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Copyright 0 1991 by the Genetics Society of America Ability of a Bacterial Chromosome Segment to Invert Is Dictated by Included Material Rather Than Flanking Sequence Michael J. Mahan’.‘ andJohn R. Roth’ Department of Biology, University of Utah, Salt Lake City, Utah 841 12 Manuscript received March6, 1989 Accepted for publication September3, 1991 ABSTRACT Homologous recombination between sequences present in inverse order within the same chromosome can result in inversion formation. We have previously shown that inverse order sequences at some sites (permissive) recombine to generate the expected inversion; no inversions are found when the same inverse order sequences flank other (nonpermissive) regionsof the chromosome. In hopes of defining how permissive and nonpermissive intervalsare determined, we have constructed a strain that carriesa large chromosomal inversion. Using this inversion mutant as the parent strain,we have determined the “permissivity”of a series of chromosomal sites for secondary inversions. For the set of intervals tested, permissivity seems to be dictated by the nature of the genetic material present within the chromosomal interval being tested rather than the flanking sequences or orientation of this material in the chromosome. Almost all permissive intervals include the origin or terminus of replication. We suggest that the rules for recovery of inversions reflect mechanistic restrictions on the occurrence of inversions rather than lethal consequencesof the completed rearrangement. I NSPECTION of the chromosomal organization in Salmonella typhimurium and Escherichia coli reveals strong conservation of gene order (BACHMAN 1983; SANDERSON and ROTH 1988)[for a discussion see ROTH and SCHMID(1981), RILEY and KRAWIEC (1987), and KRAWIECand RILEY(1990)]. This is surprising since the two organisms have diverged up to 25% in the nucleic acid sequence of coding regions (NICHOLSand YANOFSKY1979; CRAWFORD, NICHOLS and YANOFSKY1980; CARLOMAGNO et al. 1988), reducing recombination between them greater than105fold (MIDDLETON197 1; YANOFSKY,HORNand ROWE 1977). Constraints on the formation of specific types of genome rearrangements in S. typhimurium and E. coli have been observed. T h e frequency of spontaneous duplications and deletions is high. Duplications of chromosomal sites are found at a frequencyof 1O-* to 10-5 (ANDERSON and ROTH 1977, 1981); deletions of particular sites are found at a frequency of about to 10” (reviewed in FRANKLIN 197 STARLIN1; GER 1977). In contrast,rather few inversion rearrangements have been reported. T h e apparent rarity may be due in part to difficulties in identification of inversions. However for some sites where homologous sequences were provided, inversions could not be detected (KONRAD1969,1977; ZIEG and KUSHNER 1978). For other sites, inversions were found but at a low frequency (ROTHand SCHMID198 1;SCHMIDand ’ To whom correspondence should be sent. * Present address: Department of Microbiology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 021 15. To whom strain and reprint requests should be addressed. ’ Genetics 1 2 9 1021-1032 (December, 1991) ROTH 1983a,b). T o better understand the process of chromosome rearrangement, we have developed two systems for directing the formation of particular rearrangements (MAHANand ROTH 1988; SEGALL, MAHAN and ROTH 1988). Both systems involve placing homologous sequences in inverse order at separated sites in the Salmonella chromosome and selecting for recombination between them. A similar system has been developed for E. coli (FRANCOIS et al. 1987) and was used to select the same sorts of rearrangements (REBOLLO, FRANCOIS and LOUARN 1988). All of these systems are conceptual derivatives of work by KONRAD (1969, 1977). In both E. coli and Salmonella, the general results of these selections are the same. When sequences are placed at some pairs of chromosomal sites (permissive), the recombinants include the expected inversions; sequences placed at otherpairs of sites (nonpermissive) do notyield inversions. It seems likely that an understanding of the factors that dictate theseresults will lead to a better understanding of the structure, function and possibly evolution of the bacterial chromosome. Two aspects of our previous work have led us to conclude that nonpermissive intervals aredueto mechanistic problems in the recombination process, rather than to lethal consequences of the final inversion (SEGALL, MAHANand ROTH 1988). First, we have successfully used transduction crosses to directthe inversion of two intervals that are nonpermissive for inversion by intrachromosomal exchanges. The directed inversions are not lethal; thus we think it un- M. J. Mahan and J. R. Roth 1022 likely thatthefailure to detect inversion of these regions is due to lethality (A. M. SECALL,L. MIE~EL and J. R. ROTH unpublished results). Second, even for nonpermissive intervals, both of our two selection systems detect recombination events between the involved sequences. (MAHANand ROTH 1988,1989; SECALL and ROTH1989). Since these alternative sorts of recombinants (but not inversions) are formed by sequences at all nonpermissive sites, we conclude that the sequences involved can interact in some way. We have proposed that sequences at nonpermissive sites in the samechromosome cannot interact directly and therefore cannotform inversions. We suggest that such sequences can only engage in sister chromosome exchanges, which can accountfor all the noninversion recombinants detected.Results of similar experiments in E. coli have led to slightly different conclusions and will be discussed later. In the experiments presented here, we wish to test the effect of changes in the orientation and chromosomal position of a particular arc of chromosome on the permissivity of inverting other segments of the chromosome. To this end, we have constructeda strain with alarge inversion ( a m to his) involving almost halfof the bacterial chromosome and have used this strain as a parent