Colocalization of Distant Chromosomal Loci in Space in E. Coli: a Bacterial Nucleolus

Colocalization of Distant Chromosomal Loci in Space in E. Coli: a Bacterial Nucleolus

Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus Tamas Gaal,1 Benjamin P. Bratton,2 Patricia Sanchez-Vazquez,1 Alexander Sliwicki,1 Kristine Sliwicki,1 Andrew Vegel,1 Rachel Pannu,1 and Richard L. Gourse1 1Department of Bacteriology, 2Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome orga- nization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180° on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity. Colocalization of the rRNA operons required the rrn P1 promoter region but not the rrn P2 promoter or the rRNA structural genes and occurred with and without active transcription. Non-rRNA op- eron pairs did not colocalize, and the magnitude of their physical separation generally correlated with that of their genetic separation. Our results show that E. coli bacterial chromosome folding in three dimensions is not dictated entirely by genetic position but rather includes functionally related, genetically distant loci that come into close proximity, with rRNA operons forming a structure reminiscent of the eukaryotic nucleolus. [Keywords: bacterial chromosome structure; rRNA operons; nucleolus; long-distance chromosomal interactions; ParB-CFP and YFP fusions] Supplemental material is available for this article. Received September 5, 2016; revised version accepted October 5, 2016. The Escherichia coli chromosome is a single ∼4.6-mil- connected by an elongated ter region (Wang et al. 2006; lion-base-pair (bp) circular DNA molecule with a contour Wiggins et al. 2010). In a second model, the left and right length of ∼1.5 mm, ∼1000-fold longer than the bacterial arms of the chromosome are folded together with oriC at cell. One operon of ∼5 kb (the length of a ribosomal one end and ter at the other end of the filament (Youngren RNA [rRNA] operon) in theory could stretch the entire et al. 2014). In a third model, the chromosome is organized length of the bacterial cell if it were present as B-form into four macrodomains and two nonstructured regions DNA without compaction. How the chromosome is com- with a locus able to interact only with another locus in pacted and folded to form the bacterial nucleoid has been the same macrodomain or with a locus in the nonstruc- the subject of intense investigation in recent years, in part tured regions, resulting in genetic and spatial separation because it has implications for genome integrity, cell divi- between different partsof thechromosome (Espéli andBoc- sion, gene expression, and antibiotic resistance. card 2006; Espeli et al. 2008). The models are not mutually Unlike the eukaryotic nucleus, the bacterial nucleoid is exclusive, and certain models could be more applicable to not physically separated from the cytoplasm by a mem- certain times, growth conditions, or bacterial species. brane. However, like the eukaryotic chromosome, the How specific DNA loci are arranged within these broad bacterial chromosome appears to be highly organized, general outlines remains to be determined. The positions changes with the cell cycle, and responds to nutritional of individual loci in space are dynamic (Espeli et al. 2008), conditions (Thanbichler and Shapiro 2006; Dorman but systematic, genome-wide cytological analyses of both 2013; Jin et al. 2013; Dame and Tark-Dame 2016). This the E. coli and Caulobacter crescentus chromosomes con- has led to characterizations of the nucleoid as a complex cluded that the linear order of genes in space recapitulates and evolving but coherent object whose intrinsic mechan- the genetic map (Viollier et al. 2004; for review, see Wang ical features govern its structure (Fisher et al. 2013). and Rudner 2014). The linear array has been pictured as a In one model, the E. coli chromosome is described as a “bottlebrush” in which interwound loops extrude from a condensed body consisting of a compressed linear array with oriC at the center and the two ends of the array © 2016 Gaal et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publi- cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After Corresponding author: [email protected] six months, it is available under a Creative Commons License (At- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.290312. tribution-NonCommercial 4.0 International), as described at http:// 116. creativecommons.org/licenses/by-nc/4.0/. 