Initiation factor and ribosome levels are coordinately controlled in Escherichia coli growing at different rates

The levels of initiation factors and other translational components were compared in crude lysates of Escherichia coli grown at different rates. Cells were grown in media containing [35S]sulfate and different carbon sources, and harvested during mid-exponential phase after about 10 generations of balanced growth. Initiation factors (IF), elongation factors (EF), and a number of ribosomal proteins were identified unambiguously in gel patterns following two-dimensional polyacrylamide gel electrophoresis. The molar concentration of each protein was calculated from measurements of the radioactivity in excised gel spots and knowledge of the sulfur content of each protein. We found that the ribosomal proteins and elongation factors were present in equimolar amounts except for L7/L12 and EF-Tu which were 4-fold and 5-fold more abundant, respectively, and that the levels of each protein increased in proportion to growth rate. These results are similar to ones obtained previously by other methods, and serve to confirm the validity of our method. We found that the levels of IF2a and IF3 also were approximately proportional to growth rate. We also measured the levels of all three initiation factors using a radioimmune assay, showed that the factors are present in equimolar amounts, and confirmed that their abundance increased in parallel with ribosomes. We conclude that initiation factor levels are coordinately regulated with one another and with ribosome and elongation factor levels.     

Sensitivity of Escherichia coli to cephaloridine at different growth rates.

Steady-state populations of Escherichia coli B/r were treated with cephaloridine at minimal inhibitory concentrations. The antibiotic sensitivity of the cells and the localization of spheroplast emergence along the cell surface were examined as a function of cell length and growth rate. In fast-growing populations (greater than 1 division per h) the sites of cephaloridine interaction occurred preferentially at the cell pole in the smaller cells and at the cell center in dividing cells. At decreasing growth rates the cells became more resistant to cephaloridine, and a gradual shift from the cell pole toward the cell center was observed for the sphere position. A similar growth rate-dependent change in localization was found for sucrose-induced plasmolysis vacuoles.     

The inefficiency of ribosomes functioning in Escherichia coli growing at moderate rates

It is generally agreed that ribosomes function with reduced efficiency (i.e. a smaller proportion is actually engaged in protein synthesis) in bacteria growing at low growth rates (doubling times greater than 2 h). This paper examines whether the efficiency is constant in bacteria growing at various rates corresponding to doubling times of less than 2 h. Because isotopic methods cannot be used in very rich media, turbidimetric methods have been extended to follow the kinetics of growth immediately following the shift-up of cultures of Escherichia coli ML308 growing in glucose minimal medium or succinate minimal medium into a very rich medium supporting a balanced doubling time of 17.4 min. It is concluded that the efficiency of ribosome participation in protein synthesis is higher in the very rich medium than in the two minimal media, which support doubling times of 43 and 65 min, respectively, at 37 degrees C.     

Quantitative determination of FtsA at different growth rates in Escherichia coli using monoclonal antibodies

FtsA is an essential cell division protein in Escherichia coli. Its synthesis in low amounts makes the investigation of its functions difficult. Partially purified FtsA protein was obtained by solubilizing cellular inclusion bodies after overexpression of the ftsA gene for the purpose of raising monoclonal antibodies. Mice were immunized with this FtsA protein fraction and their spleen cells were fused to Sp2/0-AG14 mouse myeloma cells. Hybrid cells were screened and two clones were positively identified as FtsA monoclonal antibody producers by enzyme-linked immunosorbent assay and Western blotting. A quantitative assay using these monoclonal antibodies indicated that the average number of FtsA molecules per cell to be between 50 and 200. However, the concentration of FtsA protein normalized to total cell protein was constant over a wide range of growth rates. This finding is in agreement with the hypothesized role of FtsA protein as a stoichiometric component of the septum.     

Synthesis of RNA polymerase in Escherichia coli B-r growing at different rates

Polyacrylamide gel electrophoresis of unfractionated sodium dodecyl sulfate lysates of Escherichia coli$\text{B}\text{r}$ has been used to investigate the synthesis of β and β′ subunits of RNA polymerase as a function of bacterial growth rate. In succinate (μ = 0.67 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) supplemented media, the fraction of [¹⁴C]leucine-labeled β and β′ protein/total protein was found to be 1.05, 1.31 and 1.56%, respectively. Comparison of these values with recent estimates from this laboratory of the differential rate of synthesis of functioning RNA polymerase suggests an excess of total over functioning RNA polymerase. The significance of these data in reference to the regulation of RNA polymerase synthesis is discussed.     

Maintenance coefficients and rates of turnover of cell material in Escherichia coli ML308 at different growth temperatures

Abstract Maintenance coefficients and rates of turnover of cell protein, cell walls and total cell phosphate were determined during the growth of Escherichia coli ML308 in fed-batch culture at different temperatures. The apparent Arrhenius activation energies ( E _a) calculated for maintenance coefficients at temperatures over 30°C were 212–515 kJ· mol⁻¹, indicative of a process thermodynamically similar to denaturation. The corresponding Arrhenius values for turnover were of a different magnitude, 44–115 kJ mol⁻¹, more typical of enzyme-catalysed reactions. Addition of 0.5 M NaCl had a minor effect on maintenance at both temperatures. Turnover of cell material and salt homeostasis were therefore concluded to be minor components of the maintenance requirement of E. coli .     

