Orden de los genes y la dinámica de los cromosomas en coordenadas de expresión génica espacio-temporal durante el ciclo de crecimiento de las bacterias

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    Gene order and chromosome dynamics coordinatespatiotemporal gene expression during thebacterial growth cyclePatrick 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 Applique,Ecole Normale Suprieure 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 reviewMay 23, 2011)

    In Escherichia colicrosstalk between DNA supercoiling, nucleoid-as-

    sociated proteins and major RNA polymerase initiation factorsregulates growth phase-dependent gene transcription. We show

    that the highly conserved spatial ordering of relevant genes along

    the chromosomal replichores largely corresponds both to their tem-

    poral expression patterns during growth and to an inferred gradient

    of DNA superhelical density from the origin to the terminus. Genesimplicated in similar functions are related mainly in trans across the

    chromosomal replichores, whereas DNA-binding transcriptional reg-

    ulators interact predominantly with targets in cis along the repli-

    chores. We also demonstrate that macrodomains (the individualstructural partitions of the chromosome) are regulated differently.

    We infer that spatial and temporal variation of DNA superhelicity

    during the growth cycle coordinates oxygen and nutrient availabil-

    ity with global chromosome structure, thus providing a mechanistic

    insight into how the organization of a complete bacterial chromo-

    some encodes a spatiotemporal program integrating DNA replica-

    tion and global gene expression.

    gene order conservation | transcriptional regulatory network |protein gradients

    In Escherichia coli cells the physiological transitions induced by

    the changing growth environment are accompanied by changesin DNA superhelical density (13), nucleoid structure (46), andthe promoter selectivity of the RNA polymerase (RNAP) holo-enzyme (3, 7). During the growth cycle both the relative andabsolute concentrations of the abundant nucleoid-associatedproteins (NAPs; Table S1) change substantially and correspond-ingly generate bacterial chromatin of variable composition (8, 9).The NAPs stabilize distinct supercoil structures (1012) selec-tively favoring particular RNAP holoenzymes (1315). These

    variable nucleoprotein complexes modulate DNA topology duringthe growth cycle (Fig. 1A), optimizing the channeling of supercoilenergy into appropriate metabolic pathways (16, 17).

    The expression of the genes determining superhelical density,polymerase selectivity, and nucleoid structure is coordinated by

    cross-regulation. Thus, factor for inversion stimulation (FIS),a NAP abundant during the early exponential phase (18), regulatesexpression not only of the superhelicity determinants DNA gyrasesubunits A and B (gyrA and gyrB) and topoisomerase I (topA) (1921) but also other NAP-encoding genes including hns, subunit ofhistone-like protein from E. coli strain U93 (hupA), and DNAbinding protein from starved cells (dps) (2224) and componentsof the transcription machinery such as 38 subunit of RNA poly-merase rpoS (25). Similarly mutations affecting the selectivity ofRNAP influence NAP production (2628). Again, mutations in thegenes controlling DNA superhelicity affect the production of bothNAPs and the basal transcription machinery (27, 29). This patternof integrated control constitutes a heterarchical network co-ordinating chromosome structure with cellular metabolism (27, 28,30). A further pointer to this integrated network is the observed

    selection of mutations in fis and tRNA dihydrouridine synthase(dusB) (essential forfis expression) and also in topA (31), as well asin rpoC (the subunit of RNAP) under conditions of adaptiveevolution (32).

    Although there is substantial evidence for integrated regulationof NAPs, DNA superhelicity, and RNAP selectivity during thegrowth cycle, the mechanism by which this regulation is accom-plished remains obscure. We report here that the conserved or-

    dering of the stage-specifi

    c regulatory genes and their targets alongthe replichores corresponds with their temporal expression pat-terns during the growth cycle. We propose that this orderingreflects a gradient of DNA gyrase-binding sites and hence negativesuperhelicity from chromosomal origin (OriC) to terminus (Ter)of replication and that the generation of this superhelicity gradientis coupled to energy availability. During the growth cycle changesin local superhelicity drive morphological changes in chromosomestructure that facilitate the integration of DNA replication andgene expression.

