Escherichia coli strain BW25113 was used as a wild-type strain 28 . The plasmids used in this study are listed in Section S6 of the Additional file 1. The gfp- and lux-containing plasmids (pZEgfp and pSBluc) are derivatives of plasmids pZE1RM 17 and pSB377 29 , respectively, with a modified sequence of the multiple cloning site. The sequence between the end of the multiple cloning site (EcoR I) and the start codon (ATG) of luxC and gfp is: gaattcCCCG GGTAATTCAG GCCTGGAGGA TACGTatg and gaattcCCCG GGTAATTCAT TAAAGAGGAG AAAGGTACCG Catg, respectively. We have amplified the promoter region of fis by PCR from genomic DNA of E. coli, with oligonucleotides Fis1 and Fis2 (Fis1: ATCGCTCGAG GTGACGCGG, Fis2: TACG GAATTC GAGTTAAGAA ATGACCATAC TGTGA). Oligonucleotide Fis1 contains an XhoI restriction site, and oligonucleotide Fis2 an EcoRI restriction site, which allows cloning of the amplified DNA between these two sites on plasmids pSBluc and pZEgfp. The resulting plasmids are called pSB-fislux and pZE-fisgfp, respectively. Plasmids were verified by sequencing. They possess a colE1 origin of replication, are present at about twenty copies per cell, and do not affect bacterial growth (data not shown).

Glycerol stocks, stored at -80°C, of strains BW25113 28 carrying (or not) a plasmid-encoded reporter gene were grown overnight (≈ 15 h) at 37°C, with shaking at 200 rpm, in M9 minimal medium 30 supplemented with 0.3% glucose. For plasmid-carrying strains, the growth medium was supplemented with 100 μg·ml^-1ampicillin. The overnight culture was diluted 20-fold into the same, fresh medium. After 4 hours of growth the culture medium was changed by centrifugation and resuspension in M9 without glucose. The volume was adjusted in order to obtain an OD₆₀₀ of 0.2. The bacteria were incubated without nutrients at 37°C for an additional 15 hours. Abruptly limiting the glucose availability in this fashion assures that the bacteria are in a defined physiological state at the beginning of the experiment. For the upshift experiments, 50 μl of these growth-arrested cultures were added to 100 μl of prewarmed M9 medium, containing glucose at a final concentration of 0.15%, and grown in a microtiter plate (≈ 12 h) at 37°C. The microplates were agitated at regular intervals during growth in the Fusion microplate reader (Perkin Elmer). During a typical experimental run we acquire about 100 readings each of absorbance, luminescence, and fluorescence. Fluorescence excitation was at 485 nm and emission was monitored at 520 nm. Absorbance measurements used a 600 nm filter.

The absorbance, luminescence, and fluorescence data were fitted with regression splines, using the Spline toolbox of Matlab (Mathworks). In the absence of a specific parametric model of the data, regression splines provide a flexible, non-parametric modeling framework that allows estimation of the underlying trend in the absorbance and light intensity. In particular, we have used cubic B-splines 31 in combination with the generalized cross-validation (GCV) criterion for determining the number and the placement of the knots 32 . The optimal spline fit is the one minimizing GCV, that is, minimizing the residual sum of squares subject to a penalty term increasing with the number of knots (Section S2 of the Additional file 1). In order to find an estimate of the minimizer of GCV, and therefore of the 'best' choice of knots, we have followed a simple, stepwise knot selection schema 33 . The actual computation of the regression spline from a knot sequence is carried out by the Matlab function spap2.

A major advantage of the use of splines is that they greatly facilitate the computation of derived quantities from the primary data. Since splines are piecewise-polynomial functions, standard arithmetic operations, as well as differentiation and integration operations, can be carried out analytically 31 . This is more efficient and leads to more precise results than the use of numerical approximations. The latter cannot be completely avoided though, as some of the expressions that need to be evaluated for the computation of the host protein synthesis rate and host protein concentration involve functions that are not splines (Section S4 of the Additional file 1). In this case the integrals are computed by means of the Matlab function quad.