to test the permissivity of 11 chromosomal intervals. The results suggest that the behavior of recombining sites is a function of the genetic material located between those sites rather than orientation, position of endpoints in the chromosome, or sequences flanking the recombining sites. MATERIALS AND METHODS Bacterial strains: All strains used in this study (Table 1 are derived from Salmonellatyphimurium strain LT2. All directed transposition strains were constructed according to methods described by CHUMLEY and ROTH(1980), SCHMID and ROTH(1980). Figure la diagrams the ara-his (minute 2 to minute 42) inversion strain TT11797 described previously (MAHANand ROTH1988). [Unless otherwise specified, the mutation designations used in thisstudy are as according to SANDERSON and ROTH(1988).] This strain is unstable and can reverse itsinversion by recombination between the inverse order copies of either his or TnZO material (at ara and his). By removing these repeated sequences from the inversion join points (Figure lb), we have stabilizedthe arahis inversion, preventing it from “reflipping” to the normal chromosomal arrangement. The his and TnZO homologies were deleted from both inversion join points of TT11797 by methods described by HUGHES and ROTH(1985). Removing his and TnZOhomology at his: Mixed lysates of strains TT8269 (leuAZZ7Y::MudA) and TT7692 (hisD9953::MudA) were used to transduce ara-his inversionbearing strain TT11797 to ampicillin resistance. [MudA elements (HUGHES and ROTH1984) referto a conditionally transposition-defective derivative of the Mud prophages of CASADABAN and COHEN(1979).] The Ap’ Leu- His- transductant (TT13904) contains a deletion of genetic material (leuAZl7Y*MudA*hisDYY53) between the MudA elements in leuA (minute 3) and hisD (minute 42); the result of homologous recombination between the twoMudAele- ments, followed by recombination of this hybrid fragment with the recipient chromosome (bottom of Figure 1b). Removing his and TnlO homology at ara: Strain TT13905 is a derivative of TT13904 that contains a cobl-368::TnZOdCam insertion element [(encoding chloramphenicol resistance (ELLIOTand ROTH1988)l. This strain was transduced to Ara+ with P22 phage grown on TT13903 (ara+,zuc365Y::TnIOd-Cam) (see top of Figure lb). The Ara+ Cm’ Ap’ Leu- His- transductant (TT13906) can arise by recombination between the two TnZOd-Cam elements (one on the transduced fragment, the other in the recipient chromosome). Such events generate a deletion of genetic material (zac-3659*TnZOd-Cam*cobZ-368) between the TnZOd-Cam element in the zac region [adjacent to ara (minute 3)] and CobI (minute 41). The final recombinant is diagrammed in Figure IC).The inversion rearrangement of TT13906 was then transduced intoa wild type genetic background (TR6535) according to a two-fragment transduction method developed by SCHMID and ROTH(1983a). The resultant ara-his inversion strain (TT13909) servesas the parental strain for testing permissive and nonpermissive intervals; this strain is isogenicwith the wild-type strain (TR6535) except for the ara-his inversion, which is stable due todeleted material near the inversion join points introduced by transduction (Figure IC).The inversion join point at his (minute 42) contains deletion a (leuAI1 7Y)*MudA*(hisD9953)that fuses the leuA gene (minute 3) to thehisD gene. The inversion join point adjacent to ara (minute 3) contains a deletion (zac-365Y*TnIOd-Carn*cobl368) that fuses the zac (near ara) region to the CobI operon (minute 41). Media: The E medium of VOGEL and BONNER (1956) supplemented with 0.2% glucose was used as the defined minimal medium. Selection for growth on alternative carbon sources was done on NCE medium, described by BERKOWITZ et al. (1968), supplemented with 0.2%of the appropriate carbon source. The complex medium was nutrient broth (8 g/liter, Difco Laboratories) with added NaCl(5 g/ liter). Solid medium contained Difco agar at 1.5% final concentration. Auxotrophic requirements were included in media at final concentrations described by DAVIS, BOTSTEIN and ROTH(1980). Final concentrations of antibiotics were as follows: tetracycline hydrochloride (Sigma ChemicalCo., 16 mg/ml in rich medium, or 10 mg/ml in minimal medium); kanamycin sulfate (Sigma Chemical Co., 50 mg/ml in rich medium, or 100 mg/ml in minimal medium); chloramphenicol (Sigma ChemicalCo., 20 mg/ml inrich medium, or 5 mg/ml in minimalmedium); ampicillin (Sigma Chemical Co., 30 mg/ml in rich medium, or 15 mg/ml in minimal medium). Transductional methods: The high frequency generalized transducing mutant of bacteriophage P22 (HT 105/1, int-201) (SCHMIEGER 1972) was used for all transductional crosses. Unless otherwise specified, 0.1 ml of an overnight culture grown in complex medium (ca. 2-4 X lo9 cfu/ml) was used asa recipient of 0.1 ml transducing phage (ca. 10’lo9 pfu/ml) and plated directly on selective plates. Transductional crosses involving the selection of kanamycin or chloramphenicol resistance were preincubated overnight on solidnonselective complex medium, then replica-printed onto selective medium. Transductant clones were purified and phage-free isolates were obtained by streaking for single colonies on green indicator plates (CHANet al. 1972). Phagefree colonies were tested for phage sensitivity by crossstreaking with P22 H5 (a clear plaque mutant of phage P22). Construction of TT13913: This strain is used as a donor in transduction crosses that detect linkage disruption at both Restrictions on Inversion 1023 TABLE 1 Bacterial strains Strain" GenotvDeb TTll797 TTl3909 TT13913 TT13915 INV768[(ara-651::TnlO)*h~~*(hisC869Z::TnZO-hisOGD646), zee-P::TnZO, proAB47 DEL859[1NV768((zac-3659)*TnZOd-Cam*(cobZ-368)] DEL858[(leuA1179)*MudA*(hisD9953)]], proAB47 DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)] DEL860[(leuAlZ79)*MudJ*(hisF9954))1, proAB47 ara-65l::(TnlO-hisOGDC869Z-TnZO), proAB47, DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)] DEL858[(leuAZZ79)*MudA*(hisD9953))] pncB165::(TnlO-hisOGDC8691-TnlO), proAB47, DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)] DEL858[(leuAlZ79)*MudA*(hisD9953))1 