example Description of motility
Apolar tuft of 2 flagella
+2–4 lateral flagella
Agrobacterium tumefactions Flagella rotate clockwise or pause; consequently the
cell swims in a rather straight line or turns
Excessive flagellation
around the cell
E. Coli, S. typhimurium,
Serratia marcescens,
Proteus mirabilis
Swimming – surface motility in a colony
One flagellum at one
end, one or more
flagella subterminally
at each end. All the
flagella are contained
within the periplasmic
space
Spirochaetes The periplasmic flagella cause the cell to bend and
gyrate. The cells exhibited smooth swimming,
reversals, flexing and pausing. When the flagellar
bundles at both cell poles rotate in opposite
directions (one pulls and one pushes), the cell swims
in a rather straight line, the cell reverses.When both
bundles rotate in the same direction, the cell flexes
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Swimming without flagella
Spiroplasma are small helical bacteria lacking cell walls andflagella. They swim by changing their body helicity. Theyhave an internal cytoskeleton in the form of a flat, mono-layered ribbon, which is constructed from seven contractilefibrils connected to the inner side of the cell membrane.These fibrils change their length differentially in a coord-inated manner, thereby changing the helicity of the bodyand creating kink pairs that propagate down the length ofthe cell body, thus generating directional movement(Shaevitz et al., 2005).
Twitching
Twitching is another kind of surface motility; it involvesintermittent and jerkymovement of single bacterial cells orgroup of cells in a colony, not necessarily along the longaxis of the cell. Owing to the lengthy intermissions withoutmovement, the progressive velocity, averaged over time, isvery low (2–10 mmmin21). As in the case of slow gliders,twitching motility is powered by retraction of polar pili(Merz et al., 2000). As a matter of fact, it has been
proposed, on the basis of morphological and genetic data,that twitching motility and social gliding motility of slowgliders are essentially the same process.
Propulsion by actin filaments
A unique mode of motility, first described in 1989, is themovementofbacteria suchasListeria,ShigellaandRicettsiain host eukaryotic cells. The bacteria use a continuous actinfilament assembly for propulsion in the cytoplasm of theinfected host cell. The actin assembly at the bacterial surfaceis asymmetrical, with the filaments growing like a comet tailat one end of the bacterial cell and pushing the cell in theother direction (Lambrechts et al., 2008).See also:Actin andActin Filaments; Muscle Contraction Mechanism: Use ofSynchrotron X-ray Diffraction; Polymerization Dynamicsof Cytoskeletal Filaments
Bacterial Flagella
As mentioned above, flagella are organelles that enablebacteria to swim in an aqueous solution or swarmon ahardsurface (Figure 1). In addition to their role in motility, fla-gella are involved in bacterial colonisation; in many cases,they contribute to the bacterial virulence, and they areoften targets for antibody response. The term ‘flagellum’(pl. flagella) comes from Latin, meaning a little whip.Although this term is adequate for the eukaryotic flagel-lum, which acts like a whip, it is not adequate (and essen-tially misleading) for the bacterial flagellum, which acts byrotation. Bacterial flagella and eukaryotic flagella aretotally different organelles. Table 2 indicates the maindifferences between them, with E. coli and human sperm-atozoa representing bacterial and eukaryotic flagella,respectively. See also:Bacterial Flagella; Cilia and Flagella
Structure of flagella
Bacterial flagella consist of three major parts (Figure 2): abasal body, a hook and a filament (Kojima and Blair,2004). Although the structure of bacterial flagellamay varyin some respects between species and families (e.g. Gram-positive and Gram-negative bacteria), the main structuralaspects are common to all.
Figure 1 Flagella of E. coli observed in transmission electron microscope.
Bar, 1 mm.
Table 2 Comparison between eukaryotic and bacterial flagella
Property Sperm flagellum (human) Bacterial flagellum (E. coli)
Diameter (mm) 0.3–1 0.023
Length (mm) 60 10–15
Structure Complex structure of tubules surrounded by
an extension of the cytoplasmic membrane;
the flagellum consists of � 250 proteins
Naked filament consisting of subunits of a single protein
Function Active beating Passive rotation, driven by a rotary motor embedded in
the cytoplasmic membrane
Energy source ATP Proton-motive force across the cytoplasmic membrane
The basal body of E. coli and Salmonella is composed of acentral rod surrounded by four rings: an M ring (M formembrane, as this ring is located in the cytoplasmicmembrane), an S ring (S for supramembrane, as this ring islocated above the cytoplasmic membrane), a P (for pepti-doglycan) ring and an L (for lipopolysaccharide) ring. TheM and S rings constitute essentially one double-ring,composed of a single protein, FliF. The MS ring is astructural part of the flagellar motor (Figure 2; see below),on which the functional components of the motor aremounted. The P ring is built from the FlgI protein, and it islinked by a cylindrical wall to the L ring, built from theFlgH protein. (The L and P rings are missing in Gram-positive bacteria.) Another ring, the C ring (C for cyto-plasm), which contains the proteins FliM and FliN, isattached via the FliG protein to theMS ring from beneath,on the cytoplasmic side.