    Results

    Gene Order. We observed that the ordering, relative to OriC, ofthe genes encoding the NAPs specific to particular stages of thegrowth cycle [i.e., fis, hupA, subunit of histone-like protein from

    E. coli strain U93 (hupB), suppression of td phenotype (stpA), lrp,dps, cbpA, and subunit of integration host factor (ihfB)] ap-proximately reflects their relative abundance during the growthcycle (8, 9). With one exception, hns, the NAPs associated withthe higher overall superhelicity characteristic of exponentialgrowth are closer to the origin. Conversely, those associated withthe lower superhelical density characteristic of the stationaryphase are closer to the replication terminus (Fig. 1B). The or-dering of the genes for the transcriptional-machinery componentsexhibits a pattern similar to that of the NAPs, withrpoD, encodingthe 70 factor for vegetative growth, located closer to OriC thanrpoS, encoding the stationary phase S factor. Similarly, gyrB (butnot gyrA), encoding subunit B of DNA gyrase, is located in closeproximity to OriC. Gyrase increases negative superhelicity, es-

    pecially with the higher ATP/ADP ratios prevailing on nutritionalshift-up (33). In contrast, both topA and topoisomerase III (topB),encoding the DNA-relaxing topoisomerases, are closer to the

    Author contributions: A.T. and G.M. designed research; P.S. performed research; P.S., A.T.,

    and G.M. analyzed data; and P.S., A.T., and G.M. wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission.

    Freely available online through the PNAS open access option.

    1To whom correspondence should be addressed. E-mail: g.muskhelishvili@jacobs-university.

    de.

    See Author Summary on page 355.

    This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

    1073/pnas.1108229109/-/DCSupplemental.

    E42E50 | PNAS | January 10, 2012 | vol. 109 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1108229109

    http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=ST1mailto:[email protected]:[email protected]://www.pnas.org/content/109/2/E42/1http://www.pnas.org/content/109/2/E42/1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplementalhttp://www.pnas.org/cgi/doi/10.1073/pnas.1108229109http://www.pnas.org/cgi/doi/10.1073/pnas.1108229109http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplementalhttp://www.pnas.org/content/109/2/E42/1mailto:[email protected]:[email protected]://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=ST1
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    Ter. Spatial organization of genes modifying the transcriptionmachinery [transcriptional terminator Rho (rho), the N utiliza-tion substance gene (nus) factors, the gre factors, dnaKsuppressor

    (dksA), curly (crl)] and genes sustaining catabolism and energyproduction under aerobic [ATP synthase (atp) operon], micro-aerobic [two component signal transduction system (arcA/B)], and

    NAP abundance

    RNA polymerase

    sigma factors

    Plasmid superhelical

    density ( )

    Growth stage

    S

    -0.068 -0.043

    Shift up Early log Mid/late log Transition Stationary

    (ppGpp spike)

    TerOriC

    Ori NS Right Ter

    Ori NS Left Ter

    Ter

    TerOriC

    rpoSrpoDTerOriC

    rpoBC

    rpoA

    OriC

    Chromosomal

    macrodomains

    Aerobic/anaerobic

    metabolism,

    DNA replication,rrn genes, etc

    DNA topology

    NAPs

    RNA polymerase

    modulators

    TerOriC

    Right

    Left

    fliArpoE

    fecI

    rpoH

    rpoN

    cbpA hns

    stpA

    fnr

    atp

    arcA

    arcB

    hupA hupB

    ihfA

    ihfBlrp

    dps

    fiscrp

    hfq

    seqA

    dnaA

    rmf

    rpoZ ssrS

    crl

    rsd

    dksA

    greA

    greB

    nusA

    nusG

    rho

    gyrB gyrAparC

    parE

    yacG topA

    topBsbmC

    H

    G

    E

    D

    BAC

    A

    B

    Fig. 1. Spatiotemporal organization of chromosomal expression. (A) Temporal changes of NAPs, RNAP composition, and average plasmid DNA superhelicity ()

    during bacterial growth. Thephasesof growthcycle 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 stationaryphases. (B) Spatial ordering of regulatory genes on the E. colichromosome along the OriCTer 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 theclockwise (right) replichore are above thebar, andgenes on anti-clockwise (left)replichore are below thebar. The atp operon encodes

    ATP synthase. arcA/arcB encode a two-component systemactive under microaerobic conditions (61, 62). ArcA also represses rpoS(63). fnrhas a dominant role under

    more strictly anaerobic conditions (61). dnaA, encoding the principal initiator of DNA replication, maps close to OriC, whereas seqA, an inhibitor of replication

    initiation at OriC, maps closerto Ter. rmfdecreasesthe availability of ribosomes andmaps to a macrodomainimmediatelyadjacentto theTer 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 newlyreplicated DNA in the terminal region (64) and relaxationof negative supercoils(65).

