Active Transcription of rRNA Operons Condenses the Nucleoid inEscherichia coli: Examining the Effect of Transcription on Nucleoid Structure in the Absence of Transertion

Abstract

In Escherichia coli the genome must be compacted ϳ1,000-fold to be contained in a cellular structure termed the nucleoid. It is proposed that the structure of the nucleoid is determined by a balance of multiple compaction forces and one major expansion force. The latter is mediated by transertion, a coupling of transcription, translation, and translocation of nascent membrane proteins and/or exported proteins. In supporting this notion, it has been shown consistently that inhibition of transertion by the translation inhibitor chloramphenicol results in nucleoid condensation due to the compaction forces that remain active in the cell. Our previous study showed that during optimal growth, RNA polymerase is concentrated into transcription foci or "factories," analogous to the eukaryotic nucleolus, indicating that transcription and RNA polymerase distribution affect the nucleoid structure. However, the interpretation of the role of transcription in the structure of the nucleoid is complicated by the fact that transcription is implicated in both compacting forces and the expansion force. In this work, we used a new approach to further examine the effect of transcription, specifically from rRNA operons, on the structure of the nucleoid, when the major expansion force was eliminated. Our results showed that transcription is necessary for the chloramphenicol-induced nucleoid compaction. Further, an active transcription from multiple rRNA operons in chromosome is critical for the compaction of nucleoid induced by inhibition of translation. All together, our data demonstrated that transcription of rRNA operons is a key mechanism affecting genome compaction and nucleoid structure.An Escherichia coli cell is small, measuring approximately 2 to 4 m in length and 1 m in diameter. The bacterial genome is 4.6 million bp, which would be approximately 1.5 mm in length if stretched fully. In a rapidly growing cell, there are multiple genome equivalents. Thus, the genome must be compressed at least 1,000-fold to fit into the cell. The bacterial chromosome forms a cellular structure named the nucleoid (25, 42). Normally the E. coli nucleoid shows a characteristic "flexible doublet" shape (49) and is membrane associated (2, 46). Despite great advances being made in understanding the biochemistry and molecular biology of E. coli, the structure of the bacterial nucleoid remains poorly defined.Woldringh et al. proposed that the structure of the nucleoid is determined by a balance of expansion and compaction forces (44). Suggested compaction forces include (i) DNA binding proteins (9, 17), (ii) DNA supercoiling (29,35,38), (iii) macromolecular crowding (20,23,51), and (iv) entropy-driven depletion attraction (18). One of the proposed forces that significantly contributes to expansion of the nucleoid is called transertion. During this process, coupled transcription and translation of membrane proteins and/or periplasmic exported proteins pull and anchor the transcribed bacterial nucleoid onto the cytoplasmic membrane (3, 43). In addition,...

Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence

“…Similar to translation, the contribution of active transcription in maintaining nucleoid conformation and ribosome distribution has long been proposed (25,38,39,44). Our result with rifampicin treatment supports that model (Fig.…”

“…5B, after treatment of QC101 cultures with rifampicin, the nucleoids were condensed first (within 5 min) but soon (15-30 min) spread out through the length of the cell as uniformly fluorescent cylinders. This was consistent with earlier data on the altered ultrastructure of E. coli nucleoids after exposure to rifampicin (38,39). The ribosomes too lost their characteristic distribution in exponential phase and were spread uniformly surrounding the nucleoids.…”

“…In these situations the nucleoid cannot be the primary determinant for the ribosome distribution. Instead, it seems likely that "translation" plays an important role in ribosome and nucleoid distribution as suggested by earlier works (39,51).…”

Bacterial cytological profiling rapidly identifies the cellular pathways targeted by antibacterial molecules

“…In nearly all cases, the cytological profiles produced in response to antibiotics can be explained in terms of their physiological effects. For example, compounds that block translation lead to chromosome compaction due to the interference with coordinated translation and insertion of proteins into the membrane (31,32) whereas compounds that block transcription lead to chromosome decondensation due to the absence of active RNA polymerase (33). BCP takes advantage of the paucity of cell-cycle checkpoints in bacteria and the advent of high-resolution imaging to allow the rapid identification of the cell pathway affected by thousands of compounds, eliminating a longstanding bottleneck in academic programs and in antibiotic discovery programs.…”

Physical manipulation of the Escherichia coli chromosome reveals its soft nature

“…Furthermore, MukB binds to DNA very strongly, shows cooperativity, and acts as a macromolecular clamp (49). Other candidates may include transcription for its direct and indirect effect on chromosome compaction (50).…”