pncBZ65::(TnlO-hisOGDC8691-Tn10), nadAZZ3::MudJ,proAB47, DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)] DEL858[(leuAZZ79)*MudA*(hisD9953))1 pncAl55::(TnlO-hisOGDC869l-TnlO), proAB47, DEL859[INV768((zac-3659)*TnZOd-Cam*(cobZ-368)] DEL858[(leuAZZ79)*MudA*(hisD9953))1 pncA155::(TnlO-hisOGDC8691=TnlO), nadA213::MudJ,proAB47, DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)] DEL858[(leuAll79)*MudA*(hisD9953)]] tr~DE-243l::(TnZO-hisOGDC869Z-TnlO), proAB47, DEL859[INV768((zac-3659)*TnZOd-Cam*(cobZ-368)] DEL858[(leuAZZ79)*MudA*(hisD9953)J] zeg-74::(TnlO-hisOGDC8691-TnlO), proAB47, DEL859[INV768((zac-3659)*TnZOd-Cam*(cobZ-368)] DEL858[(leuAZZ79)*MudA*(hisD9953))1 zfa-3647:(TnlO-hisOGDC869Z-TnlO), proAB47, DEL859[INV768((zac-3659)*TnlOd-Cam*(cobl-368)] DEL858[(leuAZZ79)*MudA*(hisD9953)]] tyrA555::(TnlO-hisOGDC869Z-TnlO), proAB47, DEL859[INV768((zac-3659)*TnZOd-Cam*(cobZ-368)] DEL858[(leuAZ179)*MudA*(hisD9953))1 cysJZ-Z5Z9::(TnlO-hisOGDC8691-TnZO), proAB47, DEL859[INV768((zac3659)*TnlOd-Cam*(cobZ-368)] DEL858[(leuAZZ79)*MudA*(hisD9953))1 argA1832::(TnlO-hisOGDC8691-TnZO), proAB47, DEL859[INV768((zac-3659)*TnlOd-Cam*(cobZ-368)] DEL858[(leuAZZ79)*MudA*(hisD9953))] metE866::(TnlO-hisOGDC8691-TnlO), proAB47, DEL859[INV768((zac3659)*TnZOd-Cam*(cobZ-368)] DEL858[(leuAZZ79)*MudA*(hisD9953))I pyrB692::(TnlO-hisOGDC8691-TnlO), proAB47, DEL859[INV768((zac-3659)*TnZOd-Cam*cobl-368)] DEL858[(leuAZZ79)*MudA*(hisD9953)]] TT13916 TT13917 TT13918 TT13919 TTl3920 TT13921 TT13922 TT13923 TTl3924 TT13925 TT13926 TTl3927 * All strains are derivatives of S. typhimurium LT2 and were constructed in this laboratory. Figure l a diagrams the genotype of strain TT11797; Figure I C diagrams the genotype of strains TT13906 and TT13909. Figure 2 diagrams strain TTl3920, which is a representative example of parental ara-his inversion-bearing strain used in the inversion assay, and strain TT13930, which is a representative example of a secondary inversion recombinant. the his and non-his endpoints in inversion-bearing recombinants. Mixed lysates of strains TT8269 (leuAII79::MudA) and TT7693(hisF9954::MudA) were used to transduce the ara-his inversion strain TTl1797 (Figure la) to ampicillin resistance. The Ap' Leu- His- transductant strain TTl3910 has acquired a deletion that removes the genetic material from leuA into hisF (leuA1179)*MudA*(hisF9954);the result of homologous recombination between transduced fragments carrying the twoMudA elements and subsequent recombination of the hybrid fragment with the chromosome. The MudA element at the joinpoint of this deletion was converted to an allelic MudJ insertion by homologous recombination at the ends of Mu, replacing an ampicillin resistance marker with a kanamycin resistance marker (TT139 1 1, leuAI 179*MudJ*hisF9954) (CASADABAN and COHEN1979; CASTILHO, OLFSON and CASADABAN 1984; R. V. SONTIand J. R. ROTH, unpublished results). This deletion was introduced into ara-his inversion-bearing strain TT 13906 (see Bacterialstrains in MATERIALS AND METHODS for discussion). The inversion rearrangement of one Km' transductant (TT13912) was transduced into a wild type background (TR6535) by the two-fragment transduction method of SCHMID and ROTH (1983a). The resultant arahis inversion-bearing strain TT13913 is identical to TT13909 (Figure IC) except the large leu-his deletion, which is associated witha MudJ element, extends from leuA into hisF (leuA1179*MudJ*hisF9953). Use of this strain is described below. Linkage disruption: Linkage disruption was tested at both join points of an inversion recombinant. (1) Linkage disruption at the his endpoint. Linkage disruption atthe his endpoint, inHis+ recombinants derived from parent strains with an ara-his inversion, was diagnosed as a reduction in ability ofHis+ recombinants to inherit a large his-leu deletion associated with a Km' determinant (Figure 3); this deletion/replacement was introduced usingP22 phage grown on strain TT13913. This donor strain (TT13913) contains an ara-his inversion; the inversion join point at his (minute 42/3) contains a leuA-hisF deletion (leuA1179)*MudJ*(hisF9954) that is associatedwith a MudJ insertion element (encoding a Km' determinant), constructed according toHUGHESand ROTH (1985). Strain TT13913 is identical to TTl3909 (Figure IC) except the large his deletion, which is associated with a MudJ element, extends from leuA into hisF (leuAI 179)*MudJ*(hisF9954) (see Construction of TT 139 13 in MATERIALS AND METHODS for detailed description). When strain TT13913 is used as a donor in a transduction cross, its kanamycin resistance determinant can only be inherited by recipients that have the samejuxtaposition of his and leu sequences as the parent strain (Figure 3). Strains with a rearrangement that disrupts this arrangement show a large reduction in ability to inherit the kanamycin resistance determinant. A representative example of an inversion-bearing His+ recombinant is diagrammed in Figure 2. Linkage disruption at the non-his endpoint (at trp, minute 34) was diagnosed M. and J. Mahan 1024 using the same rationale asthat for testing thehis endpoint (above). That is, since a strain carrying a trp-his inversion contains normal trp sequence on only one side of the trp inversionjoin point (andhis material on the other), one will observe a reduction in the abilityto recover Trp' transductants (when usinga wild type donor). Nomenclature: Nomenclature is generally as described et al. (1977),and in DEMERECet al. (1966),CAMPBELL CHUMLEY, MENZELand ROTH (1979). The nomenclature "z-::TnlO" refers to a TnlO insertion in a "silent" DNA region; the"z--)' describes the map position of the insertion (SANDERSON and ROTH 1988). The nomenclature used for chromosomal rearrangementsis described in CHUMLEY and ROTH (1980),SCHMID and ROTH (1980) and HUGHES and ROTH(1 985). The nomenclature complex of chromosomal rearrangements (CRR) in which oneborder consists of multiple join points is defined as follows. Commencing with designation of the