    (Third bar) Selected genes encoding NAPs. The NAP-encoding gene closest to OriC is hupA, encoding histone-like protein from E. colistrain U93 (HU). Its early

    expression relative to hupB, encoding Hu (9), could bufferhigh negative superhelicity generatedby DNA gyrase (36). HU2 and HU, but not HU2, constrainhigh

    superhelical densities in vitro (9). A mutation in hupA both increases growth rate and antagonizes histone-like nucleoid-structuring protein (H-NS) regulation of

    certain transcription units (6). High frequency of recombination (Hfq) is a nucleic acid-bindingprotein whose major role is that of an RNAchaperone, butit also may

    act as a DNA-binding NAP (8). lrp is activated by ppGpp (38). (Fourth bar) Selected genes involved in modulating RNAP activity, including factor-utilization reg-

    ulators, secondary channel-binding proteins, termination/elongation factors, and RNAP subunits. factor-utilization regulators (light green): subunit of RNApolymerase (rpoZ), mapping close to the origin, encodes the subunit of RNAP, which confers a preference for utilization of 70 (28). Regulator of sigma D (rsd)

    encodes an anti-70 (66),whereas crlconfers a strong preferencefor S utilization(67). Note that both rpoZand crlmapcloser to OriC than do therespective factors

    whose activity they affect. Theencoded regulatory pattern thus reflects a shift from predominantly70 use close to OriCto S availability in thecentral regionof the

    chromosome. Secondary channel-binding proteins (plum): growth regulator A and B, transcription elongation factors ( greA and greB) both map in the region

    containing many genes expressed during rapid growth. GreA has been shown to stimulate initiation and transcription of genes involved in aerobic metabolism,

    including the atp operon (68, 69) as well as the rrnB P1 promoter in vitro (70, but also see ref. 71). DksA, like the plasmid-encoded quorum sensing regulator (TraR)

    protein (72), inhibits ribosomal protein promoters and rrn initiation (73, 74)and is more distant from OriC than is greA. In vivo it would act to reduce the rate of rrn

    initiation and hence antagonize transcription foci formation. Termination/elongation factors (red): The termination factor Rho is encoded by a gene located very

    close to OriC. This location may compensate for the antagonistic effect of high negative superhelicity on transcription termination, which involves the rewinding of

    DNA. RNAP subunits:The mappositions relative to OriC of rpoD and rpoS, respectively encoding70andS, correspondto theirrelative orderof temporal expression.

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    anaerobic (fnr) conditions, as well as those involved in activationand negative modulation of replication [DNA replication initiatorprotein DnaA (dnaA) and sequestration of origin (seqA)] andtranslation [ribosomal RNA (rrn) operons and ribosome matura-tion factor (rmf)], exhibit a similar pattern of chromosomal or-dering (legend of Fig. 1B and Table S2).

    We asked whether the gene order of regulatory elements as-sociated with the temporal pattern of transcription in Escherichiacoli was conserved in other -Proteobacteria. Analysis of 131-proteobacterial genomes showed that the relative distance of

    such genes from the origin and terminus was highly conserved (Fig.2A). Similar analysis of all orthologous genes demonstrated that,although the relative distance was conserved, the specific repli-chore was not conserved to the same extent (Fig. 2B). Simulationssupported this observed bias (Fig. S1). Furthermore, essentiallythe same picture emerged when we analyzed the more distantlyrelated Gram-positive bacteria (Fig. S2 A and B). We concludethat conservation of relative distance along the OriCTer axisoverrides the replichore coherence.

    Targets. Not only are the genes coordinating the major regulatorypathways ordered; their targets are ordered as well. Analyses ofthe distribution density of binding sites for E70 and ES hol-oenzymes compiled in RegulonDB (34) show opposite spatialbiases. For the vegetative 70 factor the highest percentage oftargets is found around the origin, whereas for the stationary phaseS factor the highest target density is close to the terminus (Fig. 3Aand B), consistent with both the closer location of rpoD to OriCand the temporal division of labor between 70 and S during thebacterial growth cycle (7). Similarly, the average density of bindingsites for DNA gyrase (35) diminishes byfive- to 10-fold (Fig. 3C)from OriC to Ter (36). This organization could generate a gradientof superhelical density (Fig. 3D) correlating with that of E70

    targets and anti-correlating with ES targets, as expected from the

    opposite supercoiling preferences of these holoenzymes (3, 28)and in keeping with the requirement of high negative superhelicityfor initiation of OriC replication (37).

    For the major NAPs, despite distinct chromosomal locationsand abundances during the growth cycle, a high percentage ofthe binding sites compiled in RegulonDB occurs around theorigin (Fig. 3 EH). Among the NAPs encoded in the Ter-proximal region, only integration host factor (IHF) targets acti-

    vating binding sites in the vicinity of Ter (Fig. 3F), whereas thestationary-phase regulator leucine responsive protein (LRP) (38)and the global repressor H-NS (39) both preferentially target theOriC-proximal region (Fig. 3 G and H). Additionally HU, themajor supercoil-constraining NAP for which no binding site in-formation is available, has distinct and opposite functional

    effects at the OriC and Ter ends of the chromosome, respectivelyreducing and increasing transcription (Fig. S3).To explore the relevance of this apparent chromosomal ordering

    of regulators for chromosomal expression, we used the Gene On-tology (GO) database describing the gene products in terms of as-sociated metabolic processes and investigated the spatial organi-zation of functional groups of genes. Many of these genes would bethe ultimate targets of the regulation. Using scanning windows of