Hook
The hook – built of a single protein, FlgE – is a short (only130 FlgE subunits, � 55 nm long), curved structure that
connects the basal body to the flagellar filament (Figure2). Itis believed to serve as a flexible joint that converts thetorque, generated by the flagellar motor in the plane of thecell surface, into a force having both vertical andhorizontalcomponents. See also: Bacterial Flagella: Flagellar Motor
Filament
The filament – built from � 20 000 subunits of a singleprotein, flagellin (FliC) – is a highly rigid, helical structure,10–15mm long, 23nm in diameter. (There are, however,bacteria whose filament is built from several flagellins andnot from a single one. For example, the flagellar filament ofCaulobacter crescentus is composed of six different flagellinswith distinctmolecular sizes.) It is connected to the hook viaa short junction composed of two hook-associated proteins:HAP1 (FlgK) and HAP3 (FlgL). At the other end of thefilament, there is a cap-like structure, composed of theprotein HAP2 (FliD). The filament can be in a number ofhelical forms (nine such forms have been observed experi-mentally), depending on the conditions. The defaultphysiological form is a left-handed helix. It can be convertedtooneof theother formsbyamechanical force (for example,when the direction of flagellar rotation is changed; seebelow) or by changing the pH or the ionic strength of thesuspendingmedium. The filament is passive and its rotationis totally dependent on the flagellar motor (Figure 2).
Assembly of flagella
The assembly of flagella is synchronised with the cell cycleand depends on cell division and growth phase. Approxi-mately 2% of the cell’s biosynthetic energy expenditure isfor flagellar synthesis (Macnab, 2003). The first observablestructure is the MS ring (Figure 3). Next the C ring isassembled, followed by another structure at the centre ofthe C ring – the ‘export apparatus’ – a type III secretionsystem that exports proteins necessary for flagellarassembly. Then, at the other (outward) side, the rod of thebasal body is added, subunits of the P ring are exported tothe periplasm and form the P ring, and subunits of the Lring are exported to the outer membrane and form the Lring. Subsequently, the hook is assembledwith the help of ascaffolding protein, FlgD. It is thought that the length ofthe hook (made of the FlgE protein) is determined by theprotein FliK, which acts as a molecular ruler that takesmeasurements of rod-hook length while being intermit-tently secreted during the assembly process of the hook–basal body complex (Chevance and Hughes, 2008). Fol-lowing the completion of the junction (composed of HAP1
and HAP3), the filament is assembled at its distal end in aprocess that requires the cap HAP2. The proteins that arethe building stones of the flagellum are synthesised withinthe cell. They are then pushed outward by the exportapparatus through the central channel of the flagellum,drivenby theproton-motive force.Adenosine triphosphate(ATP) hydrolysis might assist substrate delivery, thusincreasing the efficiency of assembly. The filamentis assembled by stepwise rotation of the filament cap.
Basalbody
Switch
Junction
Motor
BushingL ring
P ring
S ring (FliF)
M ring (FliF)
C ring
Out
In
OM
PL
CM
Periplasm
Cytoplasm
Rod
Central channel
MotAFliGFliNFliM
Hook
Filament
Cap
MotB
Figure 2 E. coli or Salmonella flagellum. The actual diameters of the rod, L,
P, M, S and C rings are �15, 33, 26, 29, 27 and 47 nm, respectively. CM,
cytoplasmic membrane; OM, outer membrane; and PL, peptidoglycan layer.
The sequence and timing of export of the proteins involvedin flagellar assembly are tightly controlled. Termination offilament assembly is believed to be partly regulated byFlgM – an antisigma factor secreted from the cell throughthe central channel of the flagellum. FlgM binds to thesigma factor FliA and prevents its association with ribo-nucleic acid (RNA) polymerase core enzyme. A highintracellular concentration of FlgM represses the relevantoperons and prevents or reduces the expression of theirproducts, including flagellin.
The Flagellar Motor
Structure
Like anyother electricmotor, the flagellarmotor contains arotor and a stator. The rotor is built from the MSring (FliF) and the C ring, consisting of approximately
25 molecules of FliG, 34 molecules of FliM and over100 molecules of FliN. The C ring is the motor’s gearshift,termed a switch. Its role is to shift the direction of rotationof the motor according to signals received from thereceptors on the cell surface (see below). The stator is builtfrom the proteins MotA and MotB (Figure 2). The driveshaft of themotor, termed the rod, is built from theproteinsFlgB, FlgC, FlgF and FlgG. The rod is surrounded andheld by the L and P rings, probably serving as a bushing.The helical propeller is the filament and the universaljoint that connects it to the rod of the motor is the hook(Berg, 2003; Kojima and Blair, 2004).
Function
H+-driven motors
The flagellar motor can rotate extremely fast, up to 350revolutions per second (Chen and Berg, 2000)! Thedriving force of the flagellar motor is the proton-motive
flhCflhDFliF
(i)
FliG
(ii)
FliMFliN
(iii)
FlhAFliHFlil
(iv)
FlgBFlgCFlgFFlgGFlgJFliE
(v)
Flgl
(vi)CM
RodExport
apparatus
Switchcomplex
MS ring
FlgKFlgLFliKFlhBRflH
(ix) CM
Nascent hookwith cap
PL
OM
FlgDFlgE
(viii)
FlgH
(vii)
L ringP ring
FliC/FljBFlgMFliA
(xi) CM
‘Full-length’filament
PL
OM
FliDFliC/FljB
(x)
Nascentfilamentwith cap
Hook andhook-filamentjunction zones
Figure 3 Schematic description of the stepwise assembly of E. coli and Salmonella flagella. Abbreviations: OM, outer membrane; PL, peptidoglycan layer;
and CM, cytoplasmic membrane. (Modified with permission from Aizawa, 1996.)