outward-most aspect of the rearrangement, the terminology proceedsclockwise (from low minutes to high minutes on the chromosome map) to denote, in order, subsequent aspects of the rearrangement adjacenttotheoutward border. In the case where the outward border is extended by a successive rearrangement, the CRR of the initial rearrangementis retained. Examples of strains with successive rearrangements are given below. INV768[(ara)*TnlO*(hisD)] designates an inversion ofthe geneticmaterialbetween the TnlO elements in ara and h i d ; the result of homologous recombination between the two TnlO elements. An additional exampleis a derivative of the above strain that has acquired a deletionremovinghomologyat the hisD inversion join point of strain INV768[(ara)*TnIO*(hisD)]; this strain is described as follows: INV768[(ara)*TnlO*DEL860((leuA)*MudA*(hisF)]) designates a deletion of genetic material between MudA elements in leuA and hisF, removing the TnlO homology at the inversion join point in h i d ; the result of homologous recombination between the two MudA elements. The inversion join point, which previously mapped in the hisD gene ofstrain INV768[(ara)*TnlO*(hisD)], is nowwithin the deletion: DEL860[(leuA)*MudA*(hisF)]. A derivative of the above strain that has a deletion of homology at the ara inversion join point(aswellas the leuA-hisD deletion) is designatedDEL859[INV768{(zac)*TnlOdCam*(CobI)]DEL860[(leuA)*MudA*(hisF)]]. This deletion removes genetic material between the TnlOd-Cam elements in the zac region (adjacent to ara) and the CobI operon; the result of homologous recombination between the twoTnlOd-Camelements. The inversion join point, which previously mapped in the ara genes, now maps in the deletion: DEL859[(zac)*TnlOd-Cam*(CobI)]. RESULTS The assay system: A genetic system has been developed that detects strains containing directed inversions of particular chromosome regions (MAHAN and ROTH 1988). We have previously used this system to surveyrecovery of inversions of severalparticular intervals of the bacterial chromosome(SEGAL,MAHAN and ROTH 1988; MAHAN, SECALL and ROTH 1990). T h e results of these experiments indicated that some intervals form inversions at high frequency (permissive), whereas other intervals fail to show inversion (nonpermissive). In this paper, we use a parental strain J. R. Roth that contains an ara-his inversion to examine the effects of this parental inversion on the permissivity of inversion for intervals that are either internal to the region inverted (subintervals) or external to the parental inversion. Figure I C is adiagram of strain T T l 3 9 0 9 that carries a stable ara-his inversion (see Figure Ib and MATERIALS AND METHODS for construction). T h e inversion join point adjacent toara contains a deletion that fuses material near theara region (zac, minute 3) tothe CobIgenes (normally at minute41) (zac3659*TnlOd-Cam*cobZ-368). T h e inversion join point athis contains a deletionthat fuses the hisD gene (minute 42) to the leuA gene (normally at minute 3) (leuAll79*MudA*hisD9953). T h e hisD-leuA deletion (leuAll79*MudA*hisD9953) removes the his promoter, thehisG gene and partof the hisD gene. These deletions remove all shared homology from the two join point regiowand stabilize the parental (ara-his) inversion. Into the trp region of this stable inversion strain (Figure IC), we introduced a chromosomal fragment that carries the proximal portion of the his operon (hisOGDC') flanked by TnlO elements in the same orientation (C' indicates that only a fragment of the hisC gene is present, Figure Id). The his material at trp is in an orientation opposite thatof the normal his region. This strain is able to grow on histidinol as a source of histidine (Hal+) due to the functional hisD gene present at thetrp locus. (The hisD gene product catalyzes the conversion of the intermediatehistidinol to histidine.) By selecting for His+ derivatives of this strain, one can select for recombination between the inverse order his sequences, generating a complete his' operon.Thisgeneralprocedure has been followed to construct a series of strains; each strain has his sequences placed at a particular site outside the his operon in inverse orientationvis a vis the standardhis region. For each strain, selection for His+ recombinants demands an exchange between the two separated parts of the his operon. This exchange may or may not lead to inversion formation. In cases where inversions are impossible (nonpermissive), His+ recombinants can form by excision of the TnlO-his0GDC'-TnlO material as a circle and integration of this circle at the his region (MAHANand ROTH 1988, 1989). Detection of inversions: An homologous reciprocal exchange event between inverse order sequences present in the same chromosome results in an inversion of the intervening material between the sites of exchange. Since the Hol+ His- parent strain TT 13920 contains inverse order his homologies, selection for His+ recombinants yields a recombinant(such as strain TT13930 in Figure 2) that contains an inversion of the arc of chromosomal material lying between the Restrictions on Inversion 1025 ( 1 b) Removal of shared homology from inversion join points (1 a ) Unstable inversion (TT11797) Stable inversion (Id) Parent strain (TT13909) (TT13920) (1 C) Of hlSD'MudA'leuA FIGURE .