For each of the derived quantities, we computed 95% confidence bands using a standard bootstrap method. In particular, we have followed the residual resampling scheme 34 , which constructs bootstrap data sets by repeatedly resampling the residuals of the optimal spline fit (Section S5 of the Additional file 1). For each of the 200 bootstrap data sets generated, we computed the synthesis rates and concentrations of the host and reporter proteins. From this empirically determined distribution, we obtained an estimate of the 95% confidence interval for the predicted values at evenly-spaced time-points, using so-called bootstrap percentiles 34 . The confidence bands shown in the figures in the text have been obtained by connecting the estimates of the point-wise confidence intervals.

For each type of measurement, an appropriate procedure for background correction has been developed. The absorbance background is detected by performing measurements on wells without bacteria, containing growth medium only. One would expect these background levels to be constant over time, which is confirmed by the actual measurements (data not shown). Denoting by A _u the uncorrected absorbance and by A _b the background absorbance, we define the corrected absorbance A as:

The fluorescence background is determined by measuring the fluorescence of a strain carrying the promoterless vector pZEgfp. The background fluorescence is not constant, but rather varies with the population size due to the autofluorescence of bacterial cells. In this case, direct subtraction of the background readings from the uncorrected fluorescence intensity at each time-point t is not appropriate, as the size of the bacterial population generating the uncorrected signal is generally different from the size of the population generating the background signal.

We therefore first compute the average fluorescence intensity per cell for the uncorrected signal and the background signal. We denote by B(t) the absorbance of the strain carrying the promoterless vector, A(t) the absorbance of the strain with the functional reporter system, I _u (t) the uncorrected fluorescence intensity, and I _b (t) the background fluorescence intensity. The average fluorescence intensity per cell for the uncorrected and the background signal are then given by I _u (t)/A(t) and I _b (t)/B(t), respectively. We subtract the latter from the former to obtain the corrected average fluorescence intensity per cell, which we then multiply by the population size, as estimated by the absorbance, to obtain the corrected fluorescence intensity I(t):

The background correction for the luminescent measurements could, in principle, be carried out in the same way as the one for the fluorescence measurements. However, as the luminescence background is quite low in practice, simple background subtraction is usually sufficient:

Equal quantities of protein were separated on 18% SDS-PAGE acrylamide gels and transferred onto nitrocellulose filters (Amersham Pharmacia). Filters were incubated with anti-Fis antibodies. Immunoblots were developed by using horseradish peroxidase-conjugated goat anti-rabbit antibody, followed by enhanced chemiluminescence (Amersham). The image of the blot acquired with a highly sensitive CCD camera and averaged for two minutes was quantified using the ImageJ software 35 .

Total RNA was extracted from cells using the hot phenol procedure 36 , or the Trizol procedure (Invitrogen). RNA samples were stored in DEPC water at -80°C until further use. The total RNA was loaded on a polyacrylamide (6% TBE-Urea, Invitrogen) or agarose gel (1%). After migration, the RNA was transferred to a Hybond-N membrane (Amersham Biosciences) and crosslinked with UV (1200 J). The membrane was prehybridized in Ultrahyb (Ambion) for 1 h at 42°C, followed by addition of radiolabeled oligonucleotide probe and hybridization overnight at 42°C. Membranes were washed twice with 2× SSC/0.1% SDS at room temperature followed by one wash with 2× SSC/0.1% SDS at 42°C for 2 min. Oligonucleotide probes were labelled by polynucleotide kinase according to manufacturer protocols (Fermentas) using [32P] ATP (6000 Ci/mmole; Perkin-Elmer). Probes were purified over mini quick spin columns (Roche) prior to use. Membranes were exposed on a phosphor screen, the screen revealed on a FLA-8000 (Fujifilm), and the image of the film quantified using ImageJ. The sequences of the probes used are listed in Section S6 of the Additional file 1.

To determine the degradation constant γ _q of the GFP reporter of fis, we grew a bacterial culture under the experimental conditions described above to exponential phase and added chloramphenicol to 100 μg/ml. The fluorescence data obtained after growth arrest were fitted by an exponential to yield the degradation constant. A similar procedure was followed for the luciferase reporter. A value for the degradation constant γ _p of Fis was obtained by growing cells to the same growth stage and treating them with spectinomycine (100 μg/ml). 1 ml samples were removed every hour during 5 h and treated as described in the section on Western blot analysis. An exponential fit gave the value of γ _p .