Bacterial Chemotaxis
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force across the cytoplasmic membrane and not ATP.The proton-motive force is produced by respiration or,under anaerobic conditions, by the proton ATPase at theexpense of ATP hydrolysis (Kojima and Blair, 2004). Theinwardly directed proton electrochemical potential drivesan influx of � 1000 protons per revolution through aproton-conducting channel composed of MotA andMotB. MotB anchors MotA to the peptidoglycan layer.The flux of protons through the proton channel isthought to stepwise rotate the motor (Berg, 2003). Thisrotation depends on electrostatic interactions betweensites in the stator protein MotA and the rotor proteinFliG. The molecular mechanism by which the switchreverses the direction of rotation of the motor is notknown. According to a recent model based on the 3Dstructure of full-length FliG, switching may be the out-come of conformational changes in FliG that reverse theelectrostatic charges involved in torque generation (Leeet al., 2010). See also: Ion Motive ATPases: P-typeATPases
Na+-driven motors
A few species possess flagellar motors that are driven by aflux of Na+ ions. These include alkalophilic Bacilii andVibrio species. Interestingly, under certain conditions,Vibrio can possess two types of flagella, each driven by adifferent ion: lateral flagella driven by a flux of protons andpolar flagella driven by a flux of Na+ ions. Na+-drivenmotors can rotate even faster than proton-driven motors,up to 1700 revolutions per second! The Na+ ions flowthroughNa+-conducting channels, composed of theMotXand MotY proteins or the PomA and PomB proteins(Yorimitsu and Homma, 2001).
Functional states of the motor
The flagella of bacteria such as E. coli and Salmonella canrotate counterclockwise and clockwise (the direction ofrotation defined for a flagellum viewed from its distal endtowards the bacterial cell), and they can also pause(Eisenbach, 1990). A pause seems to result from a futileswitching attempt from counterclockwise to clockwise.Under nonstimulated conditions, the flagella rotate mostlycounterclockwise with brief intermissions of clockwiserotation and pauses. Different flagella on a given cell seemto be independent of each other with respect to their dir-ection of rotation under nonstimulated conditions: theyreverse and pause asynchronously (Turner et al., 2000).
Link Between Flagellar Rotation andthe Bacterial Swimming Behaviour
Modes of swimming behaviour
Bacteria such as E. coli and Salmonella have two mainswimming patterns: smooth swimming in a rather straightline (a run) and a brief but abrupt turning motion (a tum-ble) (Berg andBrown, 1972;Macnab andKoshland, 1972).In the absence of stimuli, the tumbles usually occur onceevery 1–5 s (depending on the bacterial strain). Con-sequently, the bacterial cells execute a random walk,composed of runs and tumbles with essentially no netvectorial movement (Figure 4a).
A run
The run in E. coli and Salmonella is the consequence ofcounterclockwise rotation of the flagella. Because of the
(b)(a) Random walk Biased random walk
Run
TumbleAttractant
Figure 4 Swimming behaviour of E. coli cells: (a) nonstimulated conditions and (b) stimulated conditions.
flagellar left-handed helicity, counterclockwise rotationexerts a pushing force on the cell. Since the flagella aroundthe cell have different lengths and their distribution is notsymmetric, the net force is not zero. Consequently, the cellmoves in the direction of the net force and, due to theviscous drag of the medium, the flagella are swept to therear of the cell, amplify the net force in the direction ofmovement and form a left-handed bundle (aligned with thelong axis of the cell) that pushes the cell forward (Macnab,1977).
A tumble
The tumble in E. coli and Salmonella is the consequenceof clockwise rotation of the flagella. Unlike counter-clockwise rotation, which stabilises the left-handed formof the flagella, clockwise rotation destabilises this form.Consequently, the flagella undergo a transition from aleft-handed helix to a right-handed one, and the transi-tion propagates from the flagellum–cell body junctiontowards the distal end of the filament (Macnab andOrnston, 1977). However, because the periods of clock-wise rotation are relatively short and because of theoccasional pauses, the transformation from left- to right-handed helix is usually not complete. As a consequence,some flagella have segments of opposite handednesswithin the very same filament, resulting in a large anglebetween the segments (Figure 4a). This angle, which pro-vides angular motion to the bacterial cell, prevents bundleformation and forces each flagellum to act separately (each
exerts force in a different direction), thus causing tumbling(Eisenbach et al., 1990b; Macnab and Ornston, 1977). Thelack of synchrony between the flagella raises the question –how does a bacterial cell behave when some flagella rotatecounterclockwise and others rotate clockwise. It appearsthat tumbling occurs only when � 25% or more of theflagella on a given cell reverse to clockwise rotation.This means that, depending on the number of flagella percell, rotation of 1–2 flagella in the clockwise direction maybe sufficient to cause tumbling. If a single flagellum rotatesclockwise and at least three other flagella on the samecell rotate counterclockwise, the clockwise-rotating fla-gellum separates from the counterclockwise-rotatingbundle and moderately (without a tumble) changes theswimming direction of the cell. Generally speaking, thelarger the fraction of clockwise-rotating flagella, the largerandmore abrupt the change in swimmingdirection (Turneret al., 2000).
