Time‐dependent effects of transcription‐ and translation‐halting drugs on the spatial distributions of the Escherichia coli chromosome and ribosomes

Abstract

Summary Previously observed effects of rifampicin and chloramphenicol indicate that transcription and translation activity strongly affect the coarse spatial organization of the bacterial cytoplasm. Single-cell, time-resolved, quantitative imaging of chromosome and ribosome spatial distributions and ribosome diffusion in live E. coli provides insight into the underlying mechanisms. Monte Carlo simulations of model DNA-ribosome mixtures support a novel nucleoid-ribosome mixing hypothesis. In normal conditions, 70S-polysomes and the chromosomal DNA segregate, while 30S and 50S ribosomal subunits are able to penetrate the nucleoids. Growth conditions and drug treatments determine the partitioning of ribosomes into 70S-polysomes vs free 30S and 50S subunits. Entropic and excluded volume effects then dictate the resulting chromosome and ribosome spatial distributions. Direct observation of radial contraction of the nucleoids 0-5 min after treatment with either transcription- or translation-halting drugs supports the hypothesis that simultaneous transcription, translation, and insertion of proteins into the membrane (“transertion”) exerts an expanding force on the chromosomal DNA. Breaking of the DNA-RNA polymerase-mRNA-ribosome-membrane chain in either of two ways causes similar nucleoid contraction on a similar timescale. We suggest that chromosomal expansion due to transertion enables co-transcriptional translation throughout the nucleoids.

Chromosome-Membrane Interactions in Bacteria

“…Thus, like the protein synthesis inhibitors, the initial effect of rifampicin is consistent with disruption of the expansive force associated with transertion. The expansion at longer times following rifampicin treatment may be due to indirect effects of transcription inhibition or from entropic effects of ribosomal subunits mixing with the nucleoid (6). This latter hypothesis is also consistent with single-particle-tracking studies of bound and free ribosomal subunits that detected a large percentage of free subunits co-localized with nucleoid-associated proteins (75).…”

“…Furthermore, cells in most early studies were fixed, which could induce artifacts in nucleoid morphology. These issues were largely addressed in a recent study that followed the effects of transcription and translation inhibitors on the nucleoid in live cells and in real time using a microfluidic device (6). It was found that for late times (>20 min), translation inhibitors (chloramphenicol and kasugamycin) produced condensed nucleoids, whereas a transcription inhibitor (rifampicin) produced expanded nucleoids, consistent with earlier reports.…”

“…Antibiotic treatment, whether disrupting transcription or translation, clearly affects the expression of far more than just membrane proteins. Thus, one cannot rule out the possibility that an alternative mechanism distinct from transertion accounts for the observed rapid contraction of the nucleoid following inhibition of transcription or translation (6). Similarly, chromosome repositioning to the membrane for genetic loci that encode membrane proteins has not been shown to depend on concurrent transcription, translation, and insertion into the membrane (46).…”

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

“…Statistical models have suggested that this phase separation between the ribosome-containing cytoplasm and the DNA can be explained by simple entropic forces: The DNA polymer avoids the walls to maximize conformational entropy, and the polysomes (multiple 70S ribosomes on a single mRNA) occupy the empty space near the walls to maximize translational entropy (41). Although mRNA-bound 70S ribosomes are excluded from the nucleoid, it has recently been shown that free 50S and 30S subunits do have access to the nucleoid center (32,42). These results indicate that both transcription and translation can begin in the center of the nucleoid (Fig.…”

Structure and Membrane Topography of the Vibrio-Type Secretin Complex from the Type 2 Secretion System of Enteropathogenic Escherichia coli

“…In E. coli, the RNA transcribed from the gsp operon has been mapped (44), and multiple translation initiation events (45) ensure that many molecules of GspD (and the other subunits of the T2SS) would be synthesized in close proximity, translocated into the periplasm in close proximity, and translocated to arrive at the same or a neighboring LolB receptor site within a short window of time (46)(47)(48)(49). Computer simulations and protein localization studies confirm that transertion creates local regions of high concentration for a given membrane protein (reviewed by Matsumoto et al [48]).…”