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Chromosomal Position Shift of a Regulatory Gene Alters the Bacterial

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Chromosomal Position Shift of a Regulatory Gene Alters the Bacterial Published online 13 July 2015 Nucleic Acids Research, 2015, Vol. 43, No. 17 8215–8226 doi: 10.1093/nar/gkv709 Chromosomal position shift of a regulatory gene alters the bacterial phenotype Veneta Gerganova1, Michael Berger2, Elisabed Zaldastanishvili1, Patrick Sobetzko3, Corinne Lafon4, Michael Mourez4, Andrew Travers5 and Georgi Muskhelishvili1,* 1Jacobs University Bremen, School of Engineering and Science, Bremen, 28758, Germany, 2Institut fur¨ Hygiene, Universitatsklinikum¨ Munster,¨ Munster,¨ 48149, Germany, 3Philipps-Universitat¨ Marburg, LOEWE-Zentrum fur¨ Synthetische Mikrobiologie, Department of Chromosome Biology, Marburg, 35032, Germany, 4SANOFI/ TSU Infectious Diseases, Toulouse, 31036, France and 5MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK Received March 19, 2015; Revised June 10, 2015; Accepted June 30, 2015 ABSTRACT to reconcile the requirement of a roughly 1000-fold com- paction of the DNA with unimpeded accessibility of the Recent studies strongly suggest that in bacterial chromosomal loci to the transcription, replication and re- cells the order of genes along the chromosomal pair machineries. This implies a high degree of structural origin-to-terminus axis is determinative for regula- organization. Indeed, recent biochemical studies of the nu- tion of the growth phase-dependent gene expres- cleoids isolated from classical bacterial model organism Es- sion. The prediction from this observation is that cherichia coli (E. coli) proposed an intricate granular struc- positional displacement of pleiotropic genes will af- ture consistent with a branched DNA supercoil (1), whereas fect the genetic regulation and hence, the cellular genetic studies identified both small topologically isolated phenotype. To test this prediction we inserted the domains (2) and extended macrodomains (3). Recent imag- origin-proximal dusB-fis operon encoding the global ing of the growth phase-dependent variations of the fold- regulator FIS in the vicinity of replication terminus on ing of the bacterial chromosome revealed multiple small domains and also a global bent filament shape consistent both arms of the Escherichia coli chromosome. We with coiled organization (4). Furthermore, two independent found that the lower fis gene dosage in the strains studies, one using the chromosome conformation capture with terminus-proximal dusB-fis operons was com- and the other, the spatiotemporal mapping of functional pensated by increased fis expression such that the communications between the genomic loci, proposed that intracellular concentration of FIS was homeostati- the nucleoid is folded as a plectonemic coil with longitudi- cally adjusted. Nevertheless, despite unchanged FIS nally interwound chromosomal arms (5,6). levels the positional displacement of dusB-fis im- The organization of the nucleoid structure was shown to paired the competitive growth fitness of cells and depend on the binding effects of the small class of highly altered the state of the overarching network regulat- abundant nucleoid-associated proteins (NAPs), which not ing DNA topology, as well as the cellular response only regulate the chromosomal dynamics but also act to environmental stress, hazardous substances and as global regulators of gene expression (7–10). Changing growth conditions substantially affect the composition of antibiotics. Our finding that the chromosomal repo- the NAPs (11), which account for the bulk of protein re- sitioning of a regulatory gene can determine the cel- leased from isolated nucleoids (12,13). Accordingly, the ge- lular phenotype unveils an important yet unexplored nomic spatial transcript profiles (14), the boundaries of the facet of the genetic control mechanisms and paves chromosomal topological domains (15), as well as the struc- the way for novel approaches to manipulate bacterial ture of the nucleoid in vivo (16)andin vitro (17) are affected physiology. by mutations of the NAP genes. Furthermore, the NAPs, to- gether with DNA topoisomerases and components of tran- scription machinery, form an overarching network of inter- INTRODUCTION dependent pleiotropic genes coordinating the growth phase- dependent gene expression and DNA topology in E. coli (7). Transcriptional regulation of the bacterial growth is associ- Importantly, it was observed that distinct genetic loci oc- ated with dynamic structural changes of the unique bacte- cupy specific positions in the cell according to their physi- rial chromosome––the nucleoid. The nucleoid structure