Gene order and chromosome dynamics coordinate spatiotemporal gene expression during the bacterial growth cycle Patrick Sobetzkoa, Andrew Traversb,c, and Georgi Muskhelishvilia,1 aSchool of Engineering and Science, Jacobs University Bremen, D-28759 Bremen, Germany; bMedical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; and cFondation Pierre-Gilles de Gennes pour la Recherche, Laboratoire de Biologie et Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, 94235 Cachan, France Edited by Sankar Adhya, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved November 23, 2011 (received for review May 23, 2011) In Escherichia coli crosstalk between DNA supercoiling, nucleoid-as- selection of mutations in fis and tRNA dihydrouridine synthase sociated proteins and major RNA polymerase σ initiation factors (dusB) (essential for fis expression) and also in topA (31), as well as regulates growth phase-dependent gene transcription. We show in rpoC (the β′ subunit of RNAP) under conditions of adaptive that the highly conserved spatial ordering of relevant genes along evolution (32). the chromosomal replichores largely corresponds both to their tem- Although there is substantial evidence for integrated regulation poral expression patterns during growth and to an inferred gradient of NAPs, DNA superhelicity, and RNAP selectivity during the of DNA superhelical density from the origin to the terminus. Genes growth cycle, the mechanism by which this regulation is accom- implicated in similar functions are related mainly in trans across the plished remains obscure. We report here that the conserved or- chromosomal replichores, whereas DNA-binding transcriptional reg- dering of the stage-specific regulatory genes and their targets along ulators interact predominantly with targets in cis along the repli- the replichores corresponds with their temporal expression pat- chores. We also demonstrate that macrodomains (the individual terns during the growth cycle. We propose that this ordering structural partitions of the chromosome) are regulated differently. reflects a gradient of DNA gyrase-binding sites and hence negative We infer that spatial and temporal variation of DNA superhelicity superhelicity from chromosomal origin (OriC) to terminus (Ter) during the growth cycle coordinates oxygen and nutrient availabil- of replication and that the generation of this superhelicity gradient ity with global chromosome structure, thus providing a mechanistic is coupled to energy availability. During the growth cycle changes insight into how the organization of a complete bacterial chromo- in local superhelicity drive morphological changes in chromosome some encodes a spatiotemporal program integrating DNA replica- structure that facilitate the integration of DNA replication and tion and global gene expression. gene expression. gene order conservation | transcriptional regulatory network | Results protein gradients Gene Order. We observed that the ordering, relative to OriC, of the genes encoding the NAPs specific to particular stages of the n Escherichia coli cells the physiological transitions induced by growth cycle [i.e., fis, hupA, β subunit of histone-like protein from Ithe changing growth environment are accompanied by changes E. coli strain U93 (hupB), suppression of td phenotype (stpA), lrp, in DNA superhelical density (1–3), nucleoid structure (4–6), and dps, cbpA, and β subunit of integration host factor (ihfB)] ap- the promoter selectivity of the RNA polymerase (RNAP) holo- proximately reflects their relative abundance during the growth enzyme (3, 7). During the growth cycle both the relative and cycle (8, 9). With one exception, hns, the NAPs associated with absolute concentrations of the abundant nucleoid-associated the higher overall superhelicity characteristic of exponential proteins (NAPs; Table S1) change substantially and correspond- growth are closer to the origin. Conversely, those associated with ingly generate bacterial chromatin of variable composition (8, 9). the lower superhelical density characteristic of the stationary The NAPs stabilize distinct supercoil structures (10–12) selec- phase are closer to the replication terminus (Fig. 1B). The or- tively favoring particular RNAP holoenzymes (13–15). These dering of the genes for the transcriptional-machinery components variable nucleoprotein complexes modulate DNA topology during exhibits a pattern similar to that of the NAPs, with rpoD, encoding the growth cycle (Fig. 1A), optimizing the channeling of supercoil the σ70 factor for vegetative growth, located closer to OriC than energy into appropriate metabolic pathways (16, 17). rpoS, encoding the stationary phase σS factor. Similarly, gyrB (but The expression of the genes