    variable sizes (0.10.5 Mb), we mapped the genes in the GO data-base, identifying the significant matches between functionally com-plementing windows on the chromosome (Figs. S4 and S5). Wedetermined the ratio of all significant combinations forcis (matching

    windows located on the same replichore) and trans (matching win-dows on distinct replichores) arrangements. Although the GO treeorganization comprises 13 complexity levels from the most broad

    down to specific functions, most significant matches were observedat levels three and four (Figs. S6 and S7). Fig. 4A shows that func-tionally matching groups are localized predominantly on oppositereplichores within the rrn macrodomain (36) comprising OriC andthe nonstructured left (LNS) and right (RNS) macrodomains (40).Importantly, the predominantly trans organization of these groups

    was significant only when the chromosomal gene order was alignedalong the OriCTer axis. We infer that a majority of the functionallyrelated groups are organized at comparable distances from thecenter of symmetry at OriC and are related in trans across thereplichores. We denote these spatially coordinated matching GOgroups of genes as maGOGs.

    We next analyzed the interactions between the E. coli DNA-binding transcriptional regulators and their targets compiled inthe RegulonDB in the form of a static transcriptional regulatory

    A

    B

    Fig. 2. Arrangement of important regulatory elements according to their po-

    sition relative to OriC in the-Proteobacteria. (A) Genes located on theright and

    left replichores are indicated above and below the chart, respectively, as in

    Fig.1B. Horizontal bars show spatial distributions of orthologs; black color indi-

    cates the highest density for eachindividual distribution. The plotshows a strong

    conservation of chromosomal positions suggesting that origin-focused gene

    positioning is a major selection criterion in -Proteobacteria. (B) Relationship

    among the phylogenetic distance, the correlation of distance to origin, and the

    replichore coherence (the conserved replichore identity of orthologs) for all

    -Proteobacteria. The points represent the data on pair-wise species compar-

    isons computed using the Pearson correlation coefficient of either distances to

    origin or replichore identity (right/left) of all orthologous pairs. The points are

    color-coded in the 3D plot. Red indicates a higher correlation of distance to

    origin than replichore coherence; blue indicates higher replichore coherence.

    The predominance of red points indicates the stronger conservation of distance

    to origin. The phylogenetic distances in B were derived from the tree of -Pro-

    teobacteria (http://www.cbrg.ethz.ch/research/orthologous/speciestrees).

    E44 | www.pnas.org/cgi/doi/10.1073/pnas.1108229109 Sobetzko et al.

    http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=ST2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF3http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF4http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF5http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF6http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF7http://www.cbrg.ethz.ch/research/orthologous/speciestreeshttp://www.pnas.org/cgi/doi/10.1073/pnas.1108229109http://www.pnas.org/cgi/doi/10.1073/pnas.1108229109http://www.cbrg.ethz.ch/research/orthologous/speciestreeshttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF7http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF6http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF5http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF4http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF3http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=ST2
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    network (TRN). These analyses again revealed macrodomains inwhich spatially coordinated interactions between the regulatorsand targets were significantly enriched. However, in contrast to

    the spatial organization of maGOGs, significant TRN inter-actions occur mainly in cis along the replichores, largely corre-sponding to intermacrodomain communication (Fig. 4B).

    We also analyzed the spatial organization of couplons, entitiescorresponding to intersections of regulons of distinct factorsand NAPs and containing functionally related genes (27). Cou-plon analyses again demonstrated preferential communicationsin trans across the replichores (Fig. 4C). We infer that chromo-somal arrangement of functionally related genes and the spatialcommunications between transcriptional regulators and theirtargets are essentially orthogonal with respect to each other.

    Expression Profiles. Any valid regulatory network must describemeaningfully the changes in gene expression both during normalgrowth and when expression is perturbed by mutation. The

    analyses shown in Figs. 3 and 4 use the static E. coli Regulondatabase, which contains neither temporal nor spatial orderinginformation. To analyze the dynamic organization of communi-

    cations in the effective transcript profiles (41) obtained underdefined conditions, we integrated the maGOGs, TRN, andcouplon networks in a single combined heterarchical network(HEN). In such a network, as opposed to a hierarchical network,there is no subordination to one single dominant component.The HEN (Fig. 4D) served as a template for mapping the ef-fective transcript profiles. The observed gene ordering predictedthat relative transcription during exponential and stationarygrowth phases would be ordered in a similar spatial mannerrelative to OriC and Ter. The results fully confirmed this pre-diction, clearly demonstrating a division of labor between theOriC and Ter chromosomal ends in temporal expression profiles.In exponentially growing wild-type cells the OriC-proximal region

    was transcriptionally more active than the Ter-proximal regionwith the active regions corresponding largely to the rrn domain

    A E

    B F

    C G

    D H

    Fig. 3. Organization of binding sites for the major initiation factors, DNA, gyrase, and NAPs in the chromosome (RegulonDB). Distributions were calculated by

    using a sliding window of 400 kb and normalizing over the total gene number for each window. The replichores are organized from OriC to Ter (left to right).