-Construction 1 of a stable inversion-bearing strain. ( l a ) T T l l 7 9 7contains an inversion from ara-his (minute 2 to minute 42) as described in MAHANand ROTH (1988); homologous recombination between the inverse order his or TnfO homologies present at both inversion join points (at ara and athis) can reverse the inversion to normal gene order. (1b) Removal of his and TnfOhomologies at ara and at his. Genetic material was deleted between the leuA (minute 3) and hisD (minute 42) genes (leuAfl79*MudA*hisD9953) of TTI 1797 as described byHUGHES and ROTH (1985); the result of homologous recombination between two transduced fragments containing MudA elements (one in leuA, the other in hisD) and the subsequent recombination of the hybrid fragment (dark triangle) with the recipient chromosome. A derivative of this Ap" Leu- His- transductant strain (TT13905), which contains a TnfOd-Cam element in the cobI genes [cobl-368::TnfOd-Cam (minute 41)], was used as a recipient of P22 phage grown on TT13903 (zac-3659::TnlOd-Cam);selection for Ara+ requires recombination between the two TnlOd-Cam elements (open triangles), removing the genetic material between the zac region [adjacent to ara (minute 3)] and thecob1 genes (minute 41). (IC)Strain T T l 3 9 0 9 contains a stable ara-his inversion (constructed as described in l b , and in MATERIALS AND METHODS). The ara region contains a deletion (zac-3659*TnfOd-Cam*cobl-368)that fuses material from the LUC region (minute 3) to the cobI genes (minute 41). The his region contains a deletion ( l e u A l f 79*MudA*hisD9953)that fuses the leuA gene (minute 3) to the h i d gene (minute 42). (Id) Into the trp region (minute 34) of strain TT13909, we have introduced a chromosomal fragment that carries the hisOGDC' genes flanked by TnfO elements in the same orientation (C' indicates only a fragment of the hisC gene is present). The his material at the trp region of strain TT 13920 is in the opposite orientation vis & vis the normal his operon. his and trp loci. In the rearranged parent strain, this arc includes minutes 3-34 of the normal chromosome map (Figure 2). Noninversion recombinant types that survive this selection have been described in detail elsewhere (MAHANand ROTH 1988, 1989). The inversion structure of recombinant TT13930 (bottom of Figure 2) was identified by linkage disruption tests performed on both join points of the inversion. Since the inversion contains normal his material on only one side of each inversion join point and trp material on the other, it shouldnotbe possible to repair either inversion join point with a wild type transduced fragment. For example, it should be impossible to obtain a Trp+ transductant (using a wild type donor), since the trp region (thenon-his endpoint) is disrupted at the inversion join point. Furthermore, the his inversion join point of TT13930 should not be able to inherit large a his deletion (leuAI I79*MudJ*hisF9954) that is associated with a kanamycin resistance determinant (Figure 3).That is, the M. J. Mahan and J. R. Roth 1026 Recipient chromosome with parent gene order Parent inversion strain (TT13920) Kmr + m 2 orlg X a his+ TT13913 transduced fragment b X b e parent / / 1,, Select b with gene order Kmr c d e // Select HIS+ chromosome Kmr (HIS’) transductants +-T lnverslon breakpoint in reclplent Kmr Secondary inversion recombinant TT13913 transduced fragment b no shared (TT13930) b 2 ( homology ) Inversion chromosome 1 Select Km Linkage disruptlon No Knf tranductants FIGURE 2.-Selection for inversions. The trp region (minute 34) of parent strain TT13920 contains a chromosomal fragment that carries his material (hisOGDC’) in the opposite orientation vis ii vis the normal his operon (minute 42). Selection for His’ requires an exchange event between the shared inverse order hisDC homologies. Such recombination yields strain TT13930: a His+ derivative that carries an inversion of chromosomal material between the sites of exchange (trp-his). his region of parent strain TT13920 (Figure 2, top) is disrupted at the his inversion join point in recombinant strainTT 13930 (Figure 2, bottom). Transduction of all loci unlinked to either inversion join point should be normal.A more detailed descriptionof tests for linkage disruption at the his endpoint and non-his endpoint is presented in MATERIALS AND METHODS. In these tests one sees a 6-100-fold (typically a 30-fold) reduction in transduction frequency, instead of the expected absence of transductants (MAHAN, SEGALL and ROTH 1990). Genetic analysis performed on the few transductants that are found despite the linkage disruption suggests they are due to reversal of the inversion; these “backflips” can result from homologous recombination between the inverse order his or T n l O homologies at both inversion join points of the His+ derivatives [at trp (minute 34) and his (minute 42) of TT13830, bottom of Figure 2)] (M. J. MAHAN and J. R. ROTH, unpublished results). Transduction of loci unlinked to either join point is normal (data FIGURE3.-Linkage disruption at the his inversion join point in parent strains with an ara-his inversion. P22 phage grown on TT13913, which contains a large his deletion associated with a kanamycin resistance determinant (leuA1 179*MudJ*hisF9954),was used to transduce His+ recombinants to kanamycin resistance. Strains that contain parental gene order flanking both sides of the his region will inherit the deletion with normal transduction ability. Strains containing an inversion in which the his inversionjoin point disrupts the his region show a reduction in ability to inherit the large his deletion. Transduction ability at loci unlinked to either join point is normal. Strain TT13913 is identical to TT13909 (Figure IC) except the deletion, which is associated with a MudJ element, extends from leuA into hisF. The arrows denote the two breakpoints of the inversion. not shown, also see MATERIALS AND METHODS). The validity of the linkage disruption test was previously documented by Hfr mapping of an inversion strain that was initially characterized by linkage disruption (MAHANand ROTH 1988). T h e gradient of transfer of chromosomal markersconfirmed the inversion structure inferred by linkage disruption. Permissive and nonpermissive intervals in parental strains containing an uru-his inversion rearrangement: We have placed the his operon fragment (described above) at 11different locations in parental strains containingthe stable uru-his inversion; in each strain the two fragments of the his operon are in opposite orientation but can recombine to generate a His+ phenotype. From each of these strains, His+ recombinants were selected on minimal medium. Inversions (listed in Table 1) were identified by linkage disruption tests performed on both join pointsof the Restrictions on