INTRODUCTION

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The expression of a bacterial gene can be studied by measuring the relative abundance of either its mRNA or its protein product. Intuitively these two methods might seem to be equivalent, but in fact they are not. Using artificially constructed promoter-lacZ fusions integrated into the Escherichia coli chromosome, we have previously determined the activities of a number of constitutive mRNA promoters expressed as lacZ transcripts per minute per promoter and as units of -galactosidase activity (19). The transcriptional activities of these promoters increased with increasing growth rate, whereas the specific activity of -galactosidase decreased. The rate of translation initiation of lacZ mRNA was found to be rather constant, with no indication of growth rate-dependent translational control (17). Therefore, the discrepancy was not caused by any control on the translation of the lacZ reporter mRNA.

What causes gene expression at the transcriptional and translational levels to respond in opposite directions to changes in the growth rate? The answer to this question is rather simple: the abundance in the cytoplasm of any given protein, or the specific activity of an enzyme (activity per amount of protein), reflects the distribution of translating ribosomes between the encoding mRNA and bulk mRNA. This distribution depends on two factors: (i) the relative amounts of the encoding mRNA and bulk mRNA and (ii) the translation frequencies of the encoding mRNA and bulk mRNA (see, e.g., reference 26).

In this report, we have considered the effects of bulk mRNA and free ribosomes on the synthesis of -galactosidase expressed from three artificial promoter-lacZ fusions carrying the promoters for the spc ribosomal protein operon (P_spc), the pBR322 plasmid replication inhibitor RNAI (P_RNAI), and the pBR322 replication primer RNAII (P_RNAII). In previous studies involving measurements of transcripts by hybridization, P_spc and P_RNAI were found to be constitutive, without specific control; P_RNAII was positively regulated by ppGpp but was constitutive in the absence of ppGpp (19). The experiments presented here suggest to us that many poorly translated mRNAs (e.g., those with weak ribosome binding sites) accumulate during slow growth in poor media and, conversely, many frequently translated mRNAs (e.g., those with strong ribosome binding sites) accumulate during fast growth in rich media. This keeps the concentration of free ribosomes approximately constant as the growth rate increases, in spite of an increasing concentration of total ribosomes. Moreover, it produces an apparent stimulation in the expression of any given protein under conditions of slow growth or at increased intracellular levels of ppGpp. These results have implications for the expression of any bacterial gene, including the control of the synthesis of ribosomal RNA and ribosomal proteins, and for the interpretation of data obtained with reporter systems.

	MATERIALS AND METHODS

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Bacterial strains used. The Escherichia coli strains used in this work and their origins or constructions are described in Table 1. Fusions of lacZ with P_spc and the plasmid pBR322 promoters P_RNAI and P_RNAII were constructed as previously described (17, 19). The promoters were originally cloned in the plasmid vector pSL03 and then recombined into the mal locus of the chromosome of lac deletion derivatives of E. coli B/r (see Table 1 for details). pSL03 was derived from the W205 trp-lac operon fusion (24) from which the trp transcription terminator upstream of lacZ (25) has been deleted (17). The operon fusions carried the following promoter fragments: P_spc, from nucleotide (nt) 51 relative to the transcription start through rplN (the first gene of the spc operon, encoding ribosomal protein L14), ending at nt +453; P_RNAI, from nt 77 to +32; and P_RNAII, from nt 63 to +63. All promoter fragments carried EcoRI and BamHI sites at their 5' or 3' ends, respectively, for insertion into the multiple cloning site of pSL03. For the experiment for which results are shown in Fig. 2, a spc-lac operon fusion was used in which P_spc was directly linked to lacZ (from nt 51 to +59) without rplN. Previous studies have shown that strains in which lacZ is directly linked to P_spc may show an anomalous growth rate-dependent reduction in the accumulation of lacZ mRNA (17). The reason for this effect is not known; possibly, sequences close to the 5' end of the spc operon transcript interact with sequences in the trp-lac mRNA leader to produce a fortuitous signal which either causes transcription termination or shortens the mRNA lifetime. Inclusion of rplN in the construct ensures that such effects are absent (17). Inclusion of additional sequences upstream of P_spc (up to 105 bp upstream of position 51) had no measurable effect on -galactosidase expression; this suggests that the region upstream of P_spc is devoid of regulatory elements.

Conditions of growth. Cultures were grown at 37°C in medium C (11) supplemented with either 0.2% (vol/vol) glycerol or 0.2% (wt/vol) glucose, with or without 0.8% Difco Casamino Acids plus 50 µg of tryptophan/ml, or in Luria-Bertani (LB) medium (23) with 0.2% glucose. Minimal media were supplemented with phenylalanine and threonine as required, at 50 µg/ml. Experimental cultures were inoculated from overnight cultures in glycerol minimal medium by diluting at least 250-fold into minimal media or 2,000-fold into amino acid-supplemented media.

