Correlation between growth rates, EIIACrr phosphorylation, and intracellular cyclic AMP levels in Escherichia coli K-12

Abstract

In Escherichia coli K-12, components of the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) represent a signal transduction system involved in the global control of carbon catabolism through inducer exclusion mediated by phosphoenolpyruvate-dependent protein kinase enzyme IIA(Crr) (EIIA(Crr)) (= EIIA(Glc)) and catabolite repression mediated by the global regulator cyclic AMP (cAMP)-cAMP receptor protein (CRP). We measured in a systematic way the relation between cellular growth rates and the key parameters of catabolite repression, i.e., the phosphorylated EIIA(Crr) (EIIA(Crr) approximately P) level and the cAMP level, using in vitro and in vivo assays. Different growth rates were obtained by using either various carbon sources or by growing the cells with limited concentrations of glucose, sucrose, and mannitol in continuous bioreactor experiments. The ratio of EIIA(Crr) to EIIA(Crr) approximately P and the intracellular cAMP concentrations, deduced from the activity of a cAMP-CRP-dependent promoter, correlated well with specific growth rates between 0.3 h(-1) and 0.7 h(-1), corresponding to generation times of about 138 and 60 min, respectively. Below and above this range, these parameters were increasingly uncoupled from the growth rate, which perhaps indicates an increasing role executed by other global control systems, in particular the stringent-relaxed response system.

FIG. 1.

Correlation of EIIA^Crr phosphorylation state and growth rate during growth of E. coli LJ110 and LJ210 with various carbon sources. EIIA^Crr phosphorylation levels were determined by Western blotting as described in Materials and Methods. The data represent mean values from at least two independent cultures and from at least four samples taken during exponential growth of one culture. The numbers in the symbols refer to the carbon sources as indicated in Table 1 with error bars indicating standard deviations. Circles correspond to non-PTS substrates, while squares represent PTS substrates. The gray line connecting the data points for PTS substrates represents a trend line considering all PTS data points. The dashed gray line represents a trend line considering all non-PTS substrates with exceptions glucose-6-phosphate and acetate. The trend lines show almost linear correlations between EIIA^Crr phosphorylation levels and growth rate. The obtained R²s were 0.93 for the PTS and 0.86 for the non-PTS trend line. The gray dots at μ = 0.25 h⁻¹ are drawn to point out the two areas mentioned in the Discussion.

FIG. 2.

Correlation of the activities of the cAMP-independent scrKp and cAMP-dependent scrYp promoters to growth rates resulting from growth on different substrates. (A and B) Relative luminescence activities (in relative light units [RLU]) of E. coli LJ110 carrying either the constitutive and cAMP-independent scrKp promoter (A) or the constitutive and cAMP-dependent scrYp promoter (B) fused to luxCDABE genes of pCS26 during batch cultures with various carbon sources. (C and D) scrK and scrYp activities, respectively, after multiplication of the RLUs with the corresponding growth rate. This standardization is done to account for differences in dilution rate of the proteins that vary with growth rate. (D)Activity of the scrYp promoter after multiplication of the RLUs with growth rate. By using an exponential fit, we were able to obtain a R² of 0.95, showing good correlation. The trend line was added to this plot as a dotted gray line. Values represent mean values from at least three independent experiments, and the error bars indicate standard deviations. The numbers in symbols correspond to carbon sources as in Table 1.

FIG. 3.

Time course of an experiment with various glucose concentrations. Show are the measurements from a continuous bioreactor experiment with E. coli LJ110 with glucose as the carbon source. Bioreactor setup and measurements were as described in Materials and Methods. cAMP samples were taken by taken samples from the bioreactor and by rapid centrifugation of these samples at 4°C. Open circles indicating the glucose concentration (in micromolar) are given on the leftmost y axis as well as extracellular cAMP concentrations given in nanomolar and represented by open triangles. The EIIA^Crr phosphorylation level represented by filled squares is plotted on the rightmost y axis.

FIG. 4.

Growth experiments with changing carbohydrate concentrations. The figure shows measurements from continuous bioreactor experiments with strain LJ110 or LJ210 and with glucose, sucrose, or mannitol as the carbon source. The experimental setup and measurements were as described in Materials and Methods. The EIIA^Crr phosphorylation level is plotted semilogarithmically against the carbohydrate concentrations. Symbols: ▪, ○, and •, experiments with glucose as the carbon source; ▵ and ▴, experiments with sucrose as the carbon source; *, experiment with mannitol as the carbon source.