sequences. This organization might be sensitive to rearrange- ments of the order or orientation of the sequences involved, and in consequence the portion of the ...
Proc. Nati. Acad. Sci. USA Vol. 85, pp. 9391-9395, December 1988 Biochemistry
Detection and possible role of two large nondivisible zones on the Escherichia coli chromosome (chromosomal rearrangements/nucleoid structure)
Jost-EMILIo REBOLLO*, VINCENT FRAN§OIS, AND JEAN-MICHEL LOUARNt Centre de Biochimie et de Gdndtique Cellulaires du Centre National de la Recherche Scientifique, 118 route de Narbonne, 31062 Toulouse, France
Communicated by Marianne Grunberg-Manago, August 22, 1988
of two large nondivisible zones that coincide well with the regions polarized with respect to their replication.
Inversion of many predetermined segments ABSTRACT of the Escherichia coli chromosome was attempted by using a system for in vivo selection of genomic rearrangements. Two types of constraints on these inversions were observed: (i) a sensitivity to rich medium when the distance between oriC and the 86- to 91-min region (which carries loci essential for transcription and translation) is increased; (it) a poor viability or inviability of inversions having at least one endpoint in the one-third of the chromosome around replication terminators (with an exception for some inversions ending between these terminators). Although the first constraint is simply explained by a decreased dosage of the region Involved, the second one may result from disruption of two long-range chromosomal organizations. The nondivisible zones thus disclosed coincide remarkably well with the two zones that we have previously described, which are polarized with respect to their replication. It is proposed that the two phenomena result from a sequencedependent and polarized organization of the terminal region of the chromosome, which defines chromosome replication arms and may participate in nucleoid organization.
MATERIALS AND METHODS Selection of Chromosomal Rearrangements. The selection procedure is based upon the presence, in a given chromosome, of two TnlO transposon derivatives containing a tetracycline-resistance (TcR) gene, tetA, inactivated by insertion either of a streptomycin-resistance (SmR) determinant in the proximal part of the gene (TnJO-Tes) or of a kanamycin resistance (KmR) determinant in the distal part (TnlO-Tek) (Fig. la). An active tetA' locus can be reconstituted by homologous recombination between these two elements. This selectable event may be accompanied by a relative inversion of the two segments joining the transposons when these elements are in opposite orientations (6). Strain Construction. Chromosomal TnJO insertions used are presented in Fig. lb. Most of them were described in ref. 11 and were kindly provided by Barbara Bachmann (Yale University). The tool and procedure for Tes and Tek substitutions are described in ref. 6 and in Fig. la. After substitution, TnlO insertions were oriented by chromosome mobilization using a TnlO-containing transferable plasmid (6). Desired combinations of Tes and Tek insertions were constructed by phage P1-mediated transduction (12) and selection for antibiotic-resistant recombinants, with subsequent screening, when possible, of the phenotype associated with the insertion. Test for Inversion. In vivo: Strains to be assayed were Hfr derivatives harboring the F plasmid within the shortest segment to invert, with early transfer of one inversion endpoint. Inversion was monitored by the resulting dramatic change in the gradient of marker transmission to a recipient strain, LN1714. LN1714 is female strain (F-) W945 xyl mtl ile metA or B ara leu lac Y1 purE gal trp his argG thi rpsL (SmR) zdd230::Tn9 (CmR) (where Cm indicates chloramphenicol). Most of the Hfr strains used are described in ref. 8. Hfr VF1, VF2, and VF3 were isolated by a procedure to be published elsewhere. Origins of transfer are indicated Fig. 1. In vitro: The size distribution of Pst I fragments was determined as follows: Pst I digests of total cellular DNA were fractionated by electrophoresis in an agarose gel, denatured by alkaline treatment, transferred onto a Biodyne (Santa Monica, CA) transfer membrane, and hybridized with 32plabeled nick-translated Tn/O probe; the radioactive bands