Inversion rearrangement (where possible). An interval permissive to inversion is defined asone thatshows a 6- 100fold reduction in transductant frequency at both join points, but an undiminished transductant frequency for regions distant from either joinpoint. For some strains, absence of a selective marker prevented testing of one endpoint forlinkage disruption.Forexample, only the his endpoint of strain TT13916 (PncB) and TT13918(@A) can be tested for linkage disruption. The non-his endpoint cannot be tested because the pncA and pncB genes are involved in a salvage pathway of pyridine nucleotides and there is no positive selection for Pnc+ recombinants in this genetic background. Selection for Pnc+ can beaccomplished if a nadA213::MudJ is introduced (nadA is involved in the de novo synthesis of pyridine nucleotides); this allows testing linkage disruption at the non-his endpoint of the pncB-his (TT139 17) and pncA-his (TT13919) intervals, but (because of KanR determinant of the introduced MudJ) prevents the test for linkage disruption at his. Thus isogenic pairs of strains (nadA+ and nadA-) were used to permit detection of disruption at both join points. Further, the non-his endpoint of TT13915 (ara-his) cannot be tested because theparentstraincontainsa proBA deletion (proAB47). Selection for Ara+ on minimal medium containing arabinose requiresthe additionof proline to supplement the auxotrophy. Since proline can also serve as a carbon source, selection for Ara+ transductants is difficult in this genetic backround. Finally, the non-his endpoint of TT13921 (metG) and TT13922 (eut) cannot be tested because the non-his endpoints map at TnlO insertions near, not in, the respective genes (Table 1). Inspection of the results in Table 2 shows that some intervals show inversion (permissive = P), whereas other intervals fail to show inversion (nonpermissive = N). T o permit comparison of the new results with thoseobtained previously, the materialincluded in each interval is indicated using the map position (in minutes) of the intervening material on the wild-type Salmonella map. A “T”between the minutes at the end of the segment indicates that the terminus (or origin) of replication is included in the segment being tested for inversion. The behavior of this chromosomal segment, permissive (P) or nonpermissive (N) in the rearranged parent and in a parentwith a normal map order is indicated in the two columns at the far right in Table 2. Designation of wild-type chromosome behavior is based on previously published data (SEGALL,MAHAN and ROTH 1988). These intervals are diagrammed in Figure 4. Results obtained previously using this assay in a wild-type parent strain are presented in Figure 4a; results obtained here in the rearranged parent strain are diagrammed in Figure 4b. 1027 The entries above the horizontal line in Table 2 describe subintervals that are internal to the region inverted in the parental strain (ara-his). In the inversion parent, each of these subintervals is located in reversed orientation and at a slightly different point in the overall map; material that is clockwise of the ara locus inwild type is now immediately counterclockwise of the his locus. The ara-trp interval tested in both strains shows the same inversion behavior as it did in the wild-type parent. The two intervals that have not been previously tested do not include the terminus and proved to be nonpermissive. Below the horizontal line in Table 2 are presented results for eight intervals that include material outside of the parental inversion. The first three intervals (hismetG, his-eut and his-tyrA) are justclockwise of the his locus. These segments were nonpermissive in the wildtype background and remain so in the parent strain with the ara-his inversion. The second two intervals (cysJZ-his and argA-his) includelarger chromosomal segments clockwise from the his locus and show only slight differences in behavior that will be discussed below. The last three intervals (metE-his,pyrB-his and ara-his) are large regions (nearly half of the chromosome) that include the terminus(ororigin); these regions can be viewed as internal (for external) to the ara-his region, depending on the point of reference. Two intervals (pyrB-his and ara-his) show the same permissive character as they did in the wild type parental strain. The ara-his interval is essentially identical to theparental inversion; inversion of this interval returns chromosome structure to that of wild type. T h e third interval (metE-his) changes from nonpermissive to permissive as a consequence of the ara-his rearrangement. DISCUSSION By selecting for recombination between inverse order homologies, we have previously shown that some intervals of the Salmonella chromosome invertat high frequency, whereas others fail to show inversion (MAH A N and ROTH 1988;SEGALL, MAHANand ROTH 1988; MAHAN,SEGALL and ROTH 1990). In the present studies, we test the behavior of 11 intervals in a rearranged parent strain that alters the chromosomal position, neighboring sequences and, in some cases, the orientation of the arcs of chromosome tested for inversion. T o d o this, we constructed a parent chromosome with a large inversion (ara-his) that reverses the orientation of a 40-minute segment of chromosome (minutes 3-42). Three of the intervals tested are internal to this region. Eight other intervals include material outside of this region. With the exceptions noted below, all of these intervals maintained the same behavior they showed in the wild type parent strain, suggesting that the permissivity of inversion M. J. Mahan and J. R. Roth 1028 TABLE 2 Distribution of permissive and nonpermissive intervals in a parent strain with an ara-his inversion Strain" Non-his endpoint (position)' TT13916 TT13917 TT13918 TT13919 TTl3920 pncB (20) pncB (20) pncA (27) pncA (27) trb (341 TT13921 T T 13922 TT13923 TT13924 TT13925 TT 13926 TTI 3927 TT13915 metG (44) eut (50) tyrA (55.4) C Y ~ J(60) I argA (61.2) metE (84) PrrB (98) ara (2.5) Map units included in intervalb 3-20 3-20 3-27 