• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biophysjLink to Publisher's site
Biophys J. May 2003; 84(5): 2841–2851.
PMCID: PMC1302849

Feedback Regulation in the Lactose Operon: A Mathematical Modeling Study and Comparison with Experimental Data

Abstract

A mathematical model for the regulation of induction in the lac operon in Escherichia coli is presented. This model takes into account the dynamics of the permease facilitating the internalization of external lactose; internal lactose; β-galactosidase, which is involved in the conversion of lactose to allolactose, glucose and galactose; the allolactose interactions with the lac repressor; and mRNA. The final model consists of five nonlinear differential delay equations with delays due to the transcription and translation process. We have paid particular attention to the estimation of the parameters in the model. We have tested our model against two sets of β-galactosidase activity versus time data, as well as a set of data on β-galactosidase activity during periodic phosphate feeding. In all three cases we find excellent agreement between the data and the model predictions. Analytical and numerical studies also indicate that for physiologically realistic values of the external lactose and the bacterial growth rate, a regime exists where there may be bistable steady-state behavior, and that this corresponds to a cusp bifurcation in the model dynamics.

INTRODUCTION

The operon concept (Beckwith, 1987b), introduced by Jacob et al. (1960), has had a profound and lasting effect on the biological sciences. Not long after the operon concept was developed, Goodwin (1965) gave the first mathematical analysis of operon dynamics. Griffith then put forward a more complete analysis of simple repressible (negative feedback, Griffith (1968a)) and inducible (positive feedback, Griffith (1968b)) gene control networks, and Tyson and Othmer (1978) have summarized these results. Extensions considering the stability of inducible operons were published by Selgrade (1979, 1982) and Ji-Fa (1994), but none of these treatments considered the role of the DNA transcription and mRNA translation delays, though Tyson and Othmer pointed out that both should be considered.

Bliss et al. (1982) were some of the first to explicitly consider transcriptional and translational delays in their modeling of the tryptophan operon. Many subsequent workers studying tryptophan dynamics (Sinha, 1988; Sen and Liu, 1989; Xiu et al., 1997) ignored these delays although including other biological details, but the tryptophan delays were considered by Santillán and Mackey, (2001a,b). Maffahy and Savev (1999) modeled lac operon dynamics and included transcriptional and translational delays, but Wong et al. (1997) failed to treat these elements of the lac operon despite their inclusion of much of the relevant biological detail. Recent work on gene network regulation can be found in Tyson and Mackey (2001).

In this paper we offer a model of the induction process in the lac operon, including much of the relevant biological detail considered by Wong et al. (1997) (but neglecting catabolite repression) as well as the transcriptional and translational delays considered by Maffahy and Savev (1999). A more complete mathematical analysis of a reduced version of the model presented here is possible, and was considered by N. Yildirim, D. Horike, and M. C. Mackey (unpublished results). Section 2, “The Model”, develops the model and gives a set of realistic parameters estimated from the biological literature (presented in detail in Appendix B). It also examines the nature of the steady states (as deduced in Appendix C) and briefly considers their stability (Appendix D). Section 3, “Comparison with Experimental Data”, compares the numerical predictions of the model temporal behavior with three sets of data taken from the literature, and the paper ends with a discussion in Section 4, “Conclusions”.

THE MODEL

To develop our model for regulation of the lac operon, it will be helpful to refer to Fig. 1. This control system functions in the following manner. In the absence of glucose available for cellular metabolism, but in the presence of external lactose (Le), lactose is transported into the cell by a permease (P). Intracellular lactose (L) is then broken down into glucose, galactose, and allolactose (A) by the enzyme β-galactosidase (B). The allolactose feeds back to bind with the lactose repressor and enables the transcription process to proceed.

FIGURE 1
Schematic representation of the lactose operon regulatory system.

In more detail, the lac operon consists of a promoter/operator region and three larger structural genes, lacZ, lacY, and lacA. Preceding the lac operon is a regulatory operon responsible for producing a repressor (R) protein. In the absence of allolactose (A), the repressor R binds to the operator region and prevents the RNA polymerase (which binds to the promoter region) from transcribing the structural genes. However, if allolactose is present, a complex is formed between allolactose and the repressor that makes binding of the repressor to the operator region impossible. In that case, the RNA polymerase bound to the promoter is able to initiate transcription of the structural genes to produce mRNA.

Once the mRNA has been produced, the process of translation is initiated. The lacZ gene encodes for the mRNA responsible for the production of β-galactosidase (B) and translation of the lacY gene produces mRNA ultimately responsible for the production of a membrane permease (P). The mRNA produced by transcription of the lacA gene encodes for the production of thiogalactoside transacetylase, which is thought to not play a role in the regulation of the lac operon (Beckwith, 1987a) and will not be further considered here.

As shown in Appendix A, following Yagil and Yagil (1971), if the amount of repressor R bound to the operator O is small, then in a large population of cells the fraction of operators not bound by repressor (and therefore free to synthesize mRNA) is given by

equation M1
(1)

where n was interpreted as the number of molecules of allolactose required to inactivate the repressor, K1 is the equilibrium constant for the repressor-allolactose reaction, and K2 is the equilibrium constant for the operator-repressor reaction, K = 1 + K2 Rtot, and Rtot is the total amount of repressor. Notice that there will be maximal repression when A = 0, but even at maximal repression there will still be a basal level of mRNA production (“leakage”) proportional to K−1. We are now in a position to develop our model for the lac operon regulation.

The dynamics of mRNA production are given by Eq. 2,

equation M2
(2)

which is derived as follows. First, note that the production of mRNA from DNA via transcription is not an instantaneous process but requires a period of time, τM, for RNA polymerase to traverse the three structural genes. The rate of change of M is a balance between a production term αM f and a loss term equation M3 The argument of f in the production term is equation M4 where equation M5 to account for the time, τM, required to produce the mRNA. The factor equation M6 accounts for the growth dependent allolactose dilution during the transcriptional period. In the total absence of allolactose, on occasion repressor will transiently not be bound to the operator and RNA polymerase will initiate transcription. Γ0 denotes this spontaneous rate of mRNA production. The loss term in Eq. 2, equation M7 is made up of an mRNA degradation term (γMM) and an effective loss due to dilution (μM).

