Bacterial chemotaxis is a useful model for investigating in molecular detail the behavioral response of cells to changes in their environment. Peritrichously flagellated bacteria such as coli and typhimurium swim by rotating helical flagella in a counterclockwise direction. If flagellar rotation is briefly reversed, the bacteria tumble and change the direction of swimming. The bacteria continuously sample the environment and use a temporal sensing mechanism to compare the present and immediate past environments. Bacteria respond to a broad range of stimuli including changes in temperature, oxygen concentration, pH and osmotic strength. Bacteria are attracted to potential sources of nutrition such as sugars and amino acids and are repelled by other chemicals. In the methylation-dependent pathways for sensory transduction and adaptation in E. coli and S. typhimurium, chemoeffectors bind to transducing proteins that span the plasma membrane. The transducing proteins are postulated to control the rate of autophosphorylation of the CheA protein, which in turn phosphorylates the CheY protein. The phospho-CheY protein binds to the switch on the flagellar motor and is the signal for clockwise rotation of the motor. Adaptation to an attractant is achieved by increasing methylation of the transducing protein until the attractant stimulus is cancelled. Responses to oxygen and certain sugars involve methylation-independent pathways in which adaption occurs without methylation of a transducing protein. Taxis toward oxygen is mediated by the electron transport system and changes in the proton motive force. Recent studies have shown that the methylation-independent pathway converges with the methylation-dependent pathway at or before the CheA protein

Taylor, Barry L.

English

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'7 N90-13944
THE SENSORY TRANSDUCTION PATHWAYS
IN BACTERIAL CHEMOTAXIS
Barry L. Taylor
Department of Microbiology,
School of Medicine,
Loma Linda University
Loma Linda, California 92350
Bacterial chemotaxis is the least complex behavioral response and will
probably be the first behavioral system in which the entire sensory
transduction pathway from stimulus to response can be described in terms of a
sequence of biochemical and physical events. As such, it is a useful model for
understanding how more complex cells and organisms respond to changes in
their surroundings. Chemotaxis is the name given to the movement of motile
bacteria toward a source of nutrients and away from harmful substances,
thereby enhancing their chances of survival. Bacteria also respond to a
variety of other sensory stimuli (Koshland, 1988; Macnab, 1987b; Taylor,
1983a).
Escherichia c01i and Salmonella _yphimurium, the bacteria most commonly
investigated, swim by rotating four to nine flagella per cell. The flagellar
filament, which is composed of a single type of protein, is like a flexible
corkscrew with a left-handed helix (Macnab, 1987a). The flagellar motor is
embedded in the plasma membrane and anchored to the peptidoglycan and
outer membrane. The rod, which is the shaft of the motor, is connected to the
filament by a universal joint known as the hook.
If the flagellar motors rotate in a counterclockwise direction,
hydrodynamic forces collect the flagella into a bundle which has a
synchronized wave propagation that propels the bacterium forward (Macnab,
1987b). When the motors briefly reverse and rotate the flagella in a clockwise
direction, the flagella bundle flies apart causing a chaotic tumbling motion
that reorients the bacterium. When counterclockwise rotation is resumed, the
bacterium swims off in a different direction. The net result of the random
alternation between counterclockwise and clockwise rotation is a random walk
type of motion (Berg and Brown, 1972).
A temporal sensing mechanism is utilized by the bacteria to continuously
sample attractants and repellents in the environment and to compare the
present environment with the environment that the bacterium has just left
(Macnab and Koshland, 1972). If the difference is favorable, tumbling is
suppressed and the bacterium continues in the favorable direction. If the
difference is unfavorable, the probability of tumbling increases thereby
ensuring that the bacterium will change direction. The net effect is to bias
the random walk motility so that the bacteria migrate to a favorable
environment. A central goal of research into chemotaxis is to determine the
pathway by which external stimuli modulate the probability of clockwise
rotation of the flagellar motors.
