Changes in ribosomal activity of Escherichia coli cells during prolonged culture in sea salts medium

Abstract

The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the kmax/Ks ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the kmax/Ks value. (kmax is the apparent catalytic rate constant of peptidyl transferase, and Ks is the dissociation constant of the encounter complex between acetyl (Ac)[3H]Phe-tRNA-poly(U)-ribosome and puromycin.) Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.

FIG. 1

Levels of 70S ribosomal particles and AcPhe-tRNA binding to poly(U)-programmed ribosomes isolated from a clinical isolate of E. coli during starvation in sea salts medium. The level of 70S ribosomes was calculated by measuring at 260 nm the corresponding peak area of ribosomal profile after sucrose gradient centrifugation. Ribosomes were assayed for AcPhe-tRNA binding by the filter-binding technique. The values of radioactivity shown on the vertical axis represent the extent of Ac[³H]Phe-tRNA binding to 2.5 A₂₆₀ units of intact ribosomes (•), ribosomes depleted of L7/L12 protein (▵), and ribosomes depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells (○). Controls without poly(U) were included in each experiment, and the values obtained were subtracted. Bars represent standard deviations as calculated from four independently starved cultures.

FIG. 2

Time course of AcPhe-tRNA binding to E. coli ribosomes. The ribosomes were prepared from the clinical isolate cells harvested at time zero (○) and at 1 day (•), 3 days (▴), and 7 days (□) after the onset of starvation.

FIG. 3

Fragmentation of rRNA after incubation of E. coli cells in sea salts medium. rRNA was extracted from E. coli ribosomes and analyzed by 8% polyacrylamide gel electrophoresis in the presence of 6 M urea (A) or by 1% agarose denaturing gel electrophoresis (B). Lanes: 0, standards of 16S and 23S rRNA from E. coli W; 1, tRNA^Phe from E. coli W; 2 to 5, rRNAs (5 μg) prepared from the clinical isolate cells harvested at the mid-logarithmic phase of growth (lane 2) or after 1 day (lane 3), 3 days (lane 4), or 7 days (lane 5) of starvation; 6, rRNA from mid-logarithmic-phase cells of E. coli B strain; 7, rRNA from E. coli B cells harvested at the end of the starvation period. (C) Quantitation of 16S and 23S rRNA fragmentation during starvation. Equivalent aliquots (5 μg) of total rRNA from each sample were analyzed by 1% agarose denaturing gel electrophoresis, such as that shown in panel B. After staining with methylene blue, the gels were scanned at 550 nm in a densitometer. The relative intensity represents the absorbance of 16S (solid symbols) or 23S (open symbols) rRNA bands at several points of the starvation period, given as the percentage of the rRNA band absorbance corresponding to 5 μg of rRNA isolated from surviving E. coli B ribosomes (triangles) or from surviving ribosomes of the clinical isolate (circles), prepared from cultures harvested at time zero.

FIG. 4

Gel electrophoresis of total ribosomal proteins (TP70) isolated from the E. coli clinical isolate during starvation. Proteins were separated by 6 M urea–4% polyacrylamide gel electrophoresis. Lanes: 0, L7 and L12 reference standards; 1 to 4, TP70 from cells harvested at time zero (lane 1) or at 1 day (lane 2), 3 days (lane 3), or 7 days (lane 4) after the onset of starvation; 5 and 6, TP70 from ribosomal particles isolated from 24-h-starved cells and depleted of L7/L12 protein before (lane 5) or after (lane 6) reconstitution with L7/L12 protein from exponentially growing cells. The numbers in parentheses are the corresponding mean L12/L7 molar ratios, obtained from four independently starved cultures. The standard error of these values was found to be less than 0.05.