The dynamics of β-galactosidase are described by Eq. 3,

equation M8
(3)

Again realize that β-galactosidase production through mRNA translation is not instantaneous but requires a time, τB. We assume that the rate of production of B is proportional to the concentration of M a time τB ago equation M9 where again the exponential factor takes into account the dilution of mRNA due to cell growth. The loss rate of B is given by equation M10 where as before equation M11

For the allolactose dynamics,

equation M12
(4)

the first term in Eq. 4 gives the β-galactosidase mediated gain in allolactose from the conversion of lactose following the studies of Huber et al. (1976). The second term accounts for allolactose loss via conversion to glucose and galactose, again mediated by β-galactosidase (Martínez-Bilbao et al., 1991; Huber et al., 1994). The last term takes into account the degradation and dilution of allolactose.

The lactose dynamics are more complicated and given by Eq. 5:

equation M13
(5)

The first term in Eq. 5 accounts for the augmentation of intracellular lactose L through the permease facilitated transport of Le. The proportionality constant αL is a decreasing function of extracellular glucose (Saier, 1976). The second term deals with intracellular lactose loss to the extracellular fluid because of the reversible nature of the permease-mediated transport (Saier, 1976; Osumi and Saier, 1982; Postma et al., 1996; Saier et al., 1996). The coefficient βL is not dependent on the external glucose levels. The third term accounts for the conversion of lactose to allolactose as well as the hydrolysis of lactose to glucose and galactose via β-galactosidase (B). The fourth term accounts for the decrease in internal lactose concentration due to degradation and dilution.

To describe the permease dynamics

equation M14
(6)

we have Eq. 6. There, the first term reflects the assumption that permease production is directly proportional (proportionality constant αP) to the mRNA concentration a time (τP + τB) in the past, where τP is the translation time between mRNA and permease. The delay is taken to be the sum of the β-galactosidase and permease translation times under the assumption that permease production cannot start until β-galactosidase production is complete. The exponential factor equation M15 accounts for dilution of mRNA concentration due to cell growth. The second term accounts for the degradation and dilution of permease.

We have carried out an extensive search of the existing literature for data that would allow us to estimate the model parameters in Eqs. 26. The results of our determinations are summarized in Table 1, and the details of how we arrived at these parameter values are contained in Appendix B.

TABLE 1
The estimated parameters for the model as determined in Appendix B

The full model as formulated in Eqs. 26 can have one, two, or three steady states depending on the values of the parameters (μ, Le). The details of how these steady states are determined are contained in Appendix C. The results of these considerations are presented in Fig. 2. There we show in equation M16 space the region where a nonnegative steady state can exist. Note in particular that for a range of Le values, there may be three coexisting steady-state values of the intracellular allolactose levels, equation M17 and, consequently, of equation M18

FIGURE 2
The region in the equation M37 space where a nonnegative steady state can exist as a function of external lactose levels Le for the model when all parameters are held at the estimated values in Table 1 and when equation M38 The shaded area shows the region where a steady state ...

Whether or not there can be coexisting steady states depends on the growth rate μ, and an examination of the dependence of the criteria for the existence of steady states equation M19 on (μ, Le) reveals that in equation M20 space there is a cusp bifurcation occurring that is dependent on the growth rate μ and extracellular lactose levels Le.

This cusp bifurcation in the model in equation M21 space is of the following nature. There is a minimal growth rate equation M22 such that for values of the growth rate μ [set membership] [0, μmin], there is a unique steady-state level of allolactose equation M23 for any given value of external lactose levels Le. At μ = μmin, the concentration of external lactose required for induction is equation M24 However, as μ becomes larger, μ [set membership] [μmin, μmax, the system may have three steady-state values of equation M25 at a given external lactose level Le. This is the situation depicted in Fig. 2, where we used equation M26 For this value of μ, the lactose concentration required for induction (and marked with an asterisk in Fig. 2) is equation M27 which compares well with the results of a study using an artificial inducer of the lac operon, isopropylthiogalactoside (IPTG) (Baneyx, 1999). There it was found that 50–100 μM IPTG is sufficient as a lower bound to achieve full induction.

A full stability analysis of the steady states of this model is impossible, since the eigenvalue equation determining local stability is a fifth order quasipolynomial containing three delays. Consequently, we have contented ourselves with a numerical examination of the stability properties of the steady states. Briefly, the results of our numerical stimulations presented in Appendix D are as follows. When a single steady state exists, we have found that the numerical behavior is such that the model solutions always converge to that steady state at large times. When there are three coexisting steady states, the numerical solutions to the model either converged to the lower or upper branch of the S-shaped curve for various initial conditions. These results, as well as numerous others that are not shown, lead us to conclude that the middle branch of the S-shaped steady-state curve (see Fig. 2) corresponds to a steady state that is globally unstable. The nature of the boundary between initial conditions leading to the convergence to the upper or lower branch solution appears to be complicated, and it may well be a fractal basin boundary (Losson and Mackey, 1993).

COMPARISON WITH EXPERIMENTAL DATA

Given the parameter values determined in Table 1, we numerically solved the model equations to compare the predicted behavior with three distinct experimental data sets.

The first data set is from Knorre (1968), in which changes of the specific β-galactosidase concentration after a step change from glucose to lactose growth for Escherichia coli ML30 were measured. The second data set is from Pestka et al. (1984). In this paper, Pestka et al. studied specific inhibition of translation of single mRNA molecules and gave data for the specific activity of β-galactosidase versus time for E. coli 294 in the presence of IPTG. These two data sets and the model simulation determined using MATLAB's dde23 (Shampine and Thompson, 2000) routine are shown in Fig. 3.

FIGURE 3
β-galactosidase activity versus time when Le = 8.0 × 10−2 mM. The experimental data sets were taken from Knorre (1968) for E. coli ML30 (○) and from Pestka et al. (1984) for E. coli 294 (♦). The model simulation ...