    The frequency distributions (ordinate) are plotted above the zero in the ordinate for the right replichore and below the zero for the left replichore. (A) Genomic

    distributions for RNAP70-regulated promoters. (B) Genomic distributions for S-regulated promoters. (C) Genomic distributions of gyrase-binding sites. (D)

    Inferred gradient of negative superhelical density along the OriCTer axis in exponentially growing cells. (E) Genomic distribution of FIS-binding sites. (F)

    Genomic distribution of IHF-binding sites. (G) Genomic distribution of H-NSbinding sites. (H) Genomic distribution of LRP-binding sites. The chromosomal

    position for each regulator gene is indicated, and the direction of the effect is rainbow color-coded with blue for repression and red for activation. The Ori, Ter,

    left, and right macrodomains (green, cyan, blue, and red lines, respectively) are indicated on the chromosomal replichores above and below the distributions.

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    comprising the Ori and LNS macrodomains (Fig. 5 A and B).Similar spatiotemporal division of labor was observed in an rpoSmutant lacking S, but the pattern relative to wild-type cells wasreversed, with the Ter-proximal region being more favored duringthe exponential phase and the OriC-proximal region during thestationary phase (Fig. 5 C and D). Also, in an rpoZ mutant fa-

    voring the ES holoenzyme (28), the Ter-proximal region wasfavored during the exponential phase, an effect that could be re-

    versed by overproduction of70 activating the OriC end (Fig. 5 E

    and F). Importantly, although the effects of factors mainly in-volved trans communications, consistent with the orthogonal or-ganization of regulatory communications in HEN, the mutationsof fis and hns genes also substantially affected the cis communi-cations, which were largely buffered in wild-type cells (Fig. 5 GL). Furthermore, some of these spatiotemporal patterns could beclosely imitated by manipulating the composition of NAPs andDNA superhelicity (Fig. S8), validating the usefulness of HEN forgene-expression analyses.

    A B

    C D

    Fig. 4. Organization of functional groups and regulatory communications in the E. coli chromosome. (A) maGOGs. (B) TRN. (C) Couplons. (D) HEN. The

    circular genome is represented as a pair of multicolored parallel lines corresponding to the right ( Upper line) and left (Lower line) replichores. On thereplichores the macrodomains (colored as in Fig. 3) and the rrn functional domain (orange dashed line) are indicated. All trans communications occur between

    the upper and lower lines, whereas cis communications occur along the lines. The order of regulatory genes on the right and left replichores is indicated

    above and below each network, respectively, organized from OriC to Ter.

    E46 | www.pnas.org/cgi/doi/10.1073/pnas.1108229109 Sobetzko et al.

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    Discussion

    We have demonstrated that during the E. coli growth cycle inbatch culture the temporal pattern of stage-specific gene ex-pression corresponds largely with the gene order along the tworeplichores. This correspondence is apparent not only for theprincipal regulatory genes but also for their targets. Importantly,this property is a highly conserved characteristic of the repli-chores in both Gram-negative -Proteobacteria and Gram-posi-tive bacteria, implying that the ordering is related to DNA

    replication. Moreover, the most conserved property for a partic-ular gene appears to be not the precise location but the relativedistance of the gene from OriC and Ter, independent of repli-chore (Fig. 2 and Figs. S1 and S2 A and B). This finding suggeststhat the selective determinant is itself a variable property thatalso is dependent on the relative OriC/Ter distance and is con-sistent with macrodomains sharing many functions (Fig. S9).

    In fast-growing E. coli the bidirectional replication starting fromOriC creates a gradient of gene dosage from OriC to Ter, such thatthe resulting origin-proximal relative increase in gene dosage po-tentially could increase gene expression in that region. Indeed, it isprecisely this region that is preferentially active during exponentialgrowth (Fig. 5A). However, variation in gene dosage is likely to beonly one of a number of regulatory influences. Thus, the origin-

    proximal region contains a five- to 10-fold higher average densityof gyrase-binding sites than the Ter-proximal region (Fig. 3C) andalso is the preferred target region forE70 holoenzyme and for FISprotein (Fig. 3 A and E), implicated in evolutionary modulation ofDNA superhelicity (25). We infer that the gradient of gyrasebinding sites is indicative of potential gradients of negative su-perhelical density from OriC to Ter along the replichores (Fig.3D). The existence of such gradients corresponds well with boththe pattern of holoenzyme and NAP targets and the greater den-sity of supercoiling-sensitive genes (e.g.,rpoZ,fis,hupA, andrrn) inthe OriC end (16, 36, 42), as well as with the activation of the rrnmacrodomain under conditions of high negative superhelicityduring exponential growth (Fig. S10 A and B). The gradient ofgyrase-binding sites would provide a simple mechanism for cou-pling energy availability to superhelical density (2, 33) and, be-cause initiation of DNA replication is enhanced by high negativesuperhelicity (37), also to replication itself.