-Galactosidase assays. Assays for -galactosidase activity were performed with four to five 10-µl samples of culture taken at different times during exponential growth as described previously (18). The specific activity of -galactosidase was expressed as the increase in A₄₂₀ per hour of incubation of the assay at 30°C per OD₆₀₀ unit of culture in the assay. The specific activity values obtained from different samples of a given culture generally deviated from the average by less than 2%. Greater deviations, up to 10%, were sometimes observed in repeat experiments carried out with cultures grown on different days. The reproducibility of the assays can also be seen from Fig. 2c and d: the fact that, for growth without pseudomonic acid, all measured points lie on a straight line with a slope of 1.0 implies that the specific enzyme activity was exactly the same for all assays during threefold exponential growth of the culture.

	RESULTS

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Enzyme expression during exponential growth at different rates. The specific activities of -galactosidase expressed from P_spc, P_RNAI, and P_RNAII in the ppGpp-proficient E. coli B/r strains SL106, YX101, and YX102 (Table 1; the promoter-lacZ fusions in these strains are integrated into the mal locus of the bacterial chromosome [see Materials and Methods]), respectively, were measured during exponential growth in different media (Fig. 1). For all three promoters, the specific activity of -galactosidase decreased with increasing growth rate in the range between 1.0 and 3.0 doublings/h. The specific activities for P_spc and P_RNAI decreased in parallel, about 2.5-fold for the threefold increase in growth rate, whereas the specific activity for P_RNAII decreased more than fivefold over this range of growth rates (Fig. 1). Previous transcription assays with E. coli K-12 strains showed that the activities of P_spc and P_RNAI are not significantly affected by the presence of ppGpp at the low ("basal") levels accumulating during slow exponential growth in ppGpp-proficient (relA⁺ spoT⁺) strains. In contrast, P_RNAII was stimulated by the low level of ppGpp under those conditions (19). Since cytoplasmic levels of ppGpp also decrease with increasing growth rate (31), the steeper decrease in the enzyme activity curve for P_RNAII in comparison to the curves for the other two promoters is consistent with positive control of P_RNAII by ppGpp. Results similar to those for P_spc in Fig. 1 have been reported previously (17); the data are shown here for the purposes of comparison and further analysis.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Growth rate dependence of -galactosidase expression from Pspc, PRNAI, and PRNAII. Strains SL106, YX101, and YX102, respectively, were used, and the growth media (given in order from the lowest to the highest growth rate) were glycerol minimal medium, glucose minimal medium, glucose-amino acids, and LB-glucose (see Materials and Methods).

Enzyme expression during the stringent and relaxed response. The stimulation or reduction of the production of a protein under slow- or fast-growth conditions can be mimicked by artificially altering the intracellular level of ppGpp. Pseudomonic acid is a competitive inhibitor of isoleucyl tRNA synthetase and, at low concentrations, causes mild amino acid deprivation (14). A relA⁺ and a relA strain carrying P_spc-lacZ operon fusions (strains SL104 and SL111, respectively [Table 1]) were treated with pseudomonic acid. Culture mass accumulation was always reduced by the addition of pseudomonic acid (Fig. 2a and b). Under these conditions, expression of lacZ from P_spc was stimulated by a high ppGpp concentration (the stringent response [Fig. 2c]) and reduced by a low ppGpp concentration (the relaxed response [Fig. 2d]). As during exponential growth, the expression of lacZ from this promoter correlates with the ppGpp concentration.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   -Galactosidase expression from Pspc during the stringent and the relaxed response. The stringent (relA+) strain SL104 (a and c) and the relaxed (relA) strain SL111 (b and d) were grown exponentially () in glucose-amino acids medium. Mild amino acid deprivation was induced in part of the culture () by treatment with 3 µg of pseudomonic acid/ml. (a and b) culture mass density (OD600) as a function of time; (c and d) -galactosidase activity plotted against cell mass density. Values are normalized to the -galactosidase activity and culture mass (OD600) observed at the time of drug addition. , doubling time.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   -Galactosidase expression from PRNAI and PRNAII during the stringent and the relaxed response. Stringent and relaxed strains carrying PRNAI-lacZ and PRNAII-lacZ operon fusions were grown exponentially in glucose minimal medium and treated with pseudomonic acid (3 µg/ml), as described in the legend for Fig. 2. (a and b) PRNAI-lacZ fusion strains YX101 (relA+) and YX103 (relA); (c and d) PRNAII-lacZ fusion strains YX102 (relA+) and YX104 (relA). The culture mass (OD600) values and -galactosidase activities were normalized to the values observed at time zero (addition of pseudomonic acid); only the differential plots, of -galactosidase activity versus culture mass, are shown.