For this simulation, initial values for the variable were chosen as A0 = 3.80 × 10−2, M0 = 6.26 × 10−4, L0 = 3.72 × 10−1, P0 = 1.49 × 10−2, and B0 = 0.0 mM, all of which are close to the steady-state values given in Table 2 when Le = 8.0 × 10−2 mM. (With this value of Le, there is a single unique steady state). To compare these two sets of experimental data with the model simulation predictions, the data were scaled so the steady-state values of measured β-galactosidase activities and those produced by the simulation were equal. As seen in Fig. 3, there is relatively good agreement between both sets of experimental data and the model-predicted temporal approach of β-galactosidase activity to its steady-state value.

TABLE 2
Steady-state values when Le = 8.0 × 10−2 mM

As a third test of the model, a data set from Goodwin (1969) was used. In this paper, the dynamic behavior of β-galactosidase was studied in chemostat cultures of E. coli synchronized with respect to cell division by periodic phosphate feeding at a period equal to the bacterial doubling time. Experimentally, oscillations in β-galactosidase concentration were observed with a period equal to the feeding period.

To mimic the periodic phosphate feeding in our simulation, we assumed that the bacterial growth rate varies as a function of time in manner given by

equation M28
(7)

Here, equation M29 is the maximal growth rate for the bacteria, T is the period of the feeding and α is a positive parameter with dimension min−2. mod(t, T) is a function that gives the remainder on division of t by T. Selection of this type of function is motivated by the observation that growth rates decrease as nutrient levels fall and sharply increase after the addition of nutrient.

μ(t) takes its maximal value of equation M30 when t = T × k, (k = 1, 2, 3, …), which corresponds to the times that phosphate was added. The minimal value of this function, equation M31 at equation M32 represents the minimal amount of nutrient left in the vessel. Assuming no nutrient is left epsilon minutes before the addition of phosphate and letting equation M33 α can be estimated if the doubling time (T) is known: equation M34

With a feeding period of T = 100 min, we have equation M35 for equation M36, which is the value we have estimated for our model (cf. Appendix B). In Fig. 4, we compare the data on β-galactosidase activity from the forced culture as a function of time with the model predictions.

FIGURE 4
Oscillation in β-galactosidase activity in response to periodic phosphate feeding with period T = 100 min, which is the culture doubling time. The experimental data (*) together with the model simulation (solid line) using the ...

With respect to the predicted bistability at sufficiently large growth rates, illustrated in Fig. 2, evidence presented by Novick and Wiener (1957) and Cohn and Horibata (1959) qualitatively substantiate the existence of this behavior for values of the growth rate exceeding μmin. However, we are unable to determine if the quantitative ranges of Le for which we have predicted bistability correspond to the conditions under which it was seen in Novick and Wiener (1957) and Cohn and Horibata (1959).

CONCLUSIONS

Here we have developed, and analytically and numerically analyzed, a mathematical model for the regulation of the lac operon in E. coli. The final model consists of five nonlinear differential delay equations with delays due to the DNA transcription and mRNA translation processes. The model equations describe the dynamics of the permease (P) facilitating the internalization of external lactose (Le); internal lactose (L); β-galactosidase (B), which is involved in the conversion of lactose to allolactose, glucose, and galactose; the allolactose (A) interactions with the lac repressor, and mRNA (M). We have gone to considerable effort to make valid and reasonable estimates of the 24 parameters in the model. We were successful in identifying 22 of these parameters from published data, but were forced to determine the growth rate μ and γA by fitting the model to the data of Knorre (1968).

We have tested our model against two sets of β-galactosidase activity versus time data. These data came from the experimental work presented in Knorre (1968), in which changes of the specific β-galactosidase concentration after a step change from glucose to lactose growth for E. coli ML30 were measured, and the work of Pestka et al. (1984). In this latter paper, data were presented for the specific activity of β-galactosidase versus time for E. coli 294 in the presence of IPTG. These two data sets and the model simulation are shown in Fig. 3, and there is a remarkable degree of concordance between the data and the model predictions.

As a third test of the model, data from Goodwin (1969) giving the dynamic behavior of β-galactosidase was studied in chemostat cultures of E. coli synchronized with respect to cell division by periodic phosphate feeding at a period equal to the bacterial doubling time. Experimentally, oscillations in β-galactosidase concentration were observed with a period equal to the feeding period. Fig. 4 shows the β-galactosidase activity data as well as the model predictions. Again, there is a satisfying degree of agreement.

Analytical and numerical studies of the model also predict that for physiologically realistic values of external lactose and the bacterial growth rate, a regime exists where there may be bistable steady-state behavior, and that this corresponds to a cusp bifurcation in the model dynamics. This prediction is qualitatively confirmed by the observations of Novick and Wiener (1957) and Cohn and Horibata (1959). Although a full analysis of the stability properties of the model is not possible due to its complexity, we have found that the basic properties contained in a reduced version of this model N. Yildirim, D. Horike, and M. C. Mackey (unpublished results) (2002) (which considered only the dynamics of M, B, and A) are apparently retained in this much more complicated model as far as we are able to ascertain analytically and numerically.

This agreement between the model predictions for the lactose operon and existing data, and similar agreement between a mathematical model for the tryptophan operon (Santillán and Mackey, 2001a,b), highlight the desirability and necessity of closer cooperation between experimentalists and modelers to further validate and refine mathematical models of simple and more complicated gene regulatory networks.

To close, we wish to touch on the nature of the mathematical model presented here. The model considered in this paper is formulated with the explicit assumption that one is dealing with large numbers cells (and hence of large numbers of molecules) so that the law of large numbers is operative. However, the situation is quite different if one is interested in the dynamics of small numbers (or single) prokaryotic or eukaryotic cells, for then the numbers of molecules are small. Adequate means to analytically treat such problems do not exist in a satisfactory form as of now (Gillespie, 1992; Kepler and Elston, 2001; Swain et al., 2002), and one is often reduced to mostly numerical studies (Gillespie, 1977; McAdams and Shapiro, 1995; Arkin et al., 1998). The situation is analogous to examining the interactions between small numbers of interacting particles (where the laws of mechanics or electrodynamics hold), and then deriving from these formulations the behavior of large numbers of identical units as is done (not completely successfully even at this point) in statistical mechanics. We view this connection between the “micro” and “macro” levels as one of the major mathematical challenges facing those interested in the understanding of gene control networks.