The Escherichia coli chromosome [4700 kilobases (kb); ref. 1] is compacted into a multilooped structure called the nucleoid, which is composed of many domains and perpetually remodeled by gene expression, DNA replication, and segregation. In spite of considerable progress in the understanding of factors influencing DNA topology (2), the compaction rules are poorly understood (3, 4). We have recently raised the possibility that the nucleoid is organized in a symmetric and polarized fashion along both chromosome replication arms. The proposal rests on the observation that the regions extending from about 15 min to replication terminator T1 (close to 29 min) and from about 45 min to terminator T2 (33-34 min), respectively, are replicated with normal velocity from oriC toward T1 or T2 but very slowly from T1 or T2 toward oriC and hence are polarized with respect to their replication (5). A polarized nucleoid organization globally dependent on genome sequence would probably be implemented at the DNA level by specific organizing sequences. This organization might be sensitive to rearrangements of the order or orientation of the sequences involved, and in consequence the portion of the chromosome embedded in such a structure should behave as a nondivisible unit. We report here the results of a search for nondivisible chromosomal regions. The most appropriate genomic rearrangements for their identification are DNA inversions since they disorganize the normal chromosomal order without necessarily modifying the overall genetic content. We have recently developed a system for selection of chromosomal inversions (6). This system was applied to a large number of chromosomal segments and allowed the detection
Abbreviations: Cm, chloramphenicol; F-, female strain; Hfr, highfrequency donor strain; Km, kanamycin; ors, organizing sequences; R resistant; RTF, resistance transfer factor; Sm, streptomycin; Tc, tetracycline; Tr, temperature-resistant; Ts, temperature-sensitive; NDZ, nondivisible zone. *Present address: Departamento de Bioquimica y Biologia Molecular y Gendtica, Facultad de Ciencias, 06080 Badajoz, Spain. tTo whom reprint requests should be sent.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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FIG. 1. Strategy for search of defined genomic rearrangements. (a) Tool for Tes and Tek substitutions. A bacteriophage, / AcIII c1857 cII, in which the attP int is aroA region is replaced by a TnJO tetA fragment \ carrying the two SmA and KmR insertions has been constructed (ref 6; unpublished). It mdoA KL19pyrC is presented here after its integration within a chromosomal Tn10 insertion, rendering the fad R strain temperature-sensitive (Ts) TcS SmR -trA KmR (where s indicates sensitive). A secVF1 Ti1 tI rp ondary recombination event will lead to I VF * pyrF temperature-resistaht selectable (Tr) SmR (1) or Tr KmR derivatives (2), thus generating zci \T2s B7 the Tes or Tek substitutions. (b) Map posi\z^'xcj tion of TnJO insertions and F integration sites \ J Us rg used in the present work. The TnJO inserVF3 E\ \KL16 tions are represented by heavy arrows, the pK19l zdf zde-395M6 arrowhead indicating the conventional ISIO right (7). Genetic positions are indicated KL96 zzdh g according to ref. 11. Origins of transfer are given according to ref. 8 and to unpublished data [for high-frequency donor strains (Hfrs) piMA VF1, VF2, and VF3]. The simplified map shows also the positions of sites involved in ^* his chromosome replication, the origin oriC (9) and termini T1 and T2 (5, 10). purF
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FIG. 2. Correlation between genomic rearrangements and exchanges between TnlO-Tes and TnJO-Tek insertions. (a) Expected variations of TnJO-containing Pst I fragments. Exchange 1 leads to inversion DBCA. Fragment c is conserved and two new fragments appear; their sum, d + e, is equal to a + b. Additional exchanges 2 and 3 restore the normal chromzosomal order ABCD. In 2, three new fragments appear, with the following rules: f = b + c - 2.4 kb; e + g = a + 2.4 kb (the 2.4 kb are the remnants of the KmR determinant). In 3, fragment c is conserved and fragments h and i derive from a and b by subtraction and addition of the SmR determinant (2 kb), respectively. (b) Observed variations. The size distribution of TnlO-containing Pst I fragments was compared by Southern analysis between parental TnJO-Tes strains (fragments a), parental TnJO-Tes plus TnJO-Tek strains (fragments a, b, and c), and TcR recombinants, either inverted or noninverted. Since the rules established above were always fulfilled in the TcR recombinants, fragments were labeled accordingly. Lanes: 1, his::Tes pheA::Tek; 2, as in lane 1 but inverted TcR (a fraction of the population has undergone a type 3 secondary exchange); 3, his::Tes; 4, his:Tes purE::Tek; 5, as in lane 4 but inverted TcR; 6, his::Tes; 7, purE::Tes malE::Tek; 8, as lane 7 but inverted TcR; 9, as lane 7 but noninverted TcR (type 2 secondary exchange); 10, purE::Tes; 11, thr::Tes tna::Tek; 12, as lane 11 but inverted TcR; 13, thr::Tes; 14, pheA::Tes malA::Tek; 15, as lane 14 but inverted TcR; 16, pheA::Tes.
