Changes in Ribosomal Activity of Escherichia coli Cells during Prolonged Culture in Sea Salts Medium

Abstract

The activity of ribosomes from a clinical isolate ofEscherichia 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 thek max/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 thek max/Ks value. {k max 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.

Prokaryotic community analysis with CARD-FISH in comparison with FISH in ultra-oligotrophic ground- and drinking water

“…Our study demonstrates that FISH detection rates in ultra‐oligotrophic systems can be as low as 8–23% of DAPI‐stainable cells. The CARD‐FISH method is hardly affected by a low number of ribosomes, although hybridization results might still be influenced by modification (Kalpaxis et al 1998) or degradation (Davis et al 1986) of target sites, providing a possible explanation for the decrease of detection efficiency observed with CARD‐FISH during February and March (Fig. 3).…”

Effects of Two Photoreactive Spermine Analogues on Peptide Bond Formation and Their Application for Labeling Proteins in Escherichia coli Functional Ribosomal Complexes

“…Salt-washed (0.5 M NH 4 Cl) and polyamine-depleted ribosomes from E. coli B cells, partially purified translation factors, and crude Ac[ 3 H]Phe-tRNA charged with 15.8 pmol of [ 3 H]Phe per A 260 unit were prepared as described elsewhere (17). 70S ribosomes were isolated on a 10-30% linear sucrose gradient (18). Native 50S and 30S ribosomal subunits were prepared by incubating 70S ribosomes in dissociation buffer (10mM Tris-HCl, pH 7.5, 0.5 mM magnesium acetate, 150 mM NH 4 Cl, 6 mM 2-mercaptoethanol) at 37 °C for 15 min, followed by sedimentation in a sucrose gradient to dissociate the particles into subunits.…”

Unraveling the mechanisms of inhibition of silver‐doped bioactiveglass–ceramicparticles

“…Although MRSA is not resistant to chloramphenicol (MIC~8 μg/ml 143 ), it can tolerate a high antibiotic concentration under growth‐arrested conditions. This lack of activity may be due to the bacteriostatic nature of chloramphenicol 57 or the reduced ribosome activity in dormant bacteria 144,145 . While several reports have proven chloramphenicol does not interfere with the cell‐wall synthesis, 146,147 its presence may lead to severe accretion of cell‐wall material 148 and unbalanced membrane synthesis, causing envelope thickening 149 .…”