Acknowledgments

We thank Dr. Photini Pitsikas for her initial suggestion of this problem, Profs. Claire Cupples and Moisés Santillán for their advice on parameter estimation and general comments, and Mr. Daisuke Horike for many helpful discussions.

This work was supported by the Scientific and Technical Research Council of Turkey, the North Atlantic Treaty Organization, the Mathematics of Information Technology and Complex Systems (Canada), the Natural Sciences and Engineering Research Council (grant OGP-0036920, Canada), Le Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (grant 98ER1057, Québec), and the Leverhulme Trust (UK).

APPENDIX A: REPRESSOR DYNAMICS

Let R be the repressor, E the effector (allolactose in our case), and O the operator. The effector is known to bind with the active form R of the repressor. We assume, as do Yagil and Yagil (1971), that this reaction is of the form

equation M47
(8)

where they took n to be the effective number of molecules of effector required to inactivate the repressor R. Furthermore, the operator O and repressor R are assumed Yagil and Yagil (1971) to interact according to

equation M48
(9)

Let the total operator be Otot:

equation M49
(10)

and the total level of repressor be Rtot:

equation M50
(11)

The fraction of operators not bound by repressor (and therefore free to synthesize mRNA) is given by

equation M51
(12)

If the amount of repressor R bound to the operator O is small

equation M52
(13)

so

equation M53
(14)

and consequently

equation M54
(15)

where K = 1 + K2Rtot. Notice that there will be maximal repression when E = 0 but even at maximal repression there will still be a basal level of mRNA production proportional to K−1.

APPENDIX B: PARAMETER ESTIMATION

In the model given by Eqs. 26, there are 24 parameters in total that must be estimated to characterize the system completely. In this section, we give the estimation of each of these parameters.