	DISCUSSION

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Translational competition between bulk mRNA and specific mRNA. In the following, we consider the effect of total (bulk) mRNA and its translation on enzyme expression during exponential growth in different media. For this purpose, the expression of -galactosidase from P_spc (Fig. 1) was chosen as an example. The same logic applies to the expression of any stable protein, whether it is measured by enzyme assays, radioactive labeling, protein staining, Western blotting, or some other means. This discussion requires additional information about spc promoter activity, bulk mRNA synthesis, total RNA synthesis, and protein synthesis. This information was obtained from previous measurements in the same E. coli B/r background and is summarized (with references) in Table 2.

(1)

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Growth rate dependence of the -galactosidase specific activity expressed from Pspc in strain SL106 and of transcription and translation parameters that determine this specific activity (see the text and equation 1). (a) -Galactosidase specific activity; (b) relative abundance of lacZ mRNA as a proportion of total mRNA; (c) rate of translation initiation of lacZ mRNA in relative units; (d) average rate of translation initiation of total (bulk) mRNA in translations per minute per average mRNA molecule. This rate can be found either from the total rate of protein synthesis per amount of mRNA [(dPt/dt)/Rm] or from the peptide chain elongation rate (cp) and the average distance of ribosomes on the mRNA (dr [see Table 2, footnote u]). Data represent interpolated values taken from Table 2 (see Table 2, footnotes f, p, h, and u, for the data in panels a, b, c, and d, respectively).

Decay of bulk mRNA. In Table 2, the average life of total (bulk) mRNA (_m) has not been observed. The decay of total mRNA is expected to be complex, with higher-order kinetics, reflecting at first the fast decay of the least-stable mRNAs and later the slower decay of the more-stable mRNAs. Since no data about the growth rate dependence of the decay of total mRNA are available, we have used instead the average functional lifetimes of lacZ mRNA, which are 1.9 and 2.4 min in E. coli B/r growing at 1 and 3 doublings/h, respectively (18). These lifetimes were assumed to be representative for bulk mRNA. If the lifetimes of different mRNA species differ by constant factors from the lacZ mRNA lifetimes (i.e., a different factor for each mRNA species, but the same mRNA species-dependent factor for all growth rates), then the curves for R_lac/R_m (Fig. 4b) and (dP_t/dt)/R_m (Fig. 4d) would shift in parallel either up or down, depending on whether the average _m values for bulk mRNA are greater or smaller than those of lacZ mRNA. However, if the _m of bulk mRNA increases with the growth rate more than the _m of lacZ mRNA, then the curves in Fig. 4b and d would increase less steeply. The lifetime of ribosomal protein mRNAs does indeed appear to increase with increasing growth rate more than the lifetime of lacZ mRNA (18); therefore, the relative abundance of lacZ mRNA (Fig. 4b) and the translation of bulk mRNA (Fig. 4d) might increase somewhat less steeply with the growth rate than is shown.

Translation frequency of lacZ mRNA at different growth rates. The approximately constant rate of translation of lacZ mRNA (Fig. 4c) could have several explanations: either (i) the lacZ ribosome binding site is saturated with ribosomes and initiation factors at all growth rates, (ii) the translation of lacZ mRNA is subject to a specific control that keeps it constant at all growth rates, or (iii) the concentrations of "initiation-ready" free ribosomes are nearly constant at all growth rates. Since the rate of translation of lacZ mRNA increases severalfold in the presence of the antibiotic rifampin when the amount of total mRNA decreases due to the inhibition of transcription (18), we conclude that, during normal exponential growth, the lacZ ribosome binding site is not saturated. Moreover, despite extensive study, there is no evidence to suggest that translation of lacZ mRNA is subject to any specific control. This leads us to suggest that the concentration of initiation-ready ribosomes (i.e., mature 30S ribosomal subunits with IF3, ready to bind to an mRNA ribosome binding site in the presence of saturating or constant concentrations of IF1, IF2, and initiator tRNA) is relatively constant and subsaturating at different growth rates. This causes the observed constant frequency of lacZ mRNA translation (Fig. 4c).

Translation frequency of bulk mRNA at different growth rates. Several features could contribute to the increasing average rate of translation initiation of bulk mRNA at increasing growth rates (Fig. 4d) despite a constant concentration of free, initiation-ready ribosomes and/or factors (see above). First, the average mRNA present during growth in rich media may have more-efficient ribosome binding sites than mRNA made during growth in poor media. Alternatively or in addition, the rate of translation of some mRNAs may be controlled by special regulatory sites such that translation is favored at high growth rates. The second possibility, i.e., control that favors translation at high growth rates, has been found to occur for ribosomal protein mRNAs (reviewed in reference 15), but it is not known to be the rule for most other mRNAs. Therefore, both more-efficient ribosome binding sites on constitutive mRNAs made during growth in rich media compared to more-repressible mRNAs made during growth in poor media and translational control of certain mRNAs are inferred to contribute to the increased translation of bulk mRNA at high growth rates. Whatever the exact cause, the increased translation makes bulk mRNA an increasingly better competitor for ribosome binding to lacZ mRNA as the population of mRNAs changes with increasing growth rate.