3-27 3-T-34 42-44 42-50 42-55.4 42-60 42-61.2 42-T-84 42-T-98 42-T-2.5 Percent of His+ clones with inversions his endpoint (no. tested)' 0 (20) 0 (40) 0 (20) 0 (20) 80 (20) Permissivity Disruptiond 1 .O te n tc 1 .O n 0.1 1 0.04 1 .o 1 .O 85 (20) 0 (20) 0 (20) 10 (20Y 2 0 9 " 0.07 0.02 0.10 0.17 0.13 Non-his endpoint n te 1.o nt' n 1 .O ntl 0.06 n tC n te 1.O 0.04 co.01 0.08 0.03 ntc Inv parent Wild type N N N N P ntf ntJ tf P P N N N' N' P P P N N N N N P P P 90 (20) 80 (20) 90 (20) a Entries above the horizontal line describe subintervals internal to the region inverted in the parental strain; entries below the horizontal line describe intervals outside the parental rearrangement. (P) denotes permissive intervals, while (N) denotes nonpermissive intervals. and ROTH (1988); the his locus is at minute 42. The parent strains contain an inversion of Map positions are according to SANDERSON material adjacent to ara (rac region, minute 3) to his. To describe the material within an interval, the ends of that interval are indicated in map units on the standard wild type genetic map; a "T"is included if the segment includes the terminus (or origin) of replication. ' The frequency ofHis+ recombinants was about for all intervals tested; a random collection of these clones was tested for linkage disruption. Linkage disruption is the reduction in ability of His+ recombinants to be repaired at either inversion join point with P22 phage grown on TTI 391 3 (see Figure 3 and MATERIALS AND METHODS). For permissive intervals, the numbers, expressed as a fraction, indicate the ratio of the number of transductants obtained in a His+ clone to the number of transductants obtained in the isogenic parental strain without a secondary inversion ( i e . , show no linkage disruption). The numbers presented are an average of such ratios determined for five His+ recombinants tested. For intervals judged permissive, the ratio has a value near 1.O, reflecting the fact that all His+ clones recovered show approximately the same number of transductants (at either inversionjoin point) as the noninversion parental strain ( i e . , no linkage disruption was detected). Not tested because the genotype of the parent strain does not allow selection for repair of the endpoint indicated (see RESULTS for a detailed explanation of each endpoint in question). /These intervals were not among those tested previously in a wild-type value so no information is available as to their behavior in a strain without the ara-his inversion. g The two underlined values are unusual in that they are intermediate between the values found for permissive intervals (80-90%) and those found for nonpermissive intervals (0%)(MAHAN, SECALLand ROTH 1990). For these two intervals, inversions are found but not at the high frequency characteristic of fully permissive intervals (see DISCUSSION). The His+ recombinant strain TT13936, which carries a secondary inversion from argA-his (61-42), shows a marked reduction in growth rate as judged by colony size on nutrient broth medium. intervals is dictated by the sequences included and not by their position in the chromosome or their flanking regions. Using the parentalstrain with a stable inversion rearrangement (40% of the chromosome), we tested the ability to recover secondary inversions for a series of chromosomal intervals. The permissivity (or nonpermissivity) of each of the testedintervalsfora parent strain with the ara-his inversion is presented in Table 2; the permissivity of the same intervals for a parentstrain with a wild type chromosome is presented for comparison (data for parent strains with wild type gene order were taken from SEGALL,MAHAN and ROTH 1988; MAHAN, SEGALL and ROTH 1990). Inspection of the distribution of permissive intervals reveals thatthe ability toinvert is often correlated with inclusion of the terminus or origin of replication, regardless of the parental chromosome arrangement. One interval internal to the parental inversion 3- 34 (ara-trp) has beentested in both wild-type and inversion backgrounds. T h e permissivity of this interval is maintainedeventhough, in therearranged chromosome, the tested segment is placed in inverse order with differentflanking sequences [i.e., chromosomal minutes 3 , 4 , 5 , etc. appear near thehis locus ara(minute 42)]. In the rearranged parent, the entire trp segment is both inverted and shifted 8 minutes clockwise. The interval includes the replication terminus and is permissive for inversion regardless of the parental genome arrangement. T h e segments 3-20 (ara-pncB)and 3-27 (ara-pncA) proved nonpermissive in the inversion background but have not been testedin the wild-type background. Neither of these two nonpermissive intervals includes the terminus,thustheir nonpermissivity obeys the simplest form of theterminusrule.In wild-type strains, we have previously tested the slightly shorter region 3-17-minute interval (ara-nadA) and found it permissive (SEGALL,MAHAN and ROTH 1988). This on Restrictions b FIGURE4.-Distribution of permissive and nonpermissive intervals. Part a diagrams the distribution of permissive (and nonpermissive) intervals in parent strains with wild type gene order. Part b diagrams the distribution pattern obtained from parent strains that carry an ata-his inversion. Solid lines (permissive) indicate chromosomal segments that form inversions at high frequency (6590%of the recombinant types tested) when one selects for recombination between sequences at the his locus and homologous sequences placed in inverse orientation at a distant site; wavy lines indicate chromosomal segments that are permissive at low frequency (2-10%). Srippled lines (nonpermissive) indicate chromosomal segments that fail to show inversion. The dark arrowheads in b denote the two inversion breakpoints in parent strains that contain an ara-his inversion: one at minute 3/41, the other atminute 4213. difference in behavior can be interpreted in terms of the periterminal rule described by REBOLLO,FRANCOIS and LOUARN (1 988) according to which an interval without