  • μ: The maximal value of the dilution rate μ can be estimated from the shortest interdivision time of E. coli, which is ~20 min (Watson, 1977). Given this, μmax = ln 2/20 min−1 = 3.47 × 10−2 min−1. We have also estimated a value of μ, denoted by equation M55 together with the value of γA by least-square fitting of the experimental β-galactosidase concentration data given in Knorre (1968) using the fminsearch and dde23 (Shampine and Thompson, 2000) routines in MATLAB. We found equation M56, indicating that these cultures were growing with a doubling time of 30 min. The results of this estimation were tested for several initial starting points for μ and γA, and the estimation procedure always converged to the same values of equation M57 and γA.
  • γA: The value of γA was estimated as 5.2 × 10−1 min−1 together with the value of μ by using least-square fitting of the experimental β-galactosidase activity data given in Knorre (1968) as above.
  • γM: Leive and Kollin (1967) found that the equation M58 of β-galactosidase mRNA was 2 min to give a value of equation M59 In a comparable experiment, Blundell and Kennell (1974) found equation M60 to give equation M61 We have taken the average of these two figures to give equation M62
  • γB: The rate of breakdown of β-galactosidase was measured by Mandelstam (1957) and found to be 0.05 per h corresponding to 8.33 × 10−4 min−1. Rotman and Spiegelman (1954) also reported that the maximal rate of breakdown of β-galactoside is 0.005 min−1, and noted that it is possibly much smaller than this value. We have taken the Mandelstam value.
  • K: Yagil and Yagil (1971) analyzed a number of published data sets, and from their calculations we find the average value is equation M63
  • n: Again from Yagil and Yagil (1971), we have an average Hill coefficient of 2.09. We have taken n = 2.
  • K1: The average dissociation constant of effector-repressor complex was equation M64 from the results of Yagil and Yagil (1971).
  • αM: The steady-state value of lac mRNA in the absence of induction is thought to be one molecule per cell. This corresponds to a “concentration” of 2.08 × 10−6 mM if we take the E. coli volume to be 8 × 10−16 liter. When the cells are maximally induced, the lac mRNA level is raised a thousand times compared to this uninduced steady-state value (Savageau, 1999). From Eq. 2 at a steady state,
    equation M65
    (16)
    equation M66
    (17)
    From Eqs. 16 and 17, αM is 9.97 × 10−4 mM–min−1.
  • Γ0: The term Γ0 was included in our model for the following reason. Assume that the term in question is not there, which is equivalent to taking Γ0 [equivalent] 0. An examination of Eq. 2 in a steady state yields equation M67 Let the level of lac mRNA in the maximally induced state be given by equation M68. The maximally induced state corresponds to equation M69 and equation M70 The uninduced state corresponds to equation M71 and equation M72 Thus we have two relations,
    equation M73
    and
    equation M74
    Taking the ratio of these two relations gives Θ [equivalent] K and from above, K = 7200, which would imply a value for Θ which is 7.2 times larger than what is experimentally observed (Θ = 1000). Thus the inclusion of the term Γ0.
    To determine Γ0, note that at a steady state when equation M75 from Eq. 2 we have
    equation M76
  • αB: At a steady state, from Eq. 3 we have
    equation M77
    (18)
    Kennell and Reizman (1977) reported that steady-state value of β-galactosidase is ~20 molecules per cell, which means that equation M78 Using the value of γB reported by Mandelstam (1957) for nongrowing bacteria, equation M79
  • αA: Huber et al. (1975) studied the kinetics of β-galactosidase and found Vmax = 32.6 U/mg of β-galactosidase and KM = 0.00253 M when lactose was the substrate. whereas Vmax = 49.6 U/mg of β-galactosidase and KM = 0.00120 M when allolactose is the substrate. (U is defined as μM of glucose or galactose produced per minute.) Given that the molecular mass of β-galactosidase is 540,000 Da, and 1 Da = 1.66 × 10−21 mg, 1 mol of β-galactosidase is equivalent to 6.02 × 1023 × 5.4 × 105 × 1.66 × 10−21 = 5.39 × 108 mg of β-galactosidase, so 1 mg of β-galactosidase is equivalent to 1.85 × 10−3 μmol. Therefore,
    equation M80
  • βA: From the data of Huber et al. (1975), we have equation M81 whereas Martínez-Bilbao et al. (1991) gives equation M82 We have taken the average of equation M83
  • KL: The volume of one E. coli is ~8.0 × 10−16 liter and its mass is ~1.7 × 10−12 g to yield a density of 2.1 × 103 g/liter. The parameter KL in our model corresponds to the parameter Km,Lac/ρ in Wong et al. (1997) model and the values and the units of these two parameters reported in this paper are ρ = 3.0 × 102 g of dry cell weight per liter and Km,Lac = 1.4 × 10−4 M, which gives
    equation M84
    To obtain an estimate for KL in M, we can multiply this value by the density of the cell, which gives equation M85 in agreement with the value of 1.4 ± 0.3 × 10−3 M estimated by Martínez-Bilbao et al. (1991). We have taken the latter value.
  • KA: This parameter in our model corresponds to Km,Allo/ρ in the Wong et al. (1997) model, and they took Km,Allo [similar, equals]2.8 × 10−4 M. Hence, equation M86 Using the procedure followed in the estimation of KL, the value of KA is calculated to be 1.95 mM.
  • τM: In this model, we are considering the transcription and translation of two genes, lacZ and lacY. Translation of lacZ starts shortly after transcription initiation. For the translation of lacY to begin, lacZ must be completely transcribed. Knowing that lacZ has 1022 amino acids and DNA chain elongation rate is at least 490 nucleotides per second, which is equivalent to 9800 amino acids per minute, according to Bremmer and Dennis (1996), the time for lacZ to be completely transcribed is at most
    equation M87
    This is an upper bound on τM.
  • τB: lacZ is 1022 amino acids long and the mRNA elongation rate varies between 12 and 33 amino acids per second (Monar et al., 1969; Kennell and Reizman, 1977). Talkad et al. (1976) also reported the translation rate is between 8 and 15 amino acids per second. If we take the mRNA elongation rate as 8 amino acids per second to estimate an upper bound value for τB, we obtain
    equation M88
    If the elongation rate is 33 amino acids per second, then equation M89 Sorensen et al. (1989) estimated an average value for τB of 82 s experimentally, which is 1.37 min. We have taken the upper bound as equation M90 but used equation M91.
  • γL: We assumed equation M92 implying that the degradation rate for intracellular lactose is negligible when compared with the bacterial growth rate, as did Wong et al. (1997).
  • γP: West and Stein (1973) studied the kinetics of induction of β-galactosidase permease in E. coli and estimated the mean half life of permease ranges from 1.3 min to 1.9 min, which yields a range for 0.53–0.78 min−1 for the degradation rate of permease γP. We have taken an average of these two estimates to give equation M93
  • αL: Wright et al. (1981) studied on lactose carrier protein of E. coli and measured the active transport turnover number as 48 × 60 = 2880 min−1 in EDTA-treated cells of the strain ML308-225. We have taken the same value αL = 2880 min−1.
  • αP: Eq. 6 gives
    equation M94
    at a steady state. From Kennell and Reizman (1977), we know that equation M95 The steady-state molar ratio of β-galactosidase to permease was given as equation M96 by in Maloney and Rotman (1973). From this we estimate equation M97
    Lee and Bailey (1984) studied the growth rate effects on productivity of recombinant E. coli populations and obtained an empirical relation for the transcription rate as a function of the bacterial growth rate. From this relation, we have αP = 17.37 min−1 when μ = 2.21 × 10−2 min−1, which is the value we have estimated. We have taken an intermediate value between these two estimates: equation M98
    βL1: From Wong et al. (1997), we have βL1 = 2148 min−1. Lolkema et al. (1991) gave a range for βL1 as 840–3000 min−1. We have chosen as βL1 = 2650 min−1.
  • equation M101 From Wong et al. (1997), we have
    equation M102
    wherein
    equation M103
    from Lolkema et al. (1991), Huber et al. (1980), Page and West (1984), and Wright et al. (1981), and ρ is as given before. Thus
    equation M104
    Multiplying by the density of the cell (2.1 × 103 gm/L) gives us
    equation M105
  • τP: lacY is 399 amino acids long and the mRNA elongation rate varies between 8 and 33 amino acids per second (Monar et al., 1969; Kennell and Reizman, 1977; and Talkad et al., (1976). If we take the mRNA elongation rate to be 8 amino acids per second to estimate an upper bound value for τP, we obtain
    equation M106
    If the elongation rate is 33 amino acids per second, then equation M107 We have taken the maximal value to be equation M108

APPENDIX C: STEADY-STATE ANALYSIS OF THE MODEL

In this section, the steady-state analysis of the system is investigated and a necessary condition derived for existence of positive steady state(s). Let equation M109 be the steady state of the system given by Eqs. 26. At a steady state, by definition, there are no temporal changes and thus the steady state(s) are defined implicitly by

equation M110
(19)

equation M111
(20)

equation M112
(21)

equation M113
(22)

equation M114
(23)

where equation M115 and equation M116 Moreover, f1, f2, g1, and g2 are all monotone increasing functions given by

equation M117
(24)

equation M118
(25)

where equation M119

For a steady state to make sense in a biological context, it is necessary that it be nonnegative. Now from Eqs. 20 and 23, we easily have

equation M120
(26)

equation M121
(27)

Furthermore, from Eq. 19, equation M122 can be written in terms of equation M123 as,

equation M124
(28)

Note that equation M125 and equation M126 are nonnegative whenever equation M127 is nonnegative, and from Eq. 28 we always have equation M128 Further, from Eq. 21 we can write equation M129 in terms of equation M130:

equation M131
(29)

where

equation M132
(30)

Condition I for a nonnegative steady state

From Eq. 29, to have a nonnegative steady-state value of equation M133,

equation M134
(31)

must be satisfied.