To determine the degradation constant γ _n of the reporter mRNA, strains BW25113 containing either plasmid pZACR105 (gfp) or pZACR101 (lux) were used (Section S6 of the Additional file 1). In these plasmids, the gfp gene or the lux operon are cloned downstream of the PLtetO-1 promoter that is controlled by the TetR repressor ( 37 ; Ranquet et al., in preparation). Derepression of the promoter is achieved by adding anhydrotetracycline (aTc). The strains were grown at 37°C to mid-log phase in LB medium, and aTc (500 ng/ml final) was added for 30 min to induce transcription of gfp or lux. Rifampicine (150 g/ml final) was then added to stop transcription and samples were taken every minute during 10 min. mRNA was isolated and detected as described in the section on Northern blot analysis. The degradation constant γ _m of fis messages was determined by growing the strain BW25113 in LB to mid exponential phase, where Fis is the most abundant. Rifampicine was added and the mRNA was extracted as described above.

In order to measure the expression of the gene fis in E. coli, we have constructed two reporter plasmids with identical backbones, including the antibiotic resistance gene and the origin of replication. The first contains the gfpmut3*-asv reporter gene, a variant of the gene coding for the Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria 38 . The second plasmid carries the luxCDABE operon from Xenorhabdus luminescens, encoding the enzymes of a light-producing pathway in this bacterium 39 . Because fis has its expression controlled at the transcriptional level 26 27 40 , we prepared transcriptional fusions in which the promoter region of fis is fused to the gfp gene or the lux operon.

Figure 1 summarizes the relationship between the expression of the host gene and the reporters.

Transcription of the gene fis gives rise to fis mRNA, which is subsequently translated into Fis protein. The synthesis of mRNA and protein is counterbalanced by growth dilution and degradation of the gene products. Together these processes determine the net accumulation of mRNA and protein in the cell. The expression of the gfp reporter gene follows roughly the same stages, with an important difference though. Fluorescent activity of GFP in response to light excitation depends on post-translational modifications, notably the folding of the protein to an appropriate conformation, including the autocatalytic formation of the chromophore 41 . This maturation process gives rise to an additional reaction step from GFP to active GFP (Figure 1). In the luminescent reporter gene system, light is not emitted in response to an excitatory signal, but as a by-product of an oxidation reaction. This reaction is catalyzed by the heterodimeric enzyme luciferase and requires a substrate, a long-chain aldehyde, which is synthesized by enzymes co-expressed with luciferase from the lux operon 39 .

The expression of the host gene is modeled in the classical way 42 43 44 45 , by means of differential equations describing the evolution of the cellular mRNA concentration m(t) and protein concentration p(t) as a function of time t:

The mRNA synthesis rate in (4) is given by a maximum transcription rate κ _m multiplied by the time-varying promoter activity f(t), a nonlinear function of time normalized to a value between 0 and 1. The mRNA synthesis rate is also called promoter activity. The mRNA decay rate is the sum of the growth dilution and degradation rates, accounted for by the term (μ(t) + γ _m ) m(t). Here, μ(t) denotes the growth rate as a function of time and γ _m the degradation constant for fis mRNA. The protein synthesis rate in (5) is given by κ _p m(t), where κ _p is the translation rate constant. (The term promoter activity is sometimes also used for the protein synthesis rate, when models are used that lump together the transcription and translation steps (e.g., 13 ).) The protein decay rate is again composed of a growth-dilution and degradation contribution, with γ _p the degradation constant of protein Fis. The variables and constants used in (4)-(5) and below are summarized in Table 1.

The same model is used for the transcription and translation steps of the gfp reporter gene, with the understanding that new variables n(t) and q(t) are introduced for the mRNA and protein concentrations of the reporter, respectively:

In comparison with the model of fis expression, γ _n and γ _q denote the degradation constants for mRNA and protein, respectively. An additional differential equation accounts for the maturation of GFP:

Here, r(t) stands for the concentration of active GFP, as compared to the total GFP concentration q(t), and κ _r is the rate constant for the first-order folding reaction. κ _r (q(t) - r(t)) thus represents the folding rate and we call ln 2/κ _r the folding time of GFP. The model (6)-(8) can, with some variations, be found in other work 20 21 22 23 46 .