Varieties of stimuli
Bacteria respond to a variety of stimuli, including chemicalstimuli. The origin of the chemical stimuli may be theenvironment itself or the neighbouring cells. Table 3 lists anumber of known stimuli and the corresponding behav-ioural responses of the bacteria. (Note that in species thatgrow, rather than move, in a certain direction in responseto a stimulus, the suffix is ‘tropism’ instead of ‘taxis’:chemotropism, thermotropism, etc.) Not every speciesresponds to all stimuli. Thus, while chemotaxis and ther-motaxis are probably common in all bacterial speciescapable of movement, magnetotaxis is restricted to speciesthat contain magnetosomes (intracellular structures con-sisting of a crystal of a magnetic mineral, usually the ironoxidemagnetite (Fe3O4), or the iron sulfide greigite (Fe3S4)surrounded by a membrane), and rheotaxis has thus farbeen found only in mycoplasma gliding upstream in amoving fluid. Some behavioural responses (e.g. chemo-taxis, thermotaxis, phototaxis and osmotaxis) apparentlyshare, at least partially, a common molecular mechanism.See also:Magnetotaxis: MicrobialThe chemical stimuli for bacteria are diverse, and depend
on the habitat in which the bacteria live. Sometimes, acertain stimulant may act as a chemoattractant for one
Table 3 Known stimuli and behavioural responses in
bacteria
Stimulus Behavioural response
Chemical Chemotaxis
Elastic force Elasticotaxis
Electrical field Galvanotaxis
Gravity Geotaxis or gravitaxis
Light Phototaxis
Magnetic field Magnetotaxis
Moving fluid Rheotaxis
Osmolarity Osmotaxis
Temperature Thermotaxis
Table 4 Stimuli with different functions in different species
Reagent Chemoattractant for Chemorepellent for
Phenol Esherichia coli Salmonella
Leucine Bacillus subtilis E. coli, Salmonella
Valine Bacillus subtilis E. coli, Salmonella
Tryptophan Bacillus subtilis, Chromatium vinosum E. coli, Salmonella, Rhodobacter
sphaeroides
Acetate Chromatium vinosum E. coli, Salmonella, Rhodobacter
bacterial species and as a chemorepellent for another. Afew examples are listed in Table 4.
This entry mainly concentrates on chemotaxis of E. coliand Salmonella, the most studied bacterial species. Com-mon chemical stimuli for E. coli are listed in Table 5.
Varieties of response
A variety of responses to stimuli are observed among dif-ferent bacterial species, even when the compared bacteriabelong to the same type of motility, for example flagellarmotility. Depending on the varieties of flagellar motility(Table 1), some bacterial species react to changes in theconcentration of chemical stimuli by changing the dir-ection of flagellar rotation, and others by changing thespeed of swimming (chemokinesis) or by stopping therotation. Generally speaking, when a bacterial cell senses apositive stimulus (an increasing chemoattractant gradientor a decreasing chemorepellent gradient), it continues toswim in the same direction. When it senses a negativestimulus (a decreasing chemoattractant gradient or anincreasing chemorepellent gradient), it ceases to move inthe original direction and reorients itself. A few examplesare given in Table 6.
Swimming behaviour under stimulatedconditions
Positive stimulation of E. coli decreases the probability ofclockwise rotation, whereas negative stimulation increasesit. Consequently, positive stimulation suppresses the fre-quency of tumbles, whereas negative stimulation increasesit, and the bacterial cells execute a random walk biasedtowards the chemoattractant (Figure 4b) or away from thechemorepellent (Macnab, 1996). (Runs in the ‘right’direction are prolonged, and runs in the ‘wrong’ direc-tion are very short.) The end result is migration towards
Table 5 Common stimuli for E. coli
Class of stimuli Examples
Chemoattractants
Sugars D-Glucose, D-galactose,
D-ribose, D-mannose, maltose
Amino acids L-Serine, L-aspartate,
L-alanine
Dipeptides L-Proline-L-leucine, glycine-
L-proline
Energy-linked chemicals Oxygen at � 0.7 mmolL21
Weak organic bases Trimethylamine
Chemorepellents
Alcohols Ethanol, isopropanol
Polyalcohols Glycerol, ethylene glycol
Hydrophobic amino
acids
L-Leucine, L-valine
Inorganic ions Co2+, Ni2+
Energy-linked chemicals Oxygen at � 1mmol L21
Weak organic acids Acetate, benzoate
pH Acid, alkali
Others S22, mercaptans (e.g.
2-propanethiol), indole
Table 6 Examples of responses to chemotactic stimuli in bacteria with flagellar motility
Species Response to positive stimuli Response to negative stimuli
E. coli,
Salmonella
Increased probability of counterclockwise flagellar
rotation. Consequently, runs are prolonged
Increased probability of clockwise rotation and
pausing. Consequently, the cell tumbles and
reorients more frequently
Bacillus subtilis Increased probability of clockwise flagellar
rotation. Consequently, runs are prolonged
Increased probability of counterclockwise rotation.
Consequently, the cell tumbles and reorients more
frequently
Sinorhizobium
meliloti
Increased speed of flagellar rotation. Consequently,
runs are prolonged
Decreased speed of flagellar rotation.
Consequently, the bundle of rotating flagella
separates to individual filaments rotating at
different speeds and the cell turns
Rhodobacter
sphaeroides
Increased speed and decreased stopping probability
of flagellar rotation. Consequently, runs are
prolonged
Increased stopping probability. Consequently, the
cell reorients itself
Azospirillum
brasilense
Increased speed and decreased reversal probability
of flagellar rotation. Consequently, runs are
prolonged
Presumably increased reversal probability of
flagellar rotation. Consequently, the cell reorients
itself
Spirochaetes Flagella rotate without pausing, resulting in
coordinated rotation of the two polar bundles.