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Proc. Natl. Acad. Sci. USA 85 (1988)
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Table 1. Examples of viable and unviable inversions Transfer properties of triple-resistant clones Selective Segment medium Inverted Normal Bidirectional Total endpoints Class 1 20 0 0 20 HfrKL16 LA pheA::Tes 18 2 0 malA::Tek E/glucose 20 11 18 LA 1 6 HfrP4X II 7 7 1 15 metE::Tek E/glucose 42 13 0 55 purE::Tes E/glycerol LA 0 12 8 20 HfrVF1 III 0 10 10 20 fadR::Tek E/glucose 25 0 16 9 pyrF::Tes E/glycerol 0 IV 23 23 LA 0 HfrC 20 0 0 20 trp::Tek E/glucose 16 0 0 16 thr::Tes E/glycerol The Hfr strains used harbored the F plasmid inserted within the segment assayed. Triple-resistant clones were selected on rich medium (LA; ref. 6) or on minimal medium (E; ref. 6) with either glucose or glycerol as carbon source. TcR SmR KmR clones, obtained with a frequency ranging from 10-4 to 10-3, were assayed for donor properties by cross-streak mating with LN1714 as recipient strain. We refer here to transfer of markers located outside the segment assayed. Inverted, normal, and bidirectional transfers reflect the clonal composition with a majority of inverted bacteria, a majority of normal bacteria, and a mixed population, respectively. When the rearrangement is well-tolerated (class I), "inverted" clones are much more frequent than "noninverted" ones since the former result from a single exchange and the latter result from a double exchange (Fig. 2). The origin of mixed clones is analyzed in Table 2. Generation times supported by the medium used (wild-type strain at 370C): LA, 25 min; E/glucose, 45 min: E/glycerol, 80 min.
were subsequently visualized by autoradiography, as described (6).
RESULTS Chromosomal Inversions Are Induced by Reciprocal Exchanges Between TnlO Insertions. We have previously observed that reconstitution of a tetA+ gene may occur by way of a reciprocal exchange between the Tes and the Tek insertions or, more frequently, by way of a correction conversion mechanism. Rearrangement of external markers was Table 2. Evolution of class III recombinant clones Clonal composition Young Transfer index clone Old clone Clonal Old Young clone clone I N M I designation N M 20 ND 0 10 0 ND ND ND L, 1 0.57 10.1 0 4 6 0 L, m 9 1 0.35 6.8 0 5 5 0 L, s 9 1 25 ND E, 1 0 10 0 ND ND ND 1.0 25 0 5 5 E, m 0 10 0 >100 >100 0 10 0 E, s 0 10 0 A culture of strain LN 1859 (HfrC purE::Tes pyrC::Tek) was plated on L/glucose or E/glucose agar containing Tc (25 ,ug/ml), Sm (25 ,tg/ml), and Km (25 Ag/ml). After a 40-hr incubation at 37°C, three colonies of various sizes (large, 1; medium, m; small, s; cell number ranging between 106 and 107 per colony) were picked from each medium and suspended in the same medium without antibiotics. Either immediately ("young clone") or after a 10,000-fold increase in cell number ("old clone"), these bacteria were (i) crossed with a convenient recipient strain for 1 hr at 37°C [transfer index refers to the ratio between the numbers of recombinants for a marker transferred early by HfrC (leu, at 2 min) and another transferred late (trp, at 27 min); it is >100 for strain LN1859 in these mating conditions] or (ii) subcloned on the same medium [10 subclones of each were then assayed by cross-streak matings and classified as in Table 1 in inverted (I), noninverted (N), or mixed (M) clones]. ND, not done.