Notice that a number of implicit assumptions underlie the above models of host and reporter gene expression. First, the promoter activity κ _m f (t) characterizes the transcription of both the host and reporter genes, which is a direct consequence of the use of transcriptional fusions to measure fis expression. Second, we assume that the translation constant is the same for host and reporter gene expression. In the case of Fis this is justified by the fact that translation is not regulated 26 27 40 . In situations where this assumption is not valid, and post-transcriptional regulation occurs, translational fusions to the gfp reporter gene should be used. Third, the degradation constants of active and inactive GFP are assumed to be identical, which is reasonable in the absence of evidence to the contrary. Fourth, delays in transcription and translation are small with respect to the folding time and can safely be ignored here. Fifth, the growth characteristics of the wild-type and reporter strains are the same, an assumption that we have validated by comparing the growth rates of the two strains (data not shown).

The model of reporter gene expression was specifically developed for the case of GFP, but it can be adapted in a straightforward manner to the luminescent reporter gene system. Assuming that the substrates of the light-producing reaction are not rate limiting, the dynamics of the system is conveniently described by the temporal evolution of the luciferase concentration. We have verified this assumption for the aldehyde substrate and molecular oxygen, O₂. The third substrate of the luciferase reaction, FMNH₂, is directly related to the reducting power of the cell and can become rate-limiting in particular physiological situations, such as severe depletion of carbon sources. We observe a mild manifestation of this effect at the entry into stationary phase (see Discussion). However, during exponential growth, none of these substrates is rate-limiting. Equations (6) and (7) thus remain valid, where n(t) and q(t) now represent the lux mRNA and luciferase concentrations, respectively. Since the activity of luciferase does not require the analog of a folding reaction, we simply replace (8) by the equation:

That is, the total luciferase concentration equals the active luciferase concentration (see 47 for a more detailed model of the luminescent reporter system).

We have grown E. coli strains carrying the reporter plasmids in parallel on a microplate, in M9 minimal medium, and at a constant temperature of 37°C. The basic experiment consisted in adding glucose to a growth-arrested culture, following the protocol described in the Methods section, and repeatedly measuring the absorbance at 600 nm, as well as fluorescence and luminescence intensities. The time-series data were fitted to cubic regression splines using a minimization criterion that balances goodness of fit and parsimony (Methods and Additional file 1). The resulting spline fits of the primary data were corrected for background levels of absorbance, fluorescence, and luminescence. The background measurements were carried out on wells containing growth medium without bacteria (absorbance background), and on wells with strains carrying a reporter plasmid lacking a promoter upstream of the reporter gene (fluorescence and luminescence background) (see Methods).

The results obtained with the GFP and luciferase reporter plasmids of Fis are shown in Figure 2. At time zero, the growth-arrested bacterial cultures were diluted into fresh culture medium. The bacteria progressively reach the maximum growth rate in exponential phase, as can be seen with the logarithmic scale in the plots. The increase in fluorescence and luminescence levels accelerates after about one hour, but slows down later in exponential phase. When the culture enters stationary phase, the fluorescence and luminescence levels decrease due to the down-regulation of fis.

Panels b and d of Figure 2 show 95% confidence bands for the corrected absorbance and light intensity which were computed using the bootstrap method described in the Methods section. The confidence bands are tight, reflecting the high precision of the measurements, and the curves are reproducible (see Section S3 in the Additional file 1).