Consequently, the cell swims in a straight line
Flagella pause frequently and extensively,
disrupting the coordinated rotation of the two polar
bundles. Consequently, the cell flexes and pauses
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higher chemoattractant concentrations and avoidance ofchemorepellents. Thus, the question of how the chemotaxisprocess is carried out in bacteria can be reduced to theregulation of the direction of flagellar rotation.
Genes Controlling Chemotaxis
The genes involved in controlling chemotaxis are listed inTable7. The functionof eachof them is indicated in the tableand described in more detail below.
Two additional genes ofE. coli and Salmonella, flhC andflhD, are indirectly involved in controlling chemotaxis inthe sense that they regulate the synthesis of the chemotaxismachinery. These genes form a master operon whose geneproducts control the expression of the genes involved inflagellar synthesis, motility and chemotaxis (Macnab,1996). This master operon is itself positively regulated bythe intracellular levels of cyclic adenosine monophosphate(cAMP) and its receptor. In this manner, the metabolicstate of the cell is linked to the expression of the motilityand chemotaxis components. Thus, when the level ofcAMP goes up (e.g. when glucose availability goes down),the flhDC operon is rapidly transcribed, the motility andchemotaxis machineries are synthesised and the bacteriacan navigate themselves to better locations.
Signal Transduction Pathways ofChemotaxis
The locations of the chemotaxis receptors and the flagellaare different. For example, in E. coli the receptors areclustered at the bacterial poles, whereas the flagella arerandomly distributed around the cell. This prevents directinteraction between the receptors and the flagella, and thecommunication between them is carried out by a sophis-ticated signal transduction system (Figure 5), which belongsto the large family of two-component regulatory systems.The end result of this signal transduction is a change in thedirection of flagellar rotation. The flagellar motor has apreferred direction of rotation, a default direction, coun-terclockwise in the case of E. coli and Salmonella. Thismeans that the motor always rotates counterclockwise (inthe temperature range 20–378C), unless it receives a signalto do otherwise (Eisenbach, 1990). This alsomeans that thefunction of chemoattractants and chemorepellents is toinhibit and activate the clockwise signal, respectively. Thechemotactic excitatory signal is transduced very fast and iscompletedwithin 70ms or less (Khan et al., 1993).See also:Signal Transduction: Overview
How is a gradient of a stimulant sensed?
Bacteria like E. coli and Salmonella sense temporalgradients of stimuli (gradients over time), as opposed tospatial gradients (gradients over space) (Macnab and
Koshland, 1972). This means that bacteria compare,between sequential time points, the occupancy of theirchemotaxis receptors, that is they possess a kind of short-term memory. This arrangement is optimal for bacteria ofthis size and shape, taking into consideration that a changein receptor occupancy as small as 0.4% elicits a detectablechemotactic response. It is not impossible, however, thatbacterial species with larger dimensions or different shapessense spatial gradients.
The conventional signal transductionpathway in E. coli
The components
The components of the conventional signal transductionpathway are chemotaxis receptors, proteins involved insignal transduction and adaptation, and switch proteinsthat determine the direction of flagellar rotation. There aretwo kinds of receptors: chemotaxis-specific receptors anddual-function receptors involved in both chemotaxis andtransport of the ligand.
Chemotaxis-specific receptors
The chemotaxis-specific receptors, homodimers termedmethyl-accepting chemotaxis proteins (MCPs) andarranged in trimers of dimers, are expressed by the aer, tap,tar, tsr and trg genes (Hazelbauer et al., 2008). They areclustered at the bacterial poles (one or both of them).Recent results suggest that chemorepellents stabilise theintrinsic chemoreceptor lattice, and chemoattractantsdestabilise it (Borrok et al., 2008). The MCPs are closelyrelated to each other both in terms of amino acid sequenceand structure. They are, however, different with respect totheir abundance, the sequence of their periplasmic part andthe presence of the binding site for CheR in their cyto-plasmic part. (CheR is a specificmethyltransferase having arole in adaptation; see below. The CheR-binding site is aspecific pentapeptide at the MCP’s carboxy (C)-terminus.)Thus, the most abundant MCPs, Tsr and Tar, possess thisCheR-binding site and can therefore function independentof the otherMCPs. The otherMCPs, which do not possessthis binding site, depend on the presence of Tsr and Tar tofunction in adaptation. Therefore, to be functional inadaptation, the minor MCPs must interact with majorMCPs, and this may be one of the reasons for the organ-isation of the receptors in clusters. See also:Bacterial Taxis
Dual-function receptors
Chemoattractant sugars do not bind to theMCPs directly.They either bind to a specific periplasmic binding proteininvolved in both chemotaxis and transport of the sugar(e.g. the galactose-, maltose- and ribose-binding proteins),or they bind to a specific Enzyme II (for glucose, mannose,mannitol and others) of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Theresponses to both types of sugar chemoattractants are,however, mediated by MCPs. The periplasmic binding
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aApproximate values. The values, shownhereinmainly forE. coli strainRP437 atmid-exponential phase, varywith the strain andwith the growthphase.bThe gene cheA encodes for two polypeptides, long and short (CheAL andCheAS, respectively), as a consequence of translational initiation at twodistinct in-frame initiation sites.
