A Decrease in Transcription Capacity Limits Growth Rate upon Translation Inhibition

Qing Zhang,Marco Cosentino Lagomarsino,Elisa Brambilla,Bianca Sclavi,Hualin Shi,Rui Li,Zackery Bulman

doi:10.1128/msystems.00575-20

Abstract

In bacterial cells, inhibition of ribosomes by sublethal concentrations of antibiotics leads to a decrease in the growth rate despite an increase in ribosome content. The limitation of ribosomal activity results in an increase in the level of expression from ribosomal promoters; this can deplete the pool of RNA polymerase (RNAP) that is available for the expression of nonribosomal genes. However, the magnitude of this effect remains to be quantified. Here, we use the change in the activity of constitutive promoters with different affinities for RNAP to quantify the change in the concentration of free RNAP. The data are consistent with a significant decrease in the amount of RNAP available for transcription of both ribosomal and nonribosomal genes. Results obtained with different reporter genes reveal an mRNA length dependence on the amount of full-length translated protein, consistent with the decrease in ribosome processivity affecting more strongly the translation of longer genes. The genes coding for the β and β' subunits of RNAP are among the longest genes in the Escherichia coli genome, while the genes coding for ribosomal proteins are among the shortest genes. This can explain the observed decrease in transcription capacity that favors the expression of genes whose promoters have a high affinity for RNAP, such as ribosomal promoters.IMPORTANCE Exposure of bacteria to sublethal concentrations of antibiotics can lead to bacterial adaptation and survival at higher doses of inhibitors, which in turn can lead to the emergence of antibiotic resistance. The presence of sublethal concentrations of antibiotics targeting translation results in an increase in the amount of ribosomes per cell but nonetheless a decrease in the cells' growth rate. In this work, we have found that inhibition of ribosome activity can result in a decrease in the amount of free RNA polymerase available for transcription, thus limiting the protein expression rate via a different pathway than what was expected. This result can be explained by our observation that long genes, such as those coding for RNA polymerase subunits, have a higher probability of premature translation termination in the presence of ribosome inhibitors, while expression of short ribosomal genes is affected less, consistent with their increased concentration.

Highlights

In bacterial cells, inhibition of ribosomes by sublethal concentrations of antibiotics leads to a decrease in the growth rate despite an increase in ribosome content
Bremer and coworkers have shown that the activity of the rrnBP1 promoter is inversely proportional to the concentration of ppGpp in vivo [24] and that the activity of constitutive promoters can be used to estimate the amount of free RNA polymerase in the cell [22, 24]
Growth of these strains in a 96-well plate allowed us to measure the changes in growth rate, the green fluorescent protein (GFP) concentration, and the resulting GFP production rate (Gpr) as a function of chloramphenicol concentration (Fig. 1 and Fig. S1)

Summary

Introduction

Inhibition of ribosomes by sublethal concentrations of antibiotics leads to a decrease in the growth rate despite an increase in ribosome content. We have found that inhibition of ribosome activity can result in a decrease in the amount of free RNA polymerase available for transcription, limiting the protein expression rate via a different pathway than what was expected This result can be explained by our observation that long genes, such as those coding for RNA polymerase subunits, have a higher probability of premature translation termination in the presence of ribosome inhibitors, while expression of short ribosomal genes is affected less, consistent with their increased concentration. The cellular response, as predicted by the ppGpp feedback loop, is to produce a larger amount of ribosomes and an increased translation rate; despite this increase, the cell’s growth rate is reduced [18, 19] This has been proposed to result from a decrease in the resources available for the production of nonribosomal proteins that become limiting for cellular metabolism [18]. More recent results point to a decrease in the fraction of active ribosomes to explain the decrease in the total protein production rate [19, 21]

Methods

Results

Conclusion