A central question for the interpretation of the primary data is how the latter can be related to the model variables. Let I(t) denote the corrected fluorescence or luminescence intensity over time, in relative fluorescence units (RFU) or relative luminescence units (RLU), respectively. Similarly, the dimensionless absorbance is denoted by A(t). The absorbance is proportional to the number of cells in a bacterial population. We have verified this assumption by counting the colony-forming units in parallel with the absorbance measurements (Section S1 in the Additional file 1). As a consequence, the ratio I(t)/A(t) represents the quantity of fluorescence or luminescence per cell as a function of time (e.g., 13 21 ). The latter ratio can be related to the concentration of (active) reporter protein by making the reasonable assumption that the corrected fluorescence and luminescence intensities are proportional to the number of (active) GFP and luciferase molecules in the cells, respectively. We thus obtain:

Since we do not know the proportionality constant in (10), we express concentrations in units RFU and RLU of the ratio I(t)/A(t). Notice that this provides a relative quantification of concentrations, as is usual in this kind of experiments. For most purposes, however, the relative concentrations are informative and robust measures of the dynamics of the system, for instance when we are interested in fold changes over the time-course of the experiment (see Discussion below). When this does not lead to ambiguities, we simply speak of concentrations instead of relative concentrations when we refer to variables with units RLU and RFU.

Figure 3a-b shows how the reporter concentration, computed by means of (10), varies over time during exponential phase and after entry into stationary phase. The 95% confidence bands are obviously larger than for the light intensity and absorbance measurements in Figure 2, but they remain quite reasonable. Greater uncertainty at the beginning of the experiment is due to the larger relative errors when measuring small absorbance values. The expression profiles of GFP and luciferase are highly consistent in the sense that both reporter concentrations reach a peak around 150 min, and fall back to their initial value in stationary phase. Moreover, the fold change between the maximal concentration and the concentration reached at the entry into stationary phase is about 5 in both cases.

How is the protein synthesis rate determined from the primary data? Following (7), the translation rate is proportional to the mRNA concentration n(t) of the reporter gene with proportionality constant κ _p :

where the growth rate μ(t) can be computed from the absorbance profile in Figure 2 by means of the classical formula:

The degradation constant γ _q in (11) was measured as described in the Methods section. Its value is almost the same for the two reporters: 0.012 ± 0.001 min^-1 for GFP and 0.011 ± 0.001 min^-1 for luciferase, corresponding to a half-life of about 1 h (remember that the half-life equals ln 2/γ _q .) In the case of luciferase we have q(t) = r(t), so that the total reporter concentration and its derivative can be directly determined from the primary data by means of (10). The total GFP concentration is not generally equal to the active GFP concentration, as explained above. However, for the time being, we will assume this equality to hold for GFP as well, before considering appropriate corrections at a later stage.

In Figure 4 the protein synthesis rate computed from (11) is shown for the two reporter systems. The peak occurs two generation times after the glucose upshift, 30 min before the reporter concentration reaches its maximum. This is consistent with the fact that the protein synthesis rate is proportional to the mRNA concentration, whose peak should precede that of the protein concentration. However, contrary to what has been measured previously 25 26 48 , Fis synthesis never stops completely during exponential growth of the bacterial culture. These results agree with recent microarray data showing that Fis actively regulates numerous genes in all growth phases 24 , which is only possible if Fis is present in the cell. At first sight, one might suspect the variations around 500-600 min, at the entry into stationary phase, to be due to over-fitting. Closer inspection of the data, however, in particular when comparing the raw fluorescence and luminescence data of the experiment reported in Figure 2 with the data of its replicate in Section S4 of the Additional file 1, suggests that this is probably not the case. The rapid variations in the fluorescence and luminescence levels are small, but reproducible. In the case of the luminescence reporters, where they are most pronounced, we believe them to be partly due to metabolic changes occurring during glucose depletion, such as fluctuations in reducing power, which affect the activity of the light-producing reactions (see 39 and above).

The above analysis shows that, using (10)-(12), we are able to reconstruct the reporter concentration and the reporter synthesis rate (proportional to the mRNA concentration) from the primary data. The major question raised by this analysis is whether the reconstructed quantities for the reporter system reliably represent the corresponding quantities of the host system, that is, whether n(t) = m(t) and q(t) = p(t). As discussed above, this is a priori unlikely. Remember that in the case of GFP, we have neglected the maturation step, while the half-lives of the host and reporter mRNAs and proteins are generally different as well. On the other hand, if the expression profiles of the reporter genes turned out to be good approximations of those of the host gene, this would enormously simplify the analysis and interpretation of the data. We have therefore verified to which extent the reporter concentration and synthesis rate profiles computed from the reporter gene data deviate from direct measurements of the abundance of Fis protein and fis mRNA.