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proteins bind to a specificMCP (Tar or Trg) and thus elicita chemotactic signal. In the case of a PTS sugar, the PTSEnzyme I modulates the kinase activity of the complexMCP-CheW-CheA. Like the MCPs, at least some of thedual-function receptors (e.g. the periplasmic maltose-binding protein) are clustered at the bacterial poles, prob-ably in order to allow direct interaction with the MCPs.
Switch proteins
The switch, a complex of three proteins – FliG, FliM andFliN (Figure 2) – is the target of the signal from the recep-tors. Each of these proteins interacts with the other twoproteins, forming a complex that is linked to theMS ring ofthe flagellar motor via FliG, forming the switch–motorcomplex.
Proteins mediating receptor–switch communicationwithin the cell
Themolecule that delivers the clockwise signal to the switch–motor complex is the chemotaxis protein CheY (Figure 5).This protein – a response regulator of a two-componentregulatory system – can be in two main states: phos-phorylated and nonphosphorylated. The phosphorylatedsite is Asp57. CheY is phosphorylated by CheA, anautophosphorylatable histidine kinase. The autopho-sphorylation site on CheA is His48. It is only present in thelong form of CheA, CheAL. Phosphorylated CheY(CheY�P) is dephosphorylated spontaneously or in anenhanced manner by a specific phosphatase, CheZ (Figure
5). (The term phosphatase is used here in the broader senseand it does not imply a specificmechanismofCheZaction.)The short form of CheA, CheAS, forms complexes withCheW and CheZ and activates the latter, at least in vitro.CheY can also be phosphorylated by small phosphodonors
that reside in the cell (e.g. acetyl phosphate), but theircontribution is negligible relative to that of CheA. Whennonphosphorylated, CheY is bound to CheA, which itselfis bound to theMCP receptor via a scaffold protein, CheW(Figure 5). CheY is thus one part of the quaternary complexreceptor: CheW:CheA:CheY. See also: Bacterial Taxis
Signal transduction in response to positivestimulation
Under nonstimulated conditions, the phosphorylationlevel of CheY is relatively low. Accordingly, the extent ofCheY�P binding to the switch and, consequently, theprobability of clockwise rotation are low. This situationresults in predominant counterclockwise rotation andoccasional clockwise rotation, and the bacterial swimmingbehaviourmainly consists of runswith occasional tumbles.Positive stimulation (e.g. binding of a chemoattractant tothe receptor) shifts the receptor to a form that, togetherwith CheW, inhibits the autophosphorylation of CheAwithin the two-dimensional structure of the receptorsupramolecular complex (consisting of MCP dimers,CheW and CheA molecules). Therefore, the steady-statelevel ofCheY�Pdeclines, and the probability of clockwiserotation decreases. The outcome of this situation is pro-longed runs and rare tumbles. Under certain conditions,CheZ might be involved in lowering the CheY�P level.
Signal transduction in response to negativestimulation
A negative stimulus shifts the receptor to an activeform that, together with CheW, is thought to stimulatethe autophosphorylation of CheA. This hypothesisedactivation of CheA, however, could not be demon-strated experimentally. When CheA autophosphorylates,
CheB
CheW
CheW
CheR
Rece
pto
rs CheA
CheA
CheZ
CheZCheY
CheZ
FliM FliNFliG
Motor
MotA MotBSwitch
CheZ
Figure 5 Signal transduction in bacterial chemotaxis. The scheme is not drawn to scale. Black arrows stand for regulated interactions. CheA is a histidine
kinase that phosphorylates CheB and CheY, CheB is a specific methylesterase that demethylates the chemotaxis receptors, CheR is a specific
methyltransferase that methylates the receptors, CheW is a scaffolding protein that couples CheA to the receptors, CheY is the key response regulator in
chemotaxis of E. coli, and CheZ is a phosphatase that enhances the spontaneous dephosphorylation of CheY. (Modified with permission from Bren and
Eisenbach, 2000.)
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it instantaneously phosphorylates CheY, which in turndissociates from the quaternary complex (Schuster et al.,1993). CheY�P has a relatively high affinity for the switchprotein FliM (Welch et al., 1993) and for the phosphataseCheZ (Blat and Eisenbach, 1994). Binding to the switchresults in increased probability of clockwise rotation and,therefore, the cell tumbles frequently (Figure 5). Binding toCheZ sequentially results in CheZ oligomerisation anddelayed activation of its phosphatase activity (Blat andEisenbach, 1996). Consequently, CheY�P is depho-sphorylated after a delay and the clockwise signal is ter-minated. This termination may be required to avoidlengthy tumbling events (a brief tumble is sufficient forreorientation), to avoid persistent clockwise rotation(persistent clockwise rotation leads to the formation of abundle of right-handed flagella, resulting in a run) or foradaptation (see below). See also: Bacterial Taxis
It should be pointed out that it is not known how achemorepellent activates the receptor. No binding of anychemorepellent to any chemotaxis receptor has beendemonstrated, although, in vivo, the responses to mostchemorepellents aremediated byoneormoreMCPs. Itwasproposed that the MCPs are low-affinity receptors forchemorepellents (Eisenbach et al., 1990a).
