Specific aspects of BCT are described in greater detail in Bray et al. (1993) and Bray & Bourret (1995), and in the documentation provided in the program. The experimental background to the model, and the currently accepted view of the signal relay, are described in a number of recent reviews: Falke et al. (1997), Armitage (1999), Bren & Eisenbach (2000) and Bourret and Stock (2002). Summarising briefly: the transmembrane receptors Tar and Tsr forms a stable ternary complexes with the coupling protein CheW and the CheA kinase. Binding of aspartate to Tar or serine to Tsr alters the autophosphorylation activity of CheA. Phosphoryl groups are transferred from CheA to CheY and CheB. CheZ stimulates dephosphorylation of CheY. The signalling network controls swimming behaviour by regulating the level of phosphorylated CheY. CheYp binds to the motor and changes flagellar rotation from the default counterclockwise (CCW) direction to clockwise (CW). Attractants cause decreased phosphorylation, which results in CCW flagellar rotation and "run" behaviour. Repellents increase phosphorylation, leading to CW flagellar rotation and "tumble" behaviour. CheB and CheR implement the bacterium's adaptation response by altering the methylation state of the receptors.
The following symbols are used in the program interface:
a aspartate
s serine
i inactivated
(see Conformational spread)
TT Tar/Tsr receptor;
dimer; methylated form TTm
W CheW
AA CheA; dimer; phosphorylated
form AAp
Y CheY; phosphorylated
form Yp
ZZ CheZ; dimer
R CheR
B CheB; phosphorylated
form Bp
Symbols for other molecular species in the signal transduction
pathway are built up from these symbols. For example, WWAAp is
a complex of CheW and phosphorylated CheA, while TTmiWWAA is an
inactivated complex of methylated receptor, CheW and CheA. The
latter is a ligand-free receptor complex that is functionally
equivalent to a ligand-bound complex due to conformational spread
within a cluster of receptors (Bray
et al., 1998). The extent of conformational spread (and therefore
gain) is determined by the Conf_spread parameter in the Configuration
file. Note that only unmethylated receptor complexes generate
conformational spread, although both methylated and unmethylated
complexes are equally susceptible to being inactivated. Reactions
are presented in a similar way: for example,
TTWWAAp + Y = TTWWAA + Yp
is the reaction in which a phosphoryl group is transferred from
TTWWAAp to CheY, while
TTW + WAA = TTWWAA
is the binding reaction between TTW and WAA. Note that Tar and
Tsr are not represented explicitly in this version of the program;
TTWWAA is partitioned between serine- and aspartate-binding complexes
according to the parameter Tsr_to_Tar in the Configuration file.
Double-click the BCT icon to open the program. You will then have the following options:
"I" = Information (program
description, parameter values)
"D" = Default stimulus (wild
type, no aspartate or serine)
"C" = Change parameters before
simulation
"T" = Test run
"X" = eXit program
The simplest selection is "D" for default, which
instructs the program to run a simulation for an unstimulated,
wild-type bacterium. Starting concentrations and rate constants
are loaded automatically and the time, bias and presence/absence
of aspartate and serine displayed on the screen. With present
settings, 100000 cycles are performed, each of 0.002 s duration
(that is, 2 ms of experimental time). Records are stored every
5000 cycles (10 s). At the end of this run, you will be offered
three ways to display the data:
"Default"
shows the concentration of Yp and the bias over time.
"Snapshot"
shows all of the signalling species (proteins plus aspartate,
serine, bias and receptor occupancy) at a selected time.
"Timecourse"
shows the concentration of up to four signalling components over
time.
Data from such tests are stored in separate files (called 2SNAP.BCT
and 2TIME.BCT respectively). In addition, a more detailed
time-course of the bias is available in the file 2BIAS.BCT.
These are all text files and can be exported or printed as desired.
Sample output:
2TIME.BCT with four signals
seconds TTmWWAAp Bp Yp bias
0.00 0.00e+00 0.00e+00 0.00e+00 1.00e+00
10.00 9.68e-09 9.33e-08 3.59e-06 5.20e-01
20.00 8.77e-09 8.82e-08 3.39e-06 6.55e-01
30.00 8.22e-09 8.46e-08 3.27e-06 7.34e-01
40.00 7.88e-09 8.22e-08 3.19e-06 7.79e-01
50.00 7.67e-09 8.07e-08 3.14e-06 8.06e-01
60.00 7.53e-09 7.98e-08 3.10e-06 8.22e-01
70.00 7.44e-09 7.91e-08 3.08e-06 8.32e-01
80.00 7.38e-09 7.87e-08 3.07e-06 8.38e-01
90.00 7.35e-09 7.84e-08 3.06e-06 8.42e-01
100.00 7.32e-09 7.83e-08 3.05e-06 8.45e-01
110.00 7.31e-09 7.81e-08 3.05e-06 8.47e-01
120.00 7.30e-09 7.81e-08 3.05e-06 8.48e-01
130.00 7.29e-09 7.80e-08 3.05e-06 8.49e-01
140.00 7.28e-09 7.80e-08 3.04e-06 8.49e-01
150.00 7.28e-09 7.80e-08 3.04e-06 8.49e-01
160.00 7.28e-09 7.79e-08 3.04e-06 8.49e-01
170.00 7.28e-09 7.79e-08 3.04e-06 8.50e-01
180.00 7.28e-09 7.79e-08 3.04e-06 8.50e-01
190.00 7.28e-09 7.79e-08 3.04e-06 8.50e-01
200.00 7.28e-09 7.79e-08 3.04e-06 8.50e-01
Some changes can be made while in the program itself. Select "C" in the initial menu to specify the genotype (numbers of molecules of specific Che proteins per cell), the concentration of aspartate and/or serine, and the number and duration of the simulation time intervals.