We have measured the accumulation of Fis during growth on glucose by Western blots (see the Methods section for details). Figure 5a-c shows the projection of the Western blot measurements on the GFP and luciferase concentration profiles, both normalized with respect to the peak in mid-exponential phase. The profiles inferred from the reporter gene data using our models are in good agreement with the Western blot measurements. They reproduce the peak in mid-exponential phase, although the latter seems to slightly displaced to an earlier time-point (150 min vs 210 min). Notice, however, that the error bars of the Western blot measurements overlap with the confidence interval of the reporter gene profiles so that we cannot conclude with certainty that a discrepancy has occurred. The only significant deviation between the reporter gene and Western blot measurements occurs towards the end of exponential phase, where the curve computed from the reporter data clearly underestimates the Western blot quantification.

In a similar way, the synthesis rate of the GFP and luciferase reporters has been compared with Northern blot measurements at various stages of growth. Figure 5d-e shows the superposition of the Northern blot values and the synthesis rate profiles computed from the reporter gene data. The quantities have been normalized with respect to the value of the peak in exponential phase, as above. Following the definition of the reporter synthesis rate in (11), the normalized synthesis rate equals the normalized mRNA concentration. Again, there is a good overall correspondence between the profiles obtained from the reporter gene data and the direct measurements. Some significant deviations occur though, especially at the end of exponential phase (GFP data) and in mid-exponential phase (luciferase data).

We conclude from the agreement with direct measurements of Fis protein and fis mRNA that reporter genes are a reliable tool for tracking the shape of the expression profile of the host gene. It would be interesting to know if the local deviations that we also observe are due to the systematic biases identified above. In order to answer this question, we have developed computational procedures for correcting the profiles obtained from the reporter gene data for differences in half-life and for non-negligible folding times.

In general, the half-lives of protein and mRNA will not be the same for Fis and its reporters, that is, γ _m ≠ γ _n and γ _p ≠ γ _q . This difference in half-life will cause the mRNA concentrations computed from the reporter data to deviate from the actual concentrations of fis mRNA. For example, the inferred concentration will be underestimated if γ _n /γ _m > 1, that is, if the lux or gfp message half-life is shorter than that of fis. Through the dependence of the protein synthesis rate on the mRNA concentration, this also affects the computed protein concentrations. The latter effect is modulated by possible differences in half-life of the host and reporter proteins. In particular, if γ _q /γ _p > 1, the error in the predicted mRNA concentration will be accentuated, whereas in the case of γ _q /γ _p < 1 it will be attenuated.

In order to quantify these systematic biases in our case, we experimentally determined the degradation constants of mRNA and protein of both Fis and its reporters, as described in the Methods section. The GFP and luciferase half-lives were measured to be about 1 h. For the degradation constant of Fis we found the value γ _p = 0.0065 ± 0.0020 min^-1, corresponding to a half-life of almost 2 h, twice as long as the half-life of the reporter protein. The difference is significant, as the 95% confidence intervals are disjoint: [0.89, 1.1] h and [0.96, 1.2] h, for GFP and luciferase, respectively, and [1.4, 2.6] h for Fis. The degradation constants γ _n of gfp and lux mRNA were determined to be 0.30 ± 0.13 min^-1 and 0.33 ± 0.15 min^-1, respectively, yielding half-lives of about 2 min. This is almost twice as long as the half-life for fis mRNA, equal to 1.23 min. Notice that the measurements are relatively imprecise, so that the 95% confidence intervals of the host and reporter mRNA half-lives are overlapping ([1.6, 4.1] min and [1.4, 4.1] min for gfp and lux mRNA, respectively, and [0.88, 2.1] min for fis mRNA). The measured kinetic constants and the errors on the measurements are summarized in Table 2.

The measurements of the kinetic constants allow the correction of systematic errors, using the models introduced above. As shown in Section S4 of the the Additional file 1, the Fis synthesis rate κ _p m(t) can be numerically computed from the reporter synthesis rate κ _p n(t), defined in (11), when the values of γ _m and γ _n are known. The only additional assumption needed is that, at the beginning of the experiment, the mRNA concentrations have attained their steady-state value, that is:

This assumption is valid since in our experimental conditions the bacteria have been in stationary phase for more than 12 h before dilution into fresh growth medium.