Signal transduction in response to multiple inputs
Generally speaking, when a cell is exposed to a number ofchemotactic stimuli, there appears to be only one type ofresponse, meaning that the different inputs are integrated(although they are not necessarily additive). This alsoapplies to cases in which bacteria are exposed to a che-moattractant and a chemorepellent together; however,when the response is analysed in fast kinetics, the responseto the chemorepellent precedes the response to thechemoattractant.
Bacteria can sense stimuli over a wide concentrationrange and, in spite of the wide range, do so with very highsensitivity. The reason is the high amplification of thechemotactic signal. There are at least two steps of ampli-fication, one at the receptor cluster, and theotherwithin theswitch (Sourjik, 2004).
Nonconventional signal transductionpathways in E. coli
It was found that E. coli strains, lacking most of theMCPsand the known chemotaxis machinery but containing highlevels of CheY, have a chemotactic-like response to con-ventional chemoattractants and chemorepellents (Barakand Eisenbach, 1999). These findings raise the possibilitythat, at least when the conventional signal transductioncomponents are missing, a nonconventional chemotacticsignal transduction pathway might be functional in E. coli.The identity of the components involved in this pathway isnot known.
In other studies, it was found that E. coli strains, lackingmost of the conventional signal transduction components
but expressing CheY, are able to respond to the chemor-epellents indole and benzoate (Montrone et al., 1996). Thisphosphorylation-independent signal transduction involvesinhibition of the enzyme fumarase by these chemor-epellents, resulting in elevation of the intracellular level offumarate. Fumarate affects the function of the switch–motor complex by interacting with the enzyme fumaratereductase, which forms a reversible complex with FliG(Cohen-Ben-Lulu et al., 2008). Fumarate reduces the freeenergy difference of the counterclockwise-to-clockwisetransition and, thereby, it increases the probability of theclockwise state (Prasad et al., 1998).Other studies demonstrated that CheY is highly acety-
lated in vivo (Yan et al., 2008), with lysine residues at theC terminus (primarily residues 92 and 122) as the acetyla-tion sites. This acetylation, achieved by autocatalysis fromacetyl coenzyme A (AcCoA), by the enzyme AcCoA syn-thetase, and probably by an additional, yet unknowntransacetylase, activates CheY to generate clockwiserotation. It is also thought to be involved in the signallingresponse to repellents. On the basis of binding assays thatdemonstrated that acetylation prevents the binding ofCheY to all its target proteins, it was proposed that thebinding of CheY to the switch protein FliM may be con-trolled at two levels (Figure 6): fast, positive regulation byphosphorylation according to environmental signals (e.g.chemotactic and thermotactic stimuli); and slow, negativeregulation by acetylation according to the metabolic stateof the cell (Liarzi et al., 2010).According to this hypothesis,acetylated CheY is essentially sequestered from signalling:it cannot bind to CheA at the receptor supramolecularcomplex (and, consequently, it is probably free in thecytoplasm), and it can, therefore, neither be phosphoryl-ated by CheA nor bind to FliM and CheZ. According tothis hypothesis, themagnitude of the fraction of CheY thatis not acetylated and, therefore, free for signalling dependson the metabolic state of the cell.
FliM
Fast
CheY ~ P
CheY ~ Ac
CheY
SlowMetabolic state
Chemotactic andthermotactic stimuli
Figure 6 Suggested role for the dual covalent modification of CheY.
According to the model, the metabolic state of the cell determines the
fraction of CheY molecules that are acetylated. Only nonacetylated CheY
molecules can bind to CheA and be phosphorylated by it, and only they
can bind to FliM with a resultant change in the direction of flagellar
rotation. Thus, while acetylation, which is the slower process of these
two covalent modifications, determines the fraction of CheY molecules
that can participate in chemotactic signalling, phosphorylation, whose level
is regulated by chemotactic and thermotactic stimuli, determines the
extent of CheY binding to FliM. (Taken with permission from Liarzi et al.,
Variations on signal transduction pathwaysin other bacterial species
More than one signal transduction pathway
As the genome sequences of more and more bacterial spe-cies becomeavailable, it turns out that, unlikeE. coli, whichhas only one set of che genes, a significant proportion of thebacterial strains have two or more sets of che genes. Thissuggests that these strains possess at least two signaltransduction pathways. For example,R. sphaeroideshas 13MCPs (four ofwhich are in the cytoplasm), fourCheA, twoCheB, three CheR, four CheWand six CheY (and noCheZbut one CheD) (Porter et al., 2011). By studying propermutants of R. sphaeroides, it was demonstrated that thisspecies indeed has two (or more) pathways, as well as twodifferent kinds of flagella. The reason for having two sys-tems of signalling and flagella in this species is not clear,especially that both systems appear to function undersimilar conditions.
Lack of CheZ
Many bacterial species do not contain CheZ. Such speciesusually havemore thanoneCheY, one ofwhichmay fulfil arole analogous to that of CheZ. This was demonstrated inthe case of S. meliloti and R. sphaeroides, where one of theCheY proteins assumes the role of a ‘phosphatase’ byacting as a phosphate sink. R. sphaeroides possesses anadditional phosphatase – one of the CheAproteins has thisfunction (Porter et al., 2011). In other species, CheCfunctions as a phosphatase.
Different inputs
In contrast toE. coli, where chemoattractants are sensed onthe bacterial surface, there are bacterial species in whichchemoattractants or their metabolites are detected intra-cellularly. For example, in the case of R. sphaeroides, thesugars mannitol and fructose have to be transported intothe cell and perhaps metabolised to be detected. This mayexplain the finding that in this species four of the MCPsreside intracellularly (Porter et al., 2011).