preferentially found in the first type of event, which is also characterized by the persistence of the two initial resistance characters (6). Determination of the size distribution of Tn.O-containing Pst I fragments in TcR SmR KmR recombinants from various "Tes and Tek" strains has confirmed the previous observations: the genomes of the five TcR SmR KmR recombinants analyzed in Fig. 2 displayed the marks of (i) a single reciprocal exchange in the inter-insertion zone when the in vivo test indicated inversion achievement; (ii) the same exchange as above plus a secondary exchange between TnIO sequences outside the insertions when the in vivo test indicated a normal chromosomal order. Phenotypic Consequences of Chromosomal Inversions. The segments assayed for inversion displayed four types of behavior (Table 1). Inversion of class I segments is welltolerated and gives rise to stable clones growing well in a variety of growth media. Inversion of class II segments is observed only on minimal-gycerol medium and thus confers a rich medium sensitive (Rms) phenotype. Pure clones of bacteria inverted for class III segments are not observed, whatever the growth medium. These inversions do occur, since "young" triple-resistant clones promote an inverted or bidirectional transfer, but wild-type bacteria rapidly outgrow the mutants in the population since older clones promote normal transfer. The data presented in Table 2 are compatible with a 2-fold increase in generation time due to the inversion of the purE-pyrC segment, assuming a probability of exchanges correcting the inversion of i0-3 per cell per generation. Inversion of a class IV segment is never observed. This may result either from inviability of the inverted bacteria or from prohibition of the genetic exchange itself. Map Distribution of Harmless and Deleterious Inversion Endpoints. Fig. 3 shows the positions on the standard genetic map (9) of the 47 segments assayed, arranged in the different phenotypic classes. The Hfr character has probably no influence on the distribution, since the different segment behaviors may be found with the same Hfr strain (HfrC or KL96 for instance). A most important factor determining segment behavior is endpoint map position. In class I
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Proc. Natl. Acad. Sci. USA 85 (1988)
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FIG. 3. Segment behavior in the inversion test. On a simplified E. coli chromosome map, open at oriC, the segments assayed are indicated by heavy lines joining the TnJO endpoints assayed without passing through oriC. Depending upon the inversion effect, they were distributed in four phenotypical classes further described in the text. Vertical arrays of open squares and dotted lines indicate outer and inner limits of the nondivisible zones (NDZs), respectively. Segments assayed: 1, purE-nadA (C); 2, proAB-purE (P4X); 3, iHvK-malE (P72); 4, thr-malE (H); 5, thr-metE (H); 6, purE-malE (P4X); 7, his-pheA (KL96); 8, pheA-malA (KL16); 9, pheA-tna (KL16); 10, his-purE (PK191); 11, purE-tna (P4X); 12, his-thr (PK191); 13, thr-tna (H); 14, proAB-trg (C); 15, pheA-zcjJ52 (KL96); 16, pheA-zcjJS2 (KL96); 17, malA-ilv (AB313); 18, pheA-metE (KL16); 19, iUvK-purF (KL16); 20, purE-metE (P4X); 21, pyrF-fadR (VF1); 22, aroA-fadR (KL19); 23, purE-aroA (C); 24, purE-pyrC (C); 25, his-trg (PK191); 26, pheA-trp (KL96); 27, pyrF-mdoA (VF1); 28, nadA-fadR (KL19); 29, purE-treA (KL19); 30, trp-purE (C); 31, thr-trp (C); 32, zdg-zde4O6 (VF3); 33, his-zde395 (PK191); 34, his-zdg (PK191); 35, his-zdf (PK191); 36, his-zde395 (PK191); 37, pheA-zde4O6 (PK191); 38, zcjJS2-trg (VF2); 39, zci233-trg (VF2); 40, trg-trp (B7); 41, trg-pyrC (B7); 42, purE-zci233 (C); 43, purE-zcjJS2 (C); 44, thr-zci233 (C); 45, thr-zcj152 (C); 46, trp-zdh (B7); 47, mdo-tna (KL19). Tnl0-Tes insertions are written first, and Hfr names are bracketed.