The strongest attractants for E. coli and $, lyphimurium are the amino acids
serine and aspartate (Macnab, 1987b). Other chemical attractants include some
of the other amino acids, sugars and sugar alcohols. Chemical repellents
include short-chain fatty acids and alcohols, some hydrophobic amino acids,
indole, benzoate, sodium sulfide and the divalent cations Co 2+ and Ni 2+. Other
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tactic stimuli include oxygen, temperature, pH and osmotic strength. Many
species of phototrophic bacteria are phototactic.
The methylation-dependent pathways for bacterial chemotaxis are
represented in Figure 1. Chemoattractants either bind directly to a specific
membrane-spanning transducing protein or activate a soluble binding
protein that subsequently binds to a transducing protein (Koshland, 1988;
Macnab, 1987b). No specific receptors for repellents have been unequivocally
identified; repellents may act by perturbing the membrane domain that
surrounds the transducing protein. The four transducing proteins in E. colq
that have been identified are the products of the tsr, tar, Lr_g. and Lr4/_ genes.
Each consists of a periplasmic domain, two membrane-spanning sequences
and a cytoplasmic domain (Koshland, 1988; Krikos et al, 1983).
There is a high degree of sequence identity in the cytoplasmic domains of
the four transducing proteins (Krikos et al, 1983). Two conserved regions
contain the sites that are methylated during adaptation (see below). Another
highly conserved region is believed to be involved in transmitting
chemotactic signals to the flagellar motors. This assignment is made on the
basis of signaling-deficient bacteria that have mutations in the conserved
region. Specificity is conferred on the transducing proteins by the variable
binding domains in the periplasmic portion of the protein. This has been
verified using chimeric constructs of the tsr and tar genes that consist of a 5'
region coding for the N terminus of one transducing protein and the 3' region
coding for the C terminus of the other transducing protein (Krikos et al, 1985).
Receptor specificity of the chimeric protein is similar to that of the Tsr or Tar
transducer that has the same N terminus.
Until recently little progress had been made in identifying the post-
transducer events in signal transduction. The application of three
experimental strategies has now revealed at least the skeleton of the
transduction pathway. A novel method for depleting S. typhimurium of ATP
was used by Junichi Shioi in my laboratory to demonstrate a requirement for
ATP in chemotaxis (Shioi et al, 1982). So called "gutted" strains of E. coli that
were depleted of chemotaxis genes but had normal flagellar motors were used
to study the effect on chemotaxis of restoring a single chemotaxis gene or a
combination of genes to the gutted strain (Wolfe et al, 1987). A comparison of
the sequence of three chemotaxis genes, cheA,cheY and cheB, with gene
sequences available in gene banks revealed a striking similarity with the
structural genes for a family of bacterial regulatory proteins (Stock, 1987).
The ntrB and ntrC genes involved in nitrogen assimilation in E. coli are the
most studied members of this family.
In the gutted strain the motor rotates only in a counterclockwise direction
(Parkinson and Houts, 1982). Investigations in the laboratory of Daniel
Koshland, Jr. demonstrated that introduction of the CheY protein restored
clockwise rotation (Clegg and Koshland, 1984). The probability of clockwise
rotation was a hyperbolic function of the concentration of CheY indicating
that the binding of CheY to the switch was the signal for clockwise rotation
(Kuo and Koshland, 1987). Subsequent studies in our laboratory established
that an active form of CheY causes clockwise rotation and ATP is essential to
activate CheY (Smith et al, 1988). This and the similarity of the Che and Ntr
regulatory proteins suggested that the CheY protein was activated by
phosphorylation of the protein.
Wolfe, Conley, Kramer and Berg (1987) discovered that the minimal
additions to the gutted strain required for a chemotaxis signal from the Tar
98
transducingprotein to reach the motor were the cheA, cheW and cheY genes.
Hess, Oosawa, Matsumura and Simon (1987) found that the CheA protein is
autophosphorylated in vitr9 by ATP and then transfers the phosphate moiety
to the CheY protein. It is assumed, but not yet proven, that in vivQ the
transducing proteins control either the phosphorylation of .CheA or the
transfer of phosphate from CheA to CheY.