    Although the spatial order of selected stage-specific genescorresponds well to the temporal order of expression during thegrowth cycle, the position of certain other genes, although highlyconserved, does not. Genes in this category in E. coli include hnsand gyrA. Although in Gram-positive bacteria gyrA and gyrB mapin close proximity to OriC (Fig. S2C), a rationale for the separateconserved locations of gyrA and gyrB in -Proteobacteria is notobvious. On shift-up expression of gyrB increases to a propor-tionally much greater extent than expression of gyrA (19). GyrAand GyrB together form a heterotetramer, and if GyrB werelimiting the distinct locations would favor the use of the gyrase-binding sites in the origin-proximal region. We hope to clarify any

    requirement for separate placement of the gyrA and gyrB genes inE. coli by switching their chromosomal positions.

    Both FIS and H-NS principally target the origin-proximal re-gion, potentially delimiting short topological domains (43). How-ever, fis is expressed at maximal levels during the exponentialphase and maps in the origin-proximal region, in contrast to hns,

    which maps in the Ter macrodomain. In this context MonteroLlopis et al. (44) recently reported that both Escherichia coli andCaulobacter crescentus spatially organize translation so that themRNA product of a gene is translated in close proximity to itsposition in the nucleoid. This observation has profound implica-tions for gene regulation and chromosome structure. If NAPproduction is localized, then diffusion of the resultant DNA-binding proteins within the nucleoid will be anomalous, generatingconcentration gradients. The outcome for nucleoid organization at

    A

    B

    G

    H

    M

    C

    D

    I

    J

    E

    F

    K

    L

    N

    Fig. 5. Spatiotemporal HEN patterns. The circular genome is represented as

    a pair of thin parallel lines corresponding to the right (Upper line) and left

    (Lower line) replichores as in Fig.4. Macrodomainsare indicated in color on the

    right (Upper) and left (Lower) replichores organized from OriC to Ter. (A) Ef-

    fective HEN (eHEN) pattern of exponentially growing wild-type cells. (B) eHEN

    pattern of transcripts from stationary wild-type cells. Red indicates coherentlyenhanced and blue indicates coherently reduced numbers of expressed genes

    in matching windows; black indicates an absence of coherence. Note the

    switch in the activation of the OriC and Ter ends of the chromosome at the

    transition from exponential to stationary growth. (C) eHEN pattern of rpoS

    cells lacking S obtained during exponential phase. (D) eHEN pattern of rpoS

    cells in stationary phase. Note the spatiotemporal inversion of theactivationof

    the OriC and Ter ends of the chromosome with respect to wild-type cells. ( E)

    eHEN pattern of exponentially growing rpoZcells favoring ES. (F) As in E, but

    the cells were overproducing 70 from an episome (28). Note the 70-de-

    pendent coherent activation and repression of communications in the OriC

    and Ter ends of the chromosome, respectively. (G and H) eHEN patterns of

    wild-type cells grown under conditions of high and low superhelicity, re-

    spectively. (I and J) Patterns of hns-mutant cells grown under conditions of

    high and low superhelicity, respectively. Note the supercoiling-dependent

    coherent activation and repression of communications in the left and right

    replichores, respectively. (K and L) eHEN patterns offi

    s-mutant cells grownunder conditionsof highand lowsuperhelicity,respectively. Note thecoherent

    activation of distinct supercoiling-dependent cis and trans communications. In

    AD the cells were grown in minimal medium. In EL the cells were harvested

    during exponential growth in rich double-YT medium. (M and N) Model of

    chromosomal morphology changing with DNA superhelicity and NAP gra-

    dients on transition from exponential (M) tostationary (N) growth. For clarity,

    only the interactions between the gradients of FIS (pink) and H-NS (blue) are

    shown. Although FIS levels decline dramatically on transition to stationary

    phase, the compaction of the nucleoidalong the OriCTeraxisenables H-NS to

    establish repression. The chromosome is depicted as a plectoneme, but the

    model is equally consistent with a toroidal scaffold, which would maintain the

    separation of the replichores (49, 50). The macrodomains are indicated by

    colors, and approximate chromosomal positions of the fis and hns genes are

    shown. The expression data for mapping onto the HEN connectivity patterns

    were taken from Dong and Schellhorn (59) in AD, from Geertz et al. (28) in E

    and F, and from Blot et al. (16) in GL.