inversions, either both segment endpoints map in a large region around oriC (segments 1-13), from his (44 min) to nadA (17 min), or one maps in this region while the second one is located close to 30 min in the termination region (segments 14-16). Endpoints may belong to both replication arms or to a single one; in the latter case, the change of direction of replication within the inverted segment has apparently no deleterious consequence. Class II segment endpoints fall within class I segments. The comparison of segment 6 (class I) to segment 20 (class II) suggests that the Rms phenotype of segment 20 inversion is due to the removal of the 86- to 91-min region distant from oriC. This interpretation may also hold for the other class II segments 17, 18, and 19. All segments characterized by the presence of at least one endpoint in the one-third of the chromosome surrounding
replication termini belong to class III or class IV, with the above-mentioned exceptions of class I segments 14-16. Considering the overlapping of class III and class IV segment endpoints, we suspect that most class IV segments are readily inversible and that their inversion has simply a more extreme effect on viability than that of class III segments.
DISCUSSION The deleterious effect of certain inversions may be attributed to several possible causes, such as: (i) inactivation by inversion of essential genes, for instance those, if any, activated at replication fork passage; (ii) alteration of gene dosage; (iii) disruption of a long-range essential organization. Since the inversion procedure does not in principle alter the
Proc. Natl. Acad. Sci. USA 85 (1988)
Biochemistry: Rebollo et al. genetic content outside the transposons TnI0, no "border effect" is expected. Change of the direction of replication is probably not a decisive factor, since we have previously observed that replication in either direction of any chromosomal segment is compatible with cell viability (5). Gene dosage alteration may explain the Rms phenotype of class II inversions, since variations of marker frequency as a function of map position are reduced at slow growth rates (13). The genes whose dosage is critical are not known, but the 86- to 91-min region carries a set of loci (rrnA, rrnB, rrnE, rpoBC, rpILK; ref. 9) crucial for synthesis of the transcription and translation machinery. A lower dosage of some of these genes due to delayed replication might result in their insufficient expression for growth in rich medium. Class III and IV segments, which display at least one endpoint in the 17- to 44-min region, are frequently small and nonoverlapping. This indicates that their inversion interferes negatively with functions implicating several loci or entire regions. The absence of correction by growth medium allowing slow growth rates (Table 2) and the small size of some of these segments argue against gene dosage alterations as major causes for the detrimental effect of their inversion. Our favored interpretation is that the 17- to 44-min region is organized in long-range structures constituting functional units that are disrupted by inversion-induced modifications in the order or orientation of the sequences involved. Since long inversions ending in the central part of the region are viable, we propose that this region harbors two NDZs, NDZ1 and NDZ2. The NDZs coincide strikingly well with the two regions polarized with respect to their replication that we have previously described (5). We propose that slow replication in the unnatural direction and the detrimental character of inversion share a common basis-namely, a polarized organization of the chromosome in these regions. Our model (Fig. 4) resembles that proposed for chromatin loop organization (14). It postulates a succession of polar organizing oriC