Now consider Eq. 22, and substitute Eqs. 26 and 27. After rearrangement, we obtain

equation M135
(32)

where

equation M136
(33)

equation M137
(34)

equation M138
(35)

equation M139
(36)

The left side of Eq. 32 is always positive for all nonnegative values of equation M140, which leads to the following second condition for a biologically sensible steady state:

Condition II for a nonnegative steady state

Since the left side of Eq. 32 is always nonnegative, the condition

equation M141
(37)

must be satisfied.

Theorem 1

(Necessary condition for existence of a positive steady state.) For the existence of a positive steady state for the model given by Eqs. 26,

equation M142
(38)

is a necessary condition for equation M143 in an interval defined by the intersection of the intervals for which Eqs. 31 and 37 are satisfied.

Proof

The left side of Eq. 32 is a monotone increasing function of equation M144 when K1 > 0, and has a minimal value of (αM/K0) + Γ0 when equation M145 Further, it reaches its maximal value of αM + Γ0 as equation M146 becomes large. Furthermore, the right side of Eq. 32 is zero when equation M147 Therefore, for the existence of a positive root, it is trivial that the right of Eq. 32 must be an increasing function of equation M148 for values of equation M149 and equation M150 satisfying Eqs. 31 and 37.

To prove that the condition equation M151 is a necessary condition for the right side of Eq. 32 to be an increasing function of equation M152 let

equation M153
(39)

so that

equation M154
(40)

equation M155 and equation M156 are Michaelis-Menten type functions and equation M157 and equation M158 are positive. Since equation M159 the condition

equation M160

is satisfied whenever

equation M161

holds.

Further, from Eq. 29 we have

equation M162
(41)

and from Condition I, equation M163 must be satisfied for nonnegative steady states. Thus the right side of Eq. 41 is always positive when equation M164 Therefore, equation M165 is an increasing function when equation M166 in an interval defined by the intersection of the intervals for which Eqs. 31 and 37 are satisfied. This completes the proof.

APPENDIX D: NUMERICAL STABILITY OF THE MODEL

A full stability analysis of the steady states of this model is impossible, since the eigenvalue equation determining local stability is a fifth order quasipolynomial containing three delays. Consequently, we have contented ourselves with a numerical examination of the stability properties of the steady states.

Briefly, the results of our numerical stimulations are as follows. When a single steady state exists, we have found that the numerical behavior is such that the model solutions always converge to that steady state at large times.

When there are three coexisting steady states as illustrated in Fig. 2, the behavior is slightly more complicated and is illustrated in Fig. 5 when Le = 3.0 × 10−2. At this value, the system has the three steady states given in Table 3. As seen from the results in Fig. 5, the numerical model solutions either converged to the lower or upper branch of the S-shaped curve (see Fig. 2) for various initial conditions. These results, as well as numerous others that are not shown, lead us to conclude that the middle branch of the S-shaped steady-state curve corresponds to a steady state that is globally unstable.

FIGURE 5

An external file that holds a picture, illustration, etc.
Object name is biophysj18465f05.jpg

Semilog plot of β-galactosidase activity versus time (min) showing bifurcation in the numerical simulation with the parameters of Table 1 for five initial conditions and equation M167 when Le = 3.0 × 10−2 mM, which is in the range of lactose concentration for the existence of three steady states. The selection of the five initial conditions is described in the text.

TABLE 3

Multiple steady states and their numerical values when Le = 3.0 × 10−2 mM

Steady statesequation M168equation M169equation M170equation M171equation M172
I4.31 × 10−32.14 × 10−61.44 × 10−61.01 × 10−12.98 × 10−5
II6.43 × 10−23.46 × 10−52.34 × 10−51.36 × 10−14.83 × 10−4
III1.42 × 10−11.54 × 10−41.04 × 10−41.39 × 10−12.16 × 10−3

For this simulation, we choose five equally distributed initial β-galactosidase levels between 0.24 × 10−4 and 0.32 × 10−4 mM and kept all other variables at their steady-state values corresponding to the values on the middle branch of the S-shaped curve in Fig. 2 when Le = 3.0 × 10−2 mM, and computed the temporal evolution of the model variables. For initial β-galactosidase values equal to or greater than 3.0 × 10−5, the simulated curves converged to 1.04 × 10−4, which is the value on the upper branch of the steady-state curve, whereas for the other initial values, the β-galactosidase values converged to 1.44 × 10−6, which is the steady-state value on the lower branch.

However, the relatively simple behavior shown in Fig. 5 is deceptive, as shown in Fig. 6. There we present numerical evidence that the attractor boundary in initial function space separating behaviors where one approaches the lower or upper locally stable steady state of Fig. 2 is not totally straightforward. The potentially rich nature of the boundary is revealed by taking initial functions that oscillate about the unstable branch of the steady-state curve. The ensuing dynamical behavior is highly reminiscent of the existence of a fractal basin boundary that has been noted in other, simpler, differential delay systems (Losson and Mackey, 1993).

FIGURE 6

An external file that holds a picture, illustration, etc.
Object name is biophysj18465f06.jpg

Semilog plot of β-galactosidase activity versus time showing effects of selection of the initial condition for t [set membership][−τ, 0] in the numerical simulation with the parameters of Table 1 and various initial values of mRNA and allolactose oscillating around the unstable steady-state values corresponding to the middle branch of Fig. 2 when equation M173 and Le = 4.0 × 10−2 mM (which is in the range of lactose concentration for coexistence of three steady states). The solid lines show the β-galactosidase activity when the initial allolactose functions are equation M174 (n = 1, 2, …10), t [set membership][−τM, 0], and the other variables are at the steady-state values on the middle branch. (Here equation M175 is the unstable steady-state value of A on the middle branch). The dotted lines depict the temporal changes in β-galactosidase activity when equation M176 (n = 1, 2, …10) for t [set membership][−(τB + τP),0]. Again all the other variables are at the steady-state values when Le = 4.0 × 10−2 mM and equation M177 is also the steady-state value of A on the middle branch. The steady-state values are equation M178, equation M179, equation M180, equation M181, and equation M182, when Le = 4.0 × 10−2 mM.

Notes

Necmettin Yildirim's permanent address is Atatürk Üniversitesi, Bilgisayar Bilimleri Uygulama ve Araştırma Merkezi, 25240 Erzurum, Turkey.