The results of the correction of the systematic error in the reporter synthesis rate, and thus in the reporter mRNA concentration, are shown in Figure 6a-b. For the measured values of the ratio γ _n /γ _m , which equal 0.54 for gfp mRNA and 0.59 for lux mRNA, the difference in the normalized mRNA concentration profile is seen to be negligible, falling within the confidence band of the original predictions (corresponding to the case γ _n /γ _m = 1). This means that the local discrepancies between the Northern blot measurements in Figure 5 are not due to a difference between the half-lives of fis mRNA and that of gfp and lux mRNA.

This even holds for large half-life differences. As shown in Figure 6c-d, for values of γ _n /γ _m varying between 0.25 and 4, the predicted mRNA profile remains the same. Even when γ _n /γ _m is varied by 100-fold (see Additional file 1), the differences are quite moderate and the overall shape remains largely insensitive to this parameter. We observe that such large differences in half-life do not frequently occur in bacteria 49 , contrary to what has been observed for yeast 50 .

In a similar way, the protein concentration profile can be corrected for systematic errors due to differences in the degradation constants. The Fis concentration p(t) can be computed from the GFP or luciferase concentration q(t) when in addition to the values of γ _m and γ _n , those of γ _p and γ _q are known. As above, we need to make the further assumption that the system is initially at steady state, that is:

The formulas required for the computation of p(t) are derived in Section S4 of the Additional file 1.

The ratios of γ _q /γ _p were measured to be 1.8 (GFP) and 1.7 (luciferase). Figure 7a-b shows the effect on the predicted Fis profile. Contrary to the mRNA case, the corrections push the Fis profile locally outside the confidence band of the original, uncorrected profile. For both reporters, this leads to a better agreement with the Western blot measurements in the transition from exponential to stationary phase. The corrected profiles approach or capture the measurement at 400 min, which was missed by the original profile. This shows that the predictions can be improved by carrying out the corrections, but it also reveals that the better agreement comes at the price of slightly wider confidence bands.

Figure 7c-d shows that non-negligible differences may occur when the reporter concentration profiles are corrected for larger differences in half-life, although the profiles retain the same qualitative shape. In particular, the timing of the expression peak shifts according to the value of γ _q /γ _p . Notice that the expression profiles tend to cluster together around specific values of γ _q /γ _p , with minor variations within the clusters caused by differing values of γ _n /γ _m . The influence of the difference in mRNA stability is therefore negligible with respect to the difference in protein stability, confirming the results of Figure 6. In all of the above computations we have assumed that the folding time of GFP is negligible, implying that all GFP in the cell is active (q(t) = r(t)). This may lead to an underestimation of the amount of GFP in the cell. In order to correct for the effect of this bias, we can rewrite (8) to compute total GFP from active GFP:

The maturation time of GFP was set to 25 min, as determined experimentally for the reporter used in this study (GFPmut3) 51 , thus yielding a value κ _r = 0.023 min^-1. That is, it takes 25 min to convert half of a given pool of inactive GFP to its active form.

Figure 8a shows the concentration profiles of both active and total GFP, normalized with respect to the peak in mid-exponential phase of the active GFP concentration. As expected, active GFP represents only a fraction of total GFP. However, the qualitative shape of the profiles remains essentially the same. Using the profile of q(t) instead of r(t) for computing the normalized reporter synthesis rate, and thus the normalized mRNA concentration, yields the same conclusion (Figure 8b). In both cases we see that the expression peak is slightly shifted to an earlier time-point. This is consistent with the fact that the maturation process introduces a delay in the availability of active GFP. The agreement of the computed profiles with the Western and Northern blot measurements is not improved by correcting for the folding time (result not shown).

We have also experimented with variants of GFP, in particular a rather slow folding RFP (Red Fluorescent Protein). In this case, there are considerable differences between the expression profiles obtained with luciferase (data not shown). The corrections and the corresponding confidence bands also become much larger. We conclude that a fast-folding reporter protein is essential for reliable real-time monitoring of gene expression.