    Sobetzko et al. PNAS | January 10, 2012 | vol. 109 | no. 2 | E47

    GENETICS

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    http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF9http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF10http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF10http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF10http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF10http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF10http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF9http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108229109/-/DCSupplemental/pnas.201108229SI.pdf?targetid=nameddest=SF1
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    a given locus then depends on the relative local concentrations ofdifferent NAPs, which in turn depend on the distance from the siteof production and the total number of molecules available. In thismodel FIS and H-NS would be expected to be more dominant onnucleoid structure and function in the origin-proximal regionduring the exponential and stationary phases, respectively.

    The relation of NAP function to negative superhelicity is con-sistent with the concept that NAPs can act to buffer superhelicaldensity not only at the local level, as previously reported for the

    rrnA P1 promoter (45), but also at the more global level of thechromosome and macrodomains (Fig. 5 GL). In this respecta correlation between the gradient of gyrase-binding sites and thefunctional effect of HU (36) is particularly striking (Fig. S3).In thecontext of the transcriptional effect of HU, the apparentlyanomalous positions of the rrnG and rrnH genes approximatelyhalfway between OriC and Ter delimit the rrn functional macro-domain (36). Importantly, although all seven rRNA operons havebeen shown to be necessary for rapid adaptation, only fi ve arenecessary to support near-optimal growth (46), and it is not knownhow the rebalancing of rRNA transcription after shift-up is dis-tributed among the rrn operons. Also, in a strain lacking ppGpp(the negative regulator of rRNA transcription), the regions con-taining up-regulated genes whose expression is stimulated by highnegative superhelicity are in close proximity to rrnG and rrnH(Fig.S10 C andD). This finding together with the propensity for forming

    variable transcription foci (47) raises the possibility that, in growingcells with a full complement of rrn operons, the regulation of in-dividual rrn operons depends on their position in the chromosome.

    A related issue is how the scattered distribution of tRNAgenes around the chromosome can be reconciled with chromo-somal positioning (Fig. S11). Unlike rRNA genes, the expressionof tRNA genes must be consistent both with growth and withdifferences in the relative amounts of iso-accepting tRNAs andcodon use at different growth rates (ref. 48 and legend of Fig.S11). For both rRNA and iso-accepting tRNA genes, as for othergenes described here, distance from the origin is more conservedthan replichore (Fig. S11B).

    Topological Model for Temporal Gene Expression. The deduced logicof spatial communications in the E. coli chromosome suggestsa simple topological model of the circular chromosome folded asa negative supercoil in which close spatial proximity of replichoreseither within the rrn macrodomain comprising the Ori and flank-ing flexible macrodomains or between the rrn and Ter macro-domains supports alternative communication during exponentialand stationary growth. These morphological transitions could oc-cur as a consequence of changes in superhelical density, which

    would affect the configuration of DNA directly and also wouldalter the relative affinities of the different NAPs for DNA. Thealternative configurations, with varying OriC and Ter separation,are consistent with the formation of transcription foci (47), thereorganization and physical extension of nucleoids during expo-nential growth (4951), and the compaction of nucleoids in the

    stationary phase (51, 52). This last effect is counteracted by theactivator of rRNA operons FIS, which is abundant in exponentialphase (4, 18), suggesting that the conformational transition in thenucleoid is associated with changing gradients of competing NAPs(Fig. 5 M and N). Although the extent to which gene expressionhas a linear relationship with gene copy number is unknown, it isconceivable that the relative increase in gene dosage resultingfrom the initiation of replication would increase the competitiveadvantage of regulators encoded in the origin-proximal region,

    whereas a balanced OriC-to-Ter stoichiometry on cessation ofgrowth would abolish such an advantage (Fig. S12).

    How could such morphological transitions be coupled to en-ergy availability? A primary response to nutritional shift-up un-der aerobic conditions is an increase in the ATP/ADP ratio (2,33) that activates DNA gyrase, favoring the DnaAATP complex

    active in replication initiation (53) and coordinating RNAP ac-tivity with energy availability (54). We propose that gyrase ac-tivity creates a gradient of superhelical density corresponding tothe gradient of gyrase-binding sites (Fig. 3 C and D and Fig. S10A and B) such that the selectively increased superhelicity of theorigin-proximal region and the resultant change in chromosomemorphology precede the initiation of replication. Such selectivitysuggests a lower superhelical density in the Ter region, consistent

    with the finding that in the prereplicative state in slow-growing

    cells the Ter region is extended (55). Concomitantly the in-creased spatial separation between origin-proximal H-NS targetgenes and the Ter-proximal location of the hns gene (Fig. 5M)

    would attenuate the silencing effect H-NS (39, 56, 57) on the rrnmacrodomain. On transition to stationary phase. a dramaticdecline of FIS levels (18) in conjunction with DNA relaxation(Fig. S10 A and B) and compaction of the nucleoid would enableH-NS, as well as the stationary-phase regulator LRP (38), toreestablish efficient repression (Fig. 5N) in the vicinity of repli-cation origin. This model provides a dynamic mechanism for theconcerted impact of the NAPs and DNA superhelicity on chro-mosome structure and cellular physiology (46, 9, 10, 16, 17, 19,36, 39, 58).