References

  • Arkin, A., J. Ross, and H. H. McAdams. 1998. Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-infected Escherichia coli cells. Genetics. 149:1633–1648. [PMC free article] [PubMed]
  • Baneyx, F. 1999. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10:411–421. [PubMed]
  • Beckwith, J. 1987a. The lactose operon. In Escherichia coli and Salmonella: Cellular and Molecular Biology, Vol. 2. F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, and H. E. Umbarger, editors. American Society for Microbiology, Washington, DC. 1444–1452.
  • Beckwith, J. 1987b. The operon: An historical account. In Escherichia coli and Salmonella: Cellular and Molecular Biology, Vol. 2. F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, and H. E. Umbarger, editors. American Society for Microbiology, Washington, DC. 1439–1443.
  • Bliss, R. D., R. P. Painter, and A. G. Marr. 1982. Role of feedback inhibition in stabilizing the classical operon. J. Theor. Biol. 97:177–193. [PubMed]
  • Blundell, M., and D. Kennell. 1974. Evidence for endonucleolytic attack in decay of lac messenger RNA in Escherichia coli. J. Mol. Biol. 83:143–161. [PubMed]
  • Bremmer, H., and P. P. Dennis. 1996. Modulation of chemical composition and other parameters of the cell by growth rate. In Escherichia coli and Salmonella: Cellular and Molecular Biology, Vol. 2. F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger, editors. American Society for Microbiology, Washington, DC. 1553–1569.
  • Cohn, M., and K. Horibata. 1959. Inhibition by glucose of the induced synthesis of the β-galactosidase-enzyme system of Escherichia coli: analysis of maintenance. J. Bacteriol. 78:613–623. [PMC free article] [PubMed]
  • Gillespie, D. T. 1977. Exact stochastic simulation of coupled chemical reactions. J. Phys. Chem. 81:2340–2361.
  • Gillespie, D. T. 1992. A rigorous derivation of the chemical master equation. Physica A. 188:404–425.
  • Goodwin, B. 1965. Oscillatory behaviour in enzymatic control process. Adv. Enz. Regul. 3:425–438. [PubMed]
  • Goodwin, B. C. 1969. Control dynamics of β-galactosidase in relation to the bacterial cell cycle. Eur. J. Biochem. 10:515–522. [PubMed]
  • Griffith, J. S. 1968a. Mathematics of cellular control processes. I. Negative feedback to one gene. J. Theor. Biol. 20:202–208. [PubMed]
  • Griffith, J. S. 1968b. Mathematics of cellular control processes. II. Positive feedback to one gene. J. Theor. Biol. 20:209–216. [PubMed]
  • Huber, R. E., M. Gupta, and S. Khare. 1994. The active site and mechanism of the β-galactosidase from Escherichia coli. Int. J. Biochem. 26:309–318. [PubMed]
  • Huber, R. E., G. Kurz, and K. Wallenfels. 1976. A quantitation of the factors which affect the hydrolase and transgalactosylase activities of β-galactosidase (E. coli) on lactose. Biochemistry. 15:1994–2001. [PubMed]
  • Huber, R., R. Pisko-Dubienski, and K. Hurlburt. 1980. Immediate stoichiometric appearance of β-galactosidase products in the medium of Escherichia coli cells incubated with lactose. Biochem. Biophys. Res. Commun. 96:656–661. [PubMed]
  • Huber, R. E., W. Wallenfels, and G. Kurz. 1975. The action of β-galactosidase Escherichia coli on allolactose. Can. J. Biochem. 53:1035–1039. [PubMed]
  • Jacob, F., D. Perrin, C. Sanchez, and J. Monod. 1960. L'operon: groupe de gène à expression par un operatour. C. R. Acad. Sci. 250:1727–1729. [PubMed]
  • Ji-Fa, J. 1994. A Liapunov function for four dimensional positive feedback systems. Quar. Appl. Math. 52:601–614.
  • Kennell, D., and H. Reizman. 1977. Transcription and translation initiation frequencies of the Escherichia coli lac operon. J. Mol. Biol. 114:1–21. [PubMed]
  • Kepler, T. B., and T. C. Elston. 2001. Stochasticity in transcriptional regulation: origins, consequences, and mathematical representations. Biophys. J. 81:3116–3136. [PMC free article] [PubMed]
  • Knorre, W. A. 1968. Oscillation of the rate of synthesis of β-galactosidase in Escherichia coli ML 30 and ML 308. Biochem. Biophys. Res. Commun. 31:812–817. [PubMed]
  • Lee, S. B., and J. E. Bailey. 1984. Analysis of growth rate effects on productivity of recombinant Escherichia coli populations using molecular mechanism models. Biotechnol. Bioeng. 67:805–812. [PubMed]
  • Leive, L., and V. Kollin. 1967. Synthesis, utilization and degradation of lactose operon mRNA in Escherichia coli. J. Mol. Biol. 24:247–259. [PubMed]
  • Lolkema, J., N. Carrasco, and H. Kaback. 1991. Kinetic analysis of lactose exchange in proteoliposomes reconstituted with purified lac permease. Biochemistry. 30:1284–1290. [PubMed]
  • Losson, L., and M. C. Mackey. 1993. Solution multistability in first order nonlinear differential delay equations. Chaos. 3:167–176. [PubMed]
  • Maffahy, J. M., and E. Savev. 1999. Stability analysis for a mathematical model of the lac operon. Quar. Appl. Math. 57:37–53.
  • Maloney, P. C., and S. M. Rotman. 1973. Distribution of suboptimally induced β-D-galactosidase in Escherichia coli. J. Mol. Biol. 73:77–91. [PubMed]
  • Mandelstam, J. 1957. Turnover of protein in starved bacteria and its relationship to the induced synthesis of enzyme. Nature. 179:1179–1181. [PubMed]
  • Martínez-Bilbao, M., R. E. Holdswards, L. A. Edwards, and R. E. Huber. 1991. A highly reactive β-galactosidase Escherichia coli resulting from a substitution of an aspartic acid for Gly-794. J. Biol. Chem. 266:4979–4986. [PubMed]