Dimensions of Escherichia coli at various growth rates: model for envelope growth.

The duplication of Escherichia coli B/r is described based on two independent sequences, the replication of the genome and the growth of the envelope. It is proposed that (i) new envelope growth zones are activated coincident with the initiation of new rounds of chromosome replication; (ii) each zone is active in envelope synthesis from the time of its inauguration to the division which follows the completion of the round of chromosome replication (that is, for C + D min); and (iii) the rate of envelope synthesis at each site is constant, independent of the growth rate. Measurements of the surface areas of two E. coli B/r substrains growing at a variety of rates and during nutritional transitions are consistent with the predictions of the model.     

Chromosome segregation in Escherichia coli B/r at various growth rates.

Chromosome segregation was analyzed in three substrains of Escherichia coli B/r growing at various rates. The cultures were pulse labeled with [14C]thymidine and bound to the bottom surface of a nitrocellulose membrane filter, and the radioactivity in newborn cells released from the surface during continuous elution with growth medium was measured. Since there was a fixed orientation in the release of newborn cells, the time course of the change in radioactivity per effluent cell could be used to investigate the orientation of chromosome segregation. If the radioactive deoxyribonucleic acid strands were partitioned at random between the progenies remaining attached to the membrane filter and those released into the effluent, the radioactivity per cell would decrease twofold after each generation of elution. The decrease in radioactivity was less than twofold at C + D min of elution and larger than twofold one generation later, indicating that chromosome segregation was nonrandom.     

DnaK-facilitated ribosome assembly in Escherichia coli revisited

Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30 degrees C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37 degrees C implicates a direct or indirect role for DnaK in this process.     

Inhibitory effect of heterologous ribosome recycling factor on growth of Escherichia coli

Ribosome recycling factor (RRF) of Thermotoga maritima was expressed in Escherichia coli from the cloned T. maritima RRF gene and purified. Expression of T. maritima RRF inhibited growth of the E. coli host in a dose-dependent manner, an effect counteracted by the overexpression of E. coli RRF. T. maritima RRF also inhibited the E. coli RRF reaction in vitro. Genes encoding RRFs from Streptococcus pneumoniae and Helicobacter pylori have been cloned, and they also impair growth of E. coli, although the inhibitory effect of these RRFs was less pronounced than that of T. maritima RRF. The amino acid sequence at positions 57 to 62, 74 to 78, 118 to 122, 154 to 160, and 172 to 176 in T. maritima RRF differed totally from that of E. coli RRF. This suggests that these regions are important for the inhibitory effect of heterologous RRF. We further suggest that bending and stretching of the RRF molecule at the hinge between two domains may be critical for RRF activity and therefore responsible for T. maritima RRF inhibition of the E. coli RRF reaction.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.     

The growth rate control in Escherichia coli at near to maximum growth rates: the A-stat approach

The growth characteristics of Escherichia coli K-12 in the continuous culture with a smooth increase in the dilution rate (A-stat) of various carbon sources (glucose, acetate, succinate, glycerol, lactate, acetate + succinate, casamino, acids + glucose) were studied. For all substrates studied the maximum value of specific respiration rate, Q _O2, remained between 14–18 mmol O₂ h^-1 g dwt^-1 and the maximum growth rate varied from 0.22 h-¹ on acetate to 0.77 h^-1 on glucose + casamino acids. After the respiratory capacity of the cells was exhausted at growth rates µ < µ_crit, the growth yield Y_XO2, increased slightly when the dilution rate increased. The maximum growth rate of Escherichia coli K12 was dependent on growth yield, respiratory capacity and glycolytic capacity of the strain. Analysis of the cultivation data using a stoichiometric flux model indicated that ATP synthesis in E. coli exceeds by two-fold that (theoretically) required to build up biomass. The experimental value of m_ATP < 4 mmol ATP h^-1 g dwt^-1 determined from A-stat cultivation data was low compared with the calculated unproductive hydrolysis of ATP (64–103 mmole ATP g dwt^-1).     

Feedback control of ribosome function in Escherichia coli

We have previously proposed that the rate of ribosome function during balanced growth in E. coli, expressed as the rate of peptide chain elongation, is adjusted by a feedback mechanism: whenever that rate is submaximal (i.e. below 22 amino acid residues polymerized per active ribosome at 37 degrees C), the feedback signal ppGpp is generated by an activation of the ppGpp synthetase expressed from the spoT gene. The accumulation of ppGpp reduces the synthesis of additional ribosomes and thereby reduces the consumption of amino acids which, in turn, allows the remaining ribosomes to function at a higher rate. Here we have described with supporting evidence the proposed feedback loop in greater detail and provided a mathematical analysis which predicts that the SpoT ppGpp synthetase activity should be highest when the ribosomes function at their half-maximal rate. This prediction is consistent with reported observations and is independent of the particular (unknown) mechanism by which the rate of translation controls the ppGpp synthetase activity of SpoT.