sequences (ors) interacting orderly with a scaffold apparatus, so that (i) the sequence order must be respected (hence the noninvertibility) and (ii) each scaffold-ors dissociates and reassociates rapidly at replication fork passage in the natural direction, but slowly when fork passage is in the opposite direction (hence the overall slow replication in this direction). Replication termini T1 and T2 (5, 10) might be viewed as the terminal ors of each NDZ. The regional coincidence of two long-range phenomena, which is the basis of the above model, needs further precision. "Outer" (i.e., on the oriC side) limits of the two NDZs are not well-defined: segment 1 (ending in nadA, 17 min) and segments 7, 10, and 12 (ending in his, 44 min) were placed in class I in spite of a clear growth advantage of wild-type revertants. On the "inner" side, the behavior in the inversion test of certain segments ending in the T1-T2 region is also unclear. Why is it possible to get inversion of NDZ2 (segments 15 and 16) but not of NDZ1 (segments 42-45) from endpoints zci233 and zcjlS2? Why is inversion of segments 38 and 39, entirely comprised within a dispensable region (5, 15), not observed? Our proposal for a chromosome architecture will undoubtedly be revised and refined. Confirmation might come from identification of pausing sites (in addition to T1 and T2) for replication of the NDZs in the unnatural direction. The data reported here substantiate the hypothesis that DNA is not randomly coiled in the nucleoid but is organized in a sequence-dependent fashion. This supragenic organization, limited to the terminal region of the chromosome and favoring replication in one direction, defines the chromosome replication arms and may play a central role in the segregation process. We thank Josette Patte for technical assistance and Michael Chandler for discussion and help in revising the manuscript. J.E.R. was a recipient of a fellowship from France-Spain Cooperation. 1. Kohara, Y., Akiyama, K. & Isono, K. (1987) Cell 50, 495-508. 2. Drlica, K. & Rouviere-Yaniv, J. (1987) Microbiol. Rev. 51, 301319. 3. Bjornsti, M. A., Hobot, J. A., Kelus, A. S., Williger, W. &
4. 5. 6. NDZ 2
9395
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7.
8.
9. 10.
11.
FIG. 4. Proposal for bacterial nucleoid organization. Each NDZ is supposed to be structured by a scaffolding apparatus (SA) that anchors ors arranged in an ordered and oriented fashion along the
terminally replicated region of each replication arm. The DNA segment comprised between two adjacent ors might constitute a superhelicity domain.
12. 13. 14. 15.
Kellemberger, E. (1987) in Bacterial Chromatin, eds. Gualerzi, C. 0. & Pon, C. L. (Springer, Heidelberg), pp. 64-81. Drlica, K. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, ed. Neidhardt, F. C. (Am. Soc. Microbiol., Washington, DC), pp. 91-103. de Massy, B., Bejar, S., Louarn, J., Louarn, J. M. & Bouche, J. P. (1987) Proc. Natl. Acad. Sci. USA 84, 1759-1763. Franqois, V., Louarn, J., Patte, J. & Louarn, J. M. (1987) Gene 56, 99-108. Way, J. C., Davis, M. A., Morisato, D., Roberts, D. E. & Kleckner, N. (1984) Gene 32, 369-379. Low, K. L. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, ed. Neidhardt, F. C. (Am. Soc. Microbiol., Washington, DC), pp. 1134-1137. Bachmann, B. J. (1983) Microbiol. Rev. 47, 180-230. Hill, T. M., Henson, J. M. & Kuempel, P. L. (1987) Proc. Natl. Acad. Sci. USA 84, 1754-1758. Berg, C. M. & Berg, D. E. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, ed. Neidhardt, F. C. (Am. Soc. Microbiol., Washington, DC), pp. 1071-1109. Caro, L. & Berg, C. M. (1971) Methods Enzymol. 21, 444-458. Chandler, M., Bird, R. E. & Caro, L. (1975) J. Mol. Biol. 94, 127-132. Gasser, S. M. & Laemmli, U. K. (1986) EMBO J. 5, 511-518. Henson, J. M. & Kuempel, P. L. (1985) Proc. Natl. Acad. Sci. USA 82, 3766-3770.