In addition to responding to chemotactic stimuli, bacteria adapt to such
stimuli. This was first demonstrated when Macnab and Koshland (1972) used a
rapid-mixing device to add attractant to a culture of S. typhimurium. The cells
suppressed all tumbling and swam smoothly for a short interval, then adapted
to the attractant and returned to a random motility pattern. At the molecular
level, adaptation to an attractant occurs when the transducing protein is
multiply methylated by a protein methyltransferase that is the product of the
cheR gene (Springer et al, 1979; Springer and Koshland, 1977). The methyl
donor in this reaction is S-adenosylmethionine. Methylation precisely cancels
the signal generated by the attractant. If the attractant is subsequently
removed or if a repellent is added, the cells tumble continuously then adapt
when some of the methyl esters on the transducing proteins are hydrolyzed by
the esterase activity of the cheB gene product (Stock and Koshland, 1978).
The methylation-dependent pathways are the major chemotactic pathways
and are utilized in responding to most stimuli. However, Mitsuru Niwano
working in my laboratory discovered that adaption to oxygen and to most
sugars is independent of transducer methylation (Niwano and Taylor, 1982).
The major focus of our research has been these methylation-independent
pathways.
The attraction of E. coli or S. typhimurium to oxygen is readily observed in
the accumulation of these bacteria around a trapped air bubble in a drop of
culture beneath a cover slip (Taylor, 1983a). Some other species behave
differently in the presence of a gradient of oxygen. Beijerinck (1893)
observed in the last century that aerobic bacteria beneath a coverglass form a
band near the air-liquid interface. Microaerophilic bacteria accumulate in a
band that is some distance from the interface and anaerobic bacteria
accumulate in the center of the cover slip. This suggests that oxygen is both
an attractant and a repellent and that bacteria migrate to where the oxygen
concentration is optimal for their metabolic lifestyle (Taylor, 1983a,b). This is
not surprising in view of the toxicity of some oxygen derivatives.
To distinguish between the responses of enteric bacteria to high (K0.5 = 1.0
mM) and low (K0.5 = 0.7 I.tM) concentrations of oxygen, the responses will be
referred to as the oxygen repellent and oxygen attractant responses,
respectively (Laszlo et al, 1984; Shioi et al, 1987). We found that the attractant
response to oxygen is mediated by the proton motive force (Laszlo and Taylor,
1981; Shioi and Taylor, 1984). Oxygen binding to the terminal oxidase of the
respiratory chain increases the rate of electron transport which is coupled to
translocation of protons across the inner membrane. E. coli and S.
lyphim_lri_lm sense and respond to changes in the proton motive force. This is
also the basis of the phototactic response in photosynthetic bacteria
(Harayama and Iino, 1977). We have shown that tactic responses result from a
wide variety of phenomenon that perturb the proton motive force (Taylor,
1983a,b).
Ongoing studies in our laboratory are looking at the convergence of the
methylation-dependent and methylation-independent pathways for
chemotaxis. The gutted strain of E. coli with a functional flagellar motor did
99
not respond to oxygen or to the sugar mannose which acts via the
phosphotransferase system, another methylation-independent pathway
(Rowsell et al, 1988; Taylor et al, 1988). The addition of various chemotaxis
genes showed that a normal response to oxygen or mannose was not observed
unless the cheA, cheW and cheY genes were present. This indicates that the
methylation-independent and methylation-dependent pathways converge at
or before the cheA protein. It remains to be determined how the methylation-
independent pathways modulate the phosphorylation of the cheY protein.
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OMe
®
CH30H
Figure 1. Scheme for sensory transduction in methylation-dependent
chemotaxis in E. coli and S. typhimurium. R, B, A, W, Y and Z
represent the product of the che genes with the same letter
designation. Attr, attractant; AdoMet, S-adenosylmethionine; OMe, 7-
glutamyl carboxymethyl ester; ---> order of reactions is tentative.
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The sensory transduction pathways in bacterial chemotaxis

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