2272 GENES & DEVELOPMENT 30:2272–2285 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/16; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press A bacterial nucleolus central nucleoid scaffold (Le et al. 2013; Wang and Rudner Lewis et al. 2000; Cabrera and Jin 2003; Endesfelder 2014). The ter macrodomain is organized by the MatP and et al. 2013; Jin et al. 2013). Fluorescence-labeled “tran- YfbV proteins (Thiel et al. 2012), but the identities of the scription foci” were observed in fast-growing strains short- and long-range interactions that might organize encoding fusions of GFP to RNAP, correlating with the other parts of the chromosome remain unclear. DNA- high numbers of RNAP molecules expected to be en- binding proteins such as SeqA, SlmA, MukB, and H-NS gaged in transcribing rRNA under these conditions. have been proposed to be important for nucleoid organiza- Consistent with the idea that at least some of these fluo- tion by contributing to the interactions between different rescent foci represented rRNA operons, their intensities loci (Wang and Rudner 2014; Dame Tark-Dame 2016). declined in starved or slowly growing cells. Superresolu- Our interest in E. coli chromosome structure emerged tion imaging showed that RNAPs formed clusters that from our long-term focus on the mechanisms responsible occupied specific regions of the nucleoid under the for ribosome synthesis and its control (Paul et al. 2004). conditions examined; i.e., at fast growth rates when there rRNA synthesis rates are coordinated with the cell’s are large numbers of RNAPs actively transcribing rRNA translational capacity following nutritional shifts such operons (Bratton et al. 2011; Bakshi et al. 2013; Endes- as amino acid starvation. The primary signal molecules felder et al. 2013; Stracy et al. 2015). However, such stud- responsible for adjusting ribosome synthesis rates to ies directly addressed only the locations of RNAP and changing nutritional conditions are ppGpp, an “alar- not the positions of the different rRNA operons. Since mone” whose synthesis is induced by uncharged transfer detection depended on the presence of high numbers of RNAs (tRNAs) in the ribosomal A site (Paul et al. 2004; RNAPs, no information was obtained about the positions Potrykus and Cashel 2008; Ross et al. 2016), and the con- of the rDNA at low growth rates when rRNA transcrip- centration of the initial NTP responsible for forming the tion was low. rRNA transcript (Murray et al. 2003). In contrast to previous studies, we took a direct ap- When cells grow rapidly in rich medium, the majority of proach to address whether rRNA operons are in close the cell’s RNA polymerase (RNAP) is engaged in tran- proximity in space. Using recombineering and counterse- scribing rRNA in order to produce the large number of lection approaches, we inserted sites for different fluores- ribosomes needed to meet the cell’s translational require- cent DNA-binding proteins adjacent to the different ments. Conversely, when cells grow slowly, there is much rRNA operons as well as many other genetic loci. We less rRNA transcription in order to shift the cell’s utiliza- then compared the relative positions of rRNA operons tion of nutritional resources to other priorities (Paul et al. and other genes pairwise in live cells. 2004). The combined effects on rRNA promoters of We show here that most of the rRNA operons are in various regulators—including ppGpp, NTPs, Fis (a nucle- close proximity in space. The physical separation between oid-associated protein that activates rrn P1 promoters), six of the seven rRNA operons (all except rrnC) is in the and other factors—account for the correlation between range of ∼80 to ∼130 nm independent of the genetic dis- growth rate and ribosome synthesis rates, and these fac- tance between them (in kilobases). Our data suggest that tors can compensate for each other in mutants lacking in- E. coli rRNA operons form a structure reminiscent of a eu- dividual regulators (Paul et al. 2004). karyotic nucleolus. The promoter region is necessary A complete understanding of the control of ribosome and sufficient for this “colocalization” of rRNA operons, synthesis depends on identification of not only the cis-act- but, surprisingly, the formation and persistence of the nu- ing sites and the trans-acting factors responsible for regu- cleolus-like structure do not depend on formation of an lation but also the cellular locations of these events in open transcription initiation complex (RPO) or active space and time. In theory, changes in the spatial locations transcription. We speculate that this structure facilitates or organization of the rRNA operons could accompany or ribosome assembly, as has been proposed in eukaryotes, even contribute to the changes in rRNA synthesis rates and that it also contributes to folding of the bacterial that result from changes in nutritional and environmental chromosome.

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