Different outputs
In some species, the outcome of CheY�P interaction withthe switch is different from the outcome in E. coli. InHalobacterium salinarium, for example, CheY�P appearsto increase the switching probability rather than theclockwise probability of the motor. In Bacillus subtilis,phosphorylation of CheY apparently decreases (ratherthan increases) the clockwise probability (Garrity andOrdal, 1995). InS.meliloti orR. sphaeroides, an interactionof CheY�P with the flagellar motor slows down or evenstops, respectively, the rotation instead of changing itsdirection (the flagella of these species are unidirectional;Table 1).
Signal transduction in large bacterial species
The signal transduction pathways discussed above areessentially networks of interacting enzymes, resulting in arelatively short signalling range. They are, therefore, notsuitable for large (longer than 20 mm) bacterial species.Indirect evidence suggests that in such species (e.g. Spiril-lum volutans,Rhodospirillum rubrum, Thiospirillum jenenseand cyanobacteria), the signal is electrical in nature. Per-haps the most convincing evidence was obtained in spiro-chaetes, where neurotoxins, which affect the actionpotential in excitable eukaryotic cells, were found to inhibitchemotaxis (Goulbourne andGreenberg, 1983), andwhereclamping the membrane potential at � 0mV had a similarinhibiting effect.
Adaptation
Adaptation is the process of recovery from a stimulatedbehaviour when the stimulus is still present. Adaptation isessential for every behavioural system because it allowsdetection of small changes in the stimulus level on top of aconstant stimulation level. In the case of bacterial chemo-taxis, adaptation enables bacteria to respond tonew stimuliin the presence of constant levels of chemoattractants and/or chemorepellents. Bacterial adaptation is precise, in thesense that the post-adaptation swimming behaviour isexactly like the pre-stimulus behaviour (Alon et al., 1999).Furthermore, this precision is robust; namely, it is inde-pendent of the exact level of the proteins involved inadaptation. However, the steady-state tumbling frequencyand the adaptation time do vary with the proteinconcentrations. In E. coli, there appear to be at least twoadaptation mechanisms: methylation-dependent andmethylation-independent.
Methylation-dependent adaptation
The cytoplasmic domain of each MCP contains 4–6methylatable glutamate residues. The side-chain of each ofthese glutamate residues can be methylated by CheR – aspecific methyltransferase. (Some of these methylationsites are encoded as glutamine residues that, post-translationally, are converted to glutamate residues byCheB.) The formedmethyl ester bond canbe hydrolysed byCheB – a specific methylesterase. A methylated MCPtransmits a clockwise signal to the flagella, whereasdemethylated MCP transmits a counterclockwise signal.These signals are presumably caused by conformationalchanges in the cytoplasmic, signalling domain of theMCP.See also: Bacterial Taxis; Protein Structure: UnusualCovalent BondsIt has been shown in a cell-free system that the methyl-
ation reaction is enhanced by chemoattractants and isinhibited by chemorepellents, but the mechanism under-lying these effects is not known (Kleene et al., 1979).Conversely, the demethylation reaction is enhanced bychemorepellents and is inhibited by chemoattractants.This is mainly the consequence of modulation of the
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phosphorylation level of CheB. It turns out that there is aremarkable sequence homology between the entire lengthof theCheYprotein and the amino (N)-terminus domain ofCheB. Therefore, not only is CheY phosphorylated byCheA but also CheB, with a consequent increased methy-lesterase (demethylation) activity.
Thus, negative stimulation is believed (but not demon-strated) to result in enhanced autophosphorylation ofCheA, which, in turn, increases the steady-state phos-phorylation level of CheY and, more slowly, of CheB.Upon phosphorylation, CheB is activated, the MCPs aredemethylated and the probability of clockwise rotationdecreases to the pre-stimulus level. Positive stimulationinhibits CheA autophosphorylation. CheR, more slowly,methylates the MCP. The methylated MCP enhancesCheA autophosphorylation and the end result is increasedprobability of clockwise rotation and restoration of thepre-stimulus level. (In B. subtilis, in contrast to E. coli andSalmonella, the methyltransferase CheR is involved inadaptation to negative stimulation and the methylesteraseCheB in adaptation to positive stimulation.)
Methylation-independent adaptation
There is evidence that, although methylation-defectivemutants (cheB cheR mutants) of E. coli are defective inadaptation, they can still adapt to a certain extent (Stocket al., 1985). This suggests that there is an additional,methylation-independent adaptation mechanism. Such amechanismmay be provided by CheZ. As indicated above,both the activation and deactivation of the phosphatasefunction of CheZ are delayed. The apparent consequenceof the delay is that the modulation of the phosphataseactivity occurs only after the excitatory signal is complete.Therefore, the delayed activation and deactivation appearto constitute an adaptationmechanism, which ensures thatthe phosphorylation level is partially set back close to thepre-stimulus level. Accordingly, cheZ mutants adaptslower thanwild-typemutants. It is not knownwhether thedelayed activation and deactivation of CheZ is actually themethylation-independent adaptation mentioned above.One of the possibilities is that CheZ mediates the first stepof adaptation, whereas the second, slower step, whichincludes the precise tuning of the direction of flagellarrotation, is mediated by themethylation system (Blat et al.,1998).
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