  • McAdams, H. H., and L. Shapiro. 1995. Circuit simulation of genetic networks. Science. 269:650–656. [PubMed]
  • Monar, H., D. Goodman, and G. S. Stnet. 1969. RNA chain growth rates in Escherichia coli. J. Mol. Biol. 39:1–29. [PubMed]
  • Novick, A., and M. Wiener. 1957. Enzyme induction as an all-or-none phenomenon. Proc. Natl. Acad. Sci. USA. 43:553–566. [PMC free article] [PubMed]
  • Osumi, T., and M. H. Saier. 1982. Regulation of lactose permease activity by the phosphoenolpyruvate: sugar phosphotransferase system: Evidence for direct binding of the glucose specific enzyme III to the lactose permease. Proc. Natl. Acad. Sci. USA. 79:1457–1461. [PMC free article] [PubMed]
  • Page, M. G. P., and I. C. West. 1984. The transient kinetics of uptake of galactosides into Escherichia coli. Biochem. J. 223:723–731. [PMC free article] [PubMed]
  • Pestka, S., B. L. Daugherty, V. Jung, K. Hotta, and R. K. Pestka. 1984. Anti-mRNA: specific inhibition of translation of single mRNA molecules. Proc. Natl. Acad. Sci. USA. 81:7525–7528. [PMC free article] [PubMed]
  • Postma, P. W., J. W. Lengeler, and G. R. Jacobson. 1996. Phosphoenolpyruvate-carbohydrate phosphotransferase systems. In Escherichia coli and Salmonella: Cellular and Molecular Biology, Vol. 1. F. C. Neidhart, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger, editors. American Society for Microbiology, Washington, DC. 1149–1174.
  • Rotman, R., and S. Spiegelman. 1954. On the origin of the carbon in induced synthesis of β-galactosidase. J. Bacteriol. 68:419–429. [PMC free article] [PubMed]
  • Saier Jr., M. H. 1976. Inducer exclusion and regulation of the melibose, maltose, glycerol, and lactose transport systems by the phosphoenolpyruvate: sugar phosphotransferase system. J. Biol. Chem. 251:6606–6615. [PubMed]
  • Saier, M. H., T. M. Ramseier, and J. Reizer. 1996. Regulation of carbon utilization. In Escherichia coli and Salmonella: Cellular and Molecular Biology, Vol. 1. F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger, editors. American Society for Microbiology, Washington, DC. 1325–1343.
  • Santillán, M., and M. C. Mackey. 2001a. Dynamic behaviour in mathematical models of the tryptophan operon. Chaos. 11:261–268. [PubMed]
  • Santillán, M., and M. C. Mackey. 2001b. Dynamic regulation of the tryptophan operon: a modeling study and comparison with experimental data. Proc. Natl. Acad. Sci. USA. 98:1364–1369. [PMC free article] [PubMed]
  • Savageau, M. A. 1999. Design of gene circuitry by natural selection: analysis of the lactose catabolic system in Escherichia coli. Biochem. Soc. Trans. 27:264–270. [PubMed]
  • Selgrade, J. F. 1979. Mathematical analysis of a cellular control process with positive feedback. SIAM J. Appl. Math. 36:219–229.
  • Selgrade, J. F. 1982. A Hopf bifurcation in single loop positive feedback systems. Quar. Appl. Math. 40:347–351.
  • Sen, A. K., and W. Liu. 1989. Dynamic analysis of genetic control and regulation of amino acid synthesis: the tryptophan operon in Escherichia coli. Biotechnol. Bioeng. 35:185–194. [PubMed]
  • Shampine, L. F., and S. Thompson. 2000. Solving DDEs with MATLAB. www.radford.edu/~thompson/webddes/.
  • Sinha, S. 1988. Theoretical study of tryptophan operon: application in microbial technology. Biotechnol. Bioeng. 31:117–124. [PubMed]
  • Sorensen, M. A., C. G. Kurland, and S. Pedersen. 1989. Codon usage determines translation rate in Escherichia coli. J. Mol. Biol. 207:365–377. [PubMed]
  • Swain, P. S., M. Elowitz, and E. Siggia. 2002. Intrinsic and extrinsic contributions to stochasticity in gene expression. Proc. Natl. Acad. Sci. USA. 99:12795–12800. [PMC free article] [PubMed]
  • Talkad, V., E. Schneider, and D. Kennell. 1976. Evidence for variable rates of ribosome movement in Escherichia coli. J. Mol. Biol. 104:299–303. [PubMed]
  • Tyson, J. J., and M. C. Mackey. 2001. Molecular, metabolic and genetic control: An introduction. Chaos. 11:81–83. [PubMed]
  • Tyson, J. J., and H. G. Othmer. 1978. The dynamics of feedback control circuits in biochemical pathways. In Progress in Biophysics, Vol. 5. R. Rosen, editor. Academic Press, New York. 1–62.
  • Watson, J. D. 1977. Molecular Biology of the Gene, 3rd ed. W. A. Benjamin, New York.
  • West, I. C., and W. D. Stein. 1973. The kinetics of induction of β-galactoside permease. Biochim. Biophys. Acta. 308:161–167. [PubMed]
  • Wong, P., S. Gladney, and J. D. Keasling. 1997. Mathematical model of the lac operon: inducer exclusion, catabolite repression, and diauxic growth on glucose and lactose. Biotechnol. Prog. 13:132–143. [PubMed]
  • Wright, J. K., I. Riede, and P. Overath. 1981. Lactose carrier protein of Escherichia coli: interaction with galactosides and protons. Biochemistry. 20:6404–6415. [PubMed]
  • Xiu, Z. L., A. P. Zeng, and W. D. Deckwer. 1997. Model analysis concerning the effects of growth rate and intracellular tryptophan level on the stability and dynamics of tryptophan biosynthesis in bacteria. J. Biotech. 58:125–140.
  • Yagil, G., and E. Yagil. 1971. On the relation between effector concentration and the rate of induced enzyme synthesis. Biophys. J. 11:11–27. [PMC free article] [PubMed]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...