determining superhelical density, not gyrA), encoding subunit B of DNA gyrase, is located in close polymerase selectivity, and nucleoid structure is coordinated by proximity to OriC. Gyrase increases negative superhelicity, es- cross-regulation. Thus, factor for inversion stimulation (FIS), pecially with the higher ATP/ADP ratios prevailing on nutritional a NAP abundant during the early exponential phase (18), regulates shift-up (33). In contrast, both topA and topoisomerase III (topB), expression not only of the superhelicity determinants DNA gyrase encoding the DNA-relaxing topoisomerases, are closer to the subunits A and B (gyrA and gyrB) and topoisomerase I (topA)(19– 21) but also other NAP-encoding genes including hns, α subunit of histone-like protein from E. coli strain U93 (hupA), and DNA Author contributions: A.T. and G.M. designed research; P.S. performed research; P.S., A.T., binding protein from starved cells (dps)(22–24) and components and G.M. analyzed data; and P.S., A.T., and G.M. wrote the paper. 38 of the transcription machinery such as σ subunit of RNA poly- The authors declare no conflict of interest. merase rpoS (25). Similarly mutations affecting the selectivity of This article is a PNAS Direct Submission. fl – RNAP in uence NAP production (26 28). Again, mutations in the Freely available online through the PNAS open access option. genes controlling DNA superhelicity affect the production of both 1To whom correspondence should be addressed. E-mail: g.muskhelishvili@jacobs-university. NAPs and the basal transcription machinery (27, 29). This pattern de. of integrated control constitutes a heterarchical network co- See Author Summary on page 355. ordinating chromosome structure with cellular metabolism (27, 28, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 30). A further pointer to this integrated network is the observed 1073/pnas.1108229109/-/DCSupplemental. E42–E50 | PNAS | January 10, 2012 | vol. 109 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1108229109 Downloaded by guest on September 27, 2021 A PNAS PLUS NAP abundance RNA polymerase sigma factors S Plasmid superhelical density ( ) -0.068 -0.043 Growth stage Shift up Early log Mid/late log Transition Stationary (ppGpp spike) B Chromosomal Right Ori NS Right Ter macrodomains OriC Ter Left Ori NS Left Ter Aerobic/anaerobic metabolism, BAC E arcA H seqA rmf fnr DNA replication, OriC Ter rrn genes, etc atp arcB G dnaA D yacG topA DNA topology OriC Ter gyrB parC gyrA sbmC topB parE dps ihfB hupA hfq hupB lrp cbpA hns NAPs OriC Ter crp fis stpA ihfA rsd nusG rpoBC crl rho RNA polymerase fecI dksA OriC Ter modulators rpoZ greB rpoD ssrS rpoS rpoE fliA rpoH GENETICS rpoA nusA rpoN greA Fig. 1. Spatiotemporal organization of chromosomal expression. (A) Temporal changes of NAPs, RNAP composition, and average plasmid DNA superhelicity (σ) during bacterial growth. The phases of growth cycle are correlated with preferred expression of particular NAPs, RNAP holoenzymes and the supercoiling temporal gradient are indicated below. A transient increase in guanosine tetraphosphate (ppGpp) levels occurs at the transition between the exponential and stationary phases. (B) Spatial ordering of regulatory genes on the E. coli chromosome along the OriC–Ter axis. (Top line) Correspondence of macrodomains defined by Valens et al. (40) to linear map. (First bar) Selected genes involved in aerobic/anaerobic metabolism (dark blue), DNA replication (orange), rrn genes (red), and transition phase (brown). Genes on the clockwise (right) replichore are above the bar, and genes on anti-clockwise (left) replichore are below the bar. The atp operon encodes ATP synthase. arcA/arcB encode a two-component system active under microaerobic conditions (61, 62). ArcA also represses rpoS (63). fnr has a dominant role under more strictly anaerobic conditions (61). dnaA, encoding the principal initiator of DNA replication, maps close to OriC, whereas seqA, aninhibitorofreplication initiation at OriC, maps closer to Ter. rmf decreases the availability of ribosomes and maps to a macrodomain immediately adjacent to the Ter macrodomain. (Second bar) Selected genes involved in control of DNA topology. gyrB, a component of DNA gyrase responsible for increasing negative superhelicity, maps close to OriC, whereas the gyrase inhibitor susceptibility to B17 microcin, locus C (sbmC), and topA and topB, both responsible for relaxing DNA, map either close to or within the Ter macrodomain. DNA gyrase inhibitor (yacG), encoding an inhibitor of GyrB, maps close to the center. Chromosomal partition genes C and E (parC and parE) encode the subunits of topoisomerase IV, responsible for decatenation of newly replicated DNA in the terminal region