Effect of ppGpp on protein and enzyme expression. During a mild stringent or a mild relaxed response, the -galactosidase specific activities expressed from both P_spc-lacZ and P_RNAI-lacZ operon fusions were increased and reduced, respectively (Fig. 2 and 3). Based on the analysis of the growth rate dependence of enzyme expression above, we suggest that ppGpp affects the accumulation and quality of bulk mRNAs and thereby causes the apparent positive control of enzyme expression by ppGpp. Indirect stimulation of lacZ expression by ppGpp (Fig. 2 and 3) could be produced in a number of ways. For example, ppGpp might directly or indirectly stimulate the synthesis of mRNAs with weak ribosome binding sites, or it might inhibit the synthesis of mRNAs with strong ribosome binding sites. Indirect effects of ppGpp on transcription are expected for promoters controlled by DNA binding factors like Fis, H-NS, or Lrp (see, e.g., references 9, 29, 34 and 36), whose syntheses depend on ppGpp. LacZ expression may also be increased if ppGpp stimulates the decay of mRNAs with strong ribosome binding sites (e.g., ribosomal protein mRNAs [18]). Any one or all of these effects might contribute to the apparent stimulation of enzyme expression by ppGpp.

Combined effects of growth rate and ppGpp on bacterial gene expression. The effects of ppGpp and growth rate on bulk mRNA synthesis and translation are superimposed on the direct transcriptional control by ppGpp. Three cases are to be distinguished, as follows.

Control of rRNA synthesis: role of free RNA polymerase concentration and ppGpp. Using a lacZ reporter system and lacZ mRNA hybridization assays, the absolute activities of the rrnB P1 and P2 promoters, expressed as number of transcripts per minute per promoter, were previously found to increase with increasing growth rate (19). Since no specific control factors or factor binding sites have ever been associated with rrn P2 promoters, we have assumed that rrn P2 promoters are constitutive, so that their activity is affected only by the concentration of free RNA polymerase and the promoter-specific parameters V_max and K_m, representing the maximum promoter activity at saturation with free RNA polymerase and the free RNA polymerase concentration at half-maximal activity. In the absence of ppGpp, the rrn P1 promoters are also constitutive; accordingly, their activity also depends only on the free RNA polymerase concentration and the associated V_max and K_m values. The K_m value for P1 promoters is affected by the DNA binding factors Fis and H-NS. In addition, in normal ppGpp-proficient strains, P1 activity was found to be inhibited by ppGpp. As a result, the P1 activity was lower than the P2 activity during slow bacterial growth, when basal (exponential-growth) levels of ppGpp are highest, and higher than the P2 activity during fast bacterial growth, when basal levels of ppGpp are lowest. Thus, in our model (19), the growth rate regulation of ribosome synthesis depends both on the concentration of free RNA polymerase, which determines the frequency of transcription initiation at both P1 and P2, and on the concentration of ppGpp, which selectively reduces expression from P1 under slow-growth conditions.

Role of ppGpp in the control of ribosomal protein synthesis. It has been reported that the rate of synthesis of spc mRNA from the normal spc operon relative to the rate of total mRNA synthesis increases with increasing growth rate (8, 20). This is similar to the growth rate-dependent increase in the mRNA amounts, R_lac/R_m, determined above for the P_spc-rplN-lacZ fusion (Fig. 4b). Since the rate of ribosomal protein synthesis as a proportion of total protein synthesis (denoted by _r [3]) increases with increasing growth rate similarly to the rate of spc mRNA synthesis per total mRNA synthesis, it has been suggested that the control of ribosomal protein synthesis occurs mainly at the transcriptional level (4, 8, 20). This interpretation was based on the two implicit assumptions that (i) spc mRNA and bulk mRNA are translated approximately equally and (ii) spc mRNA and bulk mRNA have approximately equal lifetimes. Based on the considerations above (Fig. 4) and previous measurements of spc mRNA lifetimes (18), both assumptions appear to be unjustified. The increasing rate of synthesis of ribosomal proteins from the spc operon with increasing growth rate is mediated through control of the decay of spc mRNA; similar mechanisms presumably control the decay of other ribosomal protein mRNAs (18, 22). This control of the mRNA lifetime adjusts the synthesis of ribosomal proteins to the ppGpp-dependent synthesis of rRNA, overriding any transcriptional regulation of ribosomal protein operons or superimposed effects of bulk mRNA translation.