Other changes must be made in text files supplied with the program before running BCT. Text file 1CON.BCT (Configuration file) contains values for the concentration of ATP, the time duration of each cycle, the number of cycles and so on; 1ENZ.BCT (Enzyme file) has a list of primary gene products used in the simulation together with the number of molecules of each per cell; 1BIND.BCT (Binding file) contains information on all of the binding reactions used in the simulation; 1REACT.BCT (Reaction file) contains information on the methylation, demethylation, phosphorylation, dephosphorylation and phosphotransfer reactions used in the model; 1SIG_B.BCT (Signal file) contains all the signalling molecules whose activity or concentration changes during the simulation. You can modify the data or text in any of these files independently of the main program with word-processing software.
Select the "T" option to obtain a selection of six different test routines:
"B" = Bench test (bias
for a set of mutants)
"G" = Gene test (gene dosage
effects)
"F" = Further gene tests (effects
on Yp and bias)
"K" = K (or rate) test
"A" = Aspartate test (vary concentration)
"Y" = Yp test (effect on motor)
"E" = End selection (return
to main menu)
"X" = eXit program
BENCH test runs through a set of mutants, including both null mutants and overexpression mutants and gives their rotational bias (stored in 3BENCH.TST). GENE test varies a chosen gene product in a user-specified genetic background and displays up to four selected signals (stored in 3GENE.TST). FURTHER gene tests perform automatic (fixed or random) multiple changes in gene dosage. RATE test varies a chosen rate constant in a user-specified genetic background and displays up to four selected signals (stored in 3RATE.TST). ASPARTATE test varies aspartate concentration in a user-specified genetic background and displays up to four selected signals (stored in 3SIG.TST). Yp test varies the intracellular concentration of CheYp and displays the resulting motor performance (stored in 3GENE.TST).
Sample output:
BENCH test with six genotypes
T R B W A Y Z
1 1 1 1 1 1 1 wild
type bias 0.85
10 1 1 1 1 1 1 T++ bias 1.00
1 1 1 10 1 1 1 W++ bias 1.00
10 1 1 10 1 1 1 T++W++ bias 0.84
1 1 1 1 10 1 1 A++ bias 0.90
1 1 1 10 10 1 1 W++A++ bias 0.97
Rate constants for the various reactions
are available from the published literature. These experimental
values are identified in the list of reactions, accessed in Information
or in the text file 1REACT.BCT. The binding constants for interaction
between receptor dimer, CheW monomer, and CheA dimer were derived
by computer optimisation to reproduce the phenotypes of specific
mutant strains (Bray & Bourret, 1995).
The relationship between CheYp and bias is obtained through an
equation of the form
bias = 1 - YpHill / (5.667*Set_YpHill + YpHill)
where Yp = concentration of CheYp; Set_Yp
= concentration of CheYp in an unstimulated, wild-type bacterium;
Hill = the degree of cooperativity between CheYp and
the motor bias. The default value of Set_Yp is 3.042
µM, calculated from rates of phosphorylation and dephosphorylation
as described in Bray & Bourret (1995); the default value of
Hill is 10.3 (Cluzel et al., 2000). These values may
also be changed in the Configuration file (1CON.BCT).
In the current program, the calculation of the bias also allows
a small contribution of CheY to the clockwise rotation of the
motor to be made. A factor, alpha, in the Configuration
file specifies the activity of CheY as a fraction of the activity
of CheYp.
The current version of BCT fails to match the sensitivity to attractants shown by living bacteria - despite the fact that it incorporates the best values of rate constants, concentrations and Kds (Bray, 2002). This discrepancy arises because of a "front end" amplification of signals at the level of receptors of around 35 fold (Sourjik & Berg, 2002). We have suggested that additional amplification of signals could arise because the activity of receptors is coupled through an allosteric mechanism operating within clusters of receptors on the cell surface (Bray et al., 1998).
The best way to explore the spread of activity within a receptor cluster is by means of a stochastic program. Recent versions of our program StochSim, for example, are able to represent both the locations of receptors within a cluster and their conformational states. In the latest version of BCT, however, we have implemented a temporary fix that represents the spread of conformational states as the recruitment (or "infection") of a certain number of neighbouring receptors. Complexes containing affected receptors are shown in the output as "TT(m)iWWAA(p)" and included in the calculation of the gain.
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Bray, D., & Bourret, R.B. (1995) Mol. Biol. Cell 6:1367-1380
Bray, D., Levin, M. D., & Morton-Firth, C. J. (1998) Nature 393:85-88
Bray, D. (2002) Proc. Natl. Acad. Sci. USA 99:7-9
Bren, A., & Eisenbach, M. (2000) J. Bacteriol. 182:6865-6873
Cluzel, P., Surette, M., & Leibler, S. (2000) Science 287:1652-1655
Falke, J.J., Bass, R.B., Butler, S.L., Chervitz, S.A., & Danielson, M.A. (1997) Annu. Rev. Cell Dev. Biol. 13:457-512
Sourjik V., & Berg H.C. (2002) Proc. Natl. Acad. Sci. USA 99:123-127