has *To whom correspondence should be addressed. Tel: + 49 421 200 3143; Fax: + 49 421 200 3249; Email: [email protected] C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 8216 Nucleic Acids Research, 2015, Vol. 43, No. 17 cal arrangement in the chromosome (18–21). Moreover, it ter generated via classical P1 transduction. The integrity of was found that in E. coli in particular, and ␥-Proteobacteria all inserts with corresponding flanking regions was moni- in general, the order of genes is highly conserved along tored by sequencing (Eurofins MWG Operon). All the used the chromosomal replication origin-to-terminus (OriC-Ter) strains are described in Supplementary Table S3. axis (6). Several lines of evidence suggest that chromosomal position is pertinent to genetic expression. First, it was ob- Growth conditions served that the E. coli chromosome demonstrates distinct transcriptionally active and silenced regions, whereby the The movants and the control strains were grown in rich dYT latter are proposed to serve as organising centres isolating medium either in microtiter plates at 37◦C with shaking (900 chromosomal domains (22). Secondly, limited diffusion of rpm) or in flasks at 37◦C, 150 rpm, unless otherwise stated. the messenger RNAs (mRNAs) from their chromosomal These conditions were used for sample collection of pro- transcription sites was observed, suggesting a physical prox- tein, mRNA and chromosomal DNA from all the strains imity of the site of translation to the cognate gene locus (23). throughout the growth cycle at OD580 of 0.3, 0.5, transition, Thirdly, the diffusion of a DNA binding regulatory protein early and late stationary phase (24 h after the start of the was found to depend on its genomic production site (24). In growth). addition, the gene order along the chromosomal OriC-Ter axis was found to correlate with temporal gene expression Competitive growth fitness measurements during the bacterial growth cycle, such that the genes in- volved at the early and late stages of the growth are located Overnight (O/N) cultures of all experimental strains were respectively closer to the chromosomal origin and terminus diluted 1:10 000 in fresh medium and grown for 12 h. Sub- of replication (6). Finally, a recent study directly showed sequently, the TR and TL strain cultures were mixed with that the chromosomal position affects the reporter gene ex- either WTeng or fis in a 1:1 ratio. The initial mixtures were pression (25). The prediction from all these observations is diluted 1:10 000 in fresh medium and grown for 12 h. After that the effects of global regulatory genes, such as, e.g. those every 12 h the cultures were sampled for plating on selec- of the NAPs, will also depend on their position along the tive plates with kanamycin and non-selective solid medium. OriC-Ter axis of the plectonemically interwound bacterial The mixtures were again diluted 1:10 000 in fresh medium chromosome. and grown for another 12 h. This dilution/plating cycle was In this study, we tested this prediction using the dusB- repeated for at least seven times. The percentage of surviv- fis operon encoding the global regulator FIS––an abun- ing cells was determined from the ratios of colony forming dant NAP transiently expressed during the early exponen- units (CFUs) on the selective and non-selective plates. The tial phase (26,27). We moved the dusB-fis operon from its competitions were performed in triplicates. original location in the vicinity of the OriC, into close prox- imity to the replication terminus on both arms of the circu- Western blotting lar E. coli chromosome. We demonstrate that as predicted, the chromosomal repositioning of the fis gene affects the For sample collection, killing buffer is added, followed by cellular phenotype. a centrifugation at 4◦C, 5000xg, 7 min. After two 1X PBS wash steps, the pellets are solubilized and sonicated (3×30 MATERIALS AND METHODS cycle, 1 s cycles, 80% Amplitude). Protein determination was performed using BCA protein assay kit from PierceTM. Generation of strains with dislocated dusB-fis operons Fifteen microgram of protein was loaded per lane and ran The E. coli K12 strain CSH50 (ara D(lac pro) thi rpsL) on 8–20% gradient polyacrylamide sodium dodecyl sul- was used throughout these experiments. For generation phate gels together with the ColorPlus prestained protein of WTeng, a chloramphenicol cassette was amplified with marker (NEB). The separated proteins were transferred on primers 1 and 2 (Supplementary Table S2) and inserted a0.2␮m nitrocellulose membrane via semy-dry blotting. A between the fis and yhdJ ORFs via the RedE/T homol- list of antibodies and their dilutions is provided
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