    ConclusionThe conservation of gene order and spatial organization ofregulatortarget interactions in chromosomal macrodomainsreported in this paper argue that the gene order specifies thetemporal pattern of gene expression during the bacterial growthcycle. Our work thus illuminates why the timing of the expressionof a gene is linked to its position on the chromosome, providinga view of the bacterial nucleoid as a highly organized dynamicentity optimized for coordinating oxygen and nutrient availability

    with spatiotemporal gene expression during rapid growth andits cessation.

    Materials and MethodsIdentification of Functional Groups in the GO Tree and Modeling of the TFGs. To

    address the similarity of chromosomal regions, we applied a sliding-window

    approach using a 500-kb window size and a 4-kb window shift creating 1,160windows, each containing about 500 genes. All genes within a window were

    assigned to theirspecific locations ontheGO tree(http://www.geneontology.

    org/). For a fixed level of the GO tree we summed up all genes assigned to

    the specific subtree, determining a unique one-dimensional pattern of GO-

    subtree (cluster) sizes for each window. Subsequently the similarity score of

    two windows (i and j) was determined by the equation:

    si;j

    c

    k1

    ni;knj;k

    c

    k1

    ni;kc

    k1

    nj;k

    where c is the number of clusters on the current GO-tree level, and n is the

    number of genes in a certain branch of this level, reflecting their covariance

    with a mean that equals zero. Hence, high similarity scores (s values) indicate

    a high correlation of the functional composition. All s values were comparedwith scores of 10,000 random genomes, generated by a random remapping

    of gene (operon) IDs to gene (operon) positions. For subsequent analyses

    window pairs with a P value < 0.05 were considered significant, and all

    others, including scores of overlapping windows [distance(i,j)

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    suffered from varying resolutions among distinct functional clusters because

    of a nonuniform depth of the GO tree.

    Modeling of the TRN. In correspondence with the GO-tree function analysis,

    we parsed the TRN communication along the chromosome by counting the

    number of connections between the subnetworks of any two 500-kb win-

    dows normalized by the total number of connections spreading from both

    windows into the genome. Using procedures analogous to GO similarity

    analysis, we determined highly connected regions on the chromosome and

    determined the peaks for the cis/trans ratio by shifting the origin.

    Couplon Similarity. As a measure of couplon similarity, we determined for

    each couplon (27) whether the number of contained genes for the current

    window was greater or less than the expected number of genes. Hence, the

    similarity of two windows equals the number of couplons coherently over-

    or underrepresented in both windows. The determination of the signifi-

    cance of matches and cis/trans ratio peaks was carried out as for the GO-tree

    similarity with random sets of both genes and operons.

    Regulator Targets. To determine target site frequency distributions, the rel-

    evant data derived from the RegulonDB database were analyzed using

    a sliding 400-kb window with the map position at the window midpoint.

    Expression Patterns. The expression data for mapping onto the HEN con-

    nectivity patterns were taken from Dong and Schellhorn (59) in Fig. 5 AD,

    from Geertz et al. (28) in Fig. 5 Eand F, and from Blot et al. (16) in Fig. 5 GL.

    Phylogenetic Analysis. In the phylum of -Proteobacteria the majority of reg-

    ulators is conserved in a plethora of species including human and plant

    pathogens such as Vibrio cholerae and Pseudomonas syringae. In total we in-

    vestigated 131 species (using a reciprocal best blast hit approach at the protein

    sequence level followed by the determination of orthologous clusters by

    a modified Girvan

    Newman algorithm, resulting in densely connected clusters(node degree n/2), where n denotes the cluster size. Species information

    containing protein sequences was derived from the RefSeq sequence database

    and origin positions using the DoriC database (60). The phylogenetic distances

    in Fig. 2B were derived from the tree of -Proteobacteria (http://www.cbrg.

    ethz.ch/research/orthologous/speciestrees). The same methodology was ap-

    plied for analysis of all the annotated genomes of Gram-positive bacteria.

    ACKNOWLEDGMENTS. Theauthorsthank MalcolmBuckleand Sylvie Rimsky forfruitful discussions. This work was supported by grants from the Ecole NormaleSuprieure de Cachan and Deutsche Forschungsgemeinschaft (to G.M.). A.T. re-ceived a Chaire dExcellence award from lAgence Nationale de la Recherche.

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