aterminuscouldinvert if it doesnot disrupt the periterminal regions. Both of the first two Inversion 1029 inversions have one endpoint within one of the suggested nondivisible zones (NDZ). In contrast, the permissive 3-1 7 minute interval does not disrupt one of these zones. Thus,the behavior of these intervals agrees with the model of REBOLLO,FRANCOISand LOUARN(1988). However,as discussed below, we prefer to think that endpoints within a NDZ render inversion mechanistically impossible rather than leading to lethal consequences of the final rearrangement. If the above intervalsare described in terms of their endpoints, the his-trp endpoints (42-34), which are nonpermissive in wild type background, become permissive in the inversion background (compare Figure 4, a and b). Conversely, the his-pncB (42-20) and hispncA (42-27) endpoints are permissive inwild type and become nonpermissive in the inversion parent. However, it should be kept in mind that the different backgrounds cause very different chromosomal material to be located between each set of endpoints. It seems that the nature of the sequences within each interval dictates the permissivity of the interval. This interpretation is reinforced by the behavior of intervals including material outsideof the ara-his region. The three segments immediately clockwise of the his operon are not included in the parental ara-his inversion and that parental inversion does not alter their nonpermissive character. Thus, despite the fact that the chromosome has been rearranged and sequences at one end of the tested interval are different, these three regions maintain their nonpermissive character. None of these intervals includes a terminus or origin region. In discussing the above inversions, the shortest chromosomalsegment between endpoints wasassumed to be undergoing inversion. However, since the chromosome is circular, the small segment might be used as a point of reference for inversion of the rest of the circle. This point becomes troublesome when discussing larger inversions. For the very large intervals 42-84, 42-98 and 42-2.5 (last three lines of Table 2), the smaller arcs of chromosome are 42, 44, 39.5 minutes, respectively, so the endpointsdivide the chromosomeinto two rather comparabledomains, making it problematical to discuss these intervals in terms of alterations of the content of the inverting segment. The most striking change observed in these experiments is that shown by the his-metE interval (42-84); it is converted from nonpermissive (in a wildtype background) to permissive by the parental arahis inversion, which alters the slightly larger arc of chromosome. Since the metE-his interval includes the terminus or origin region in both configurations, the nonpermissivity observed in the wild-type chromosome is an exception to the terminus or origin rule. The other two large intervals (his-pyrB and his-ara) are not altered in behavior by the parental ara-his 1030 M. J. Mahan and J. R. Roth inversion, which slightly modifies the smaller arc of chromosome;boththeseregions are permissive in both the wild type and inversion strains. The behavior of the cysJZ-his (42-60) and argA-his (42-61) intervals show at most slight effects of the parental ara-his inversion. Both intervals were classified as nonpermissive in a wild type background, since no inversions were recovered (0/20). In therearranged background, both intervalsyielded inversions but did so at a low frequency (2/20 and 1/40). While the difference in behavior is not statistically significant, we recovered no inversions from any ofthe other intervals classified as nonpermissive. Permissive intervals (for the assay used here) typically gave 80% inversions among the His+ recombinants. This suggests that the presence of the parental inversion may have a slight effect on the two intervals clockwise of the his locus. T o pursue this possibility, we tested 120 more His+ recombinants for the cysJI-his interval arising in the wild type background and found noinversions. The one (1/40) argA-his inversion observed in the rearranged parentdisplayed a slow-growth phenotype as judged by colony size on solid nutrient broth medium. It is possible that poor growth of inversionbearingrecombinantscontributes totheapparent nonpermissivity of the argA-his interval.However, such a slow-growth phenotype is unusual among the inversion recombinants we have seen in Salmonella. Of over 40 other intervals tested, only the ara-trp region (tested in a wild type parent) yielded inversion recombinants with a reduced growth rate. Thesearatrp inversions were recovered at ahighfrequency despite their growth impairment. In any event, the presence of the ara-his inversion in a parental strain has at best a small effect on the recovery of inversion of the adjacent 42-60 and 42-61 minute regions. It seems likely thatthecorrectexplanationfor nonpermissive intervals must account for the observations in both Salmonella and E. coli. There is striking agreement regarding the general pattern of permissive and nonpermissive intervals in the two organisms. REBOLLO,FRANCOISand LOUARN(1 988) have tested several intervals that extend from his toward the terminus within a periterminal region that does not include the terminus; they judged these intervals to be nonpermissive. They have also tested several intervals that extend fromhis across the terminus and found them permissive for inversion. They have suggested the existence of critical regions (NDZ) on either side of the terminusregion; inversions disrupting these periterminal regions,they suggest, may be lethal due to difficulties in replicating this material, when it is presented in the abnormal orientation.We feel our data may reflect some importance of the terminus region, but we are less enthusiastic about lethality as an explanation of nonpermissive intervals. By a transductional method, we have constructed inversions of two intervals that are nonpermissive for inversion by intrachromosomal exchanges. Both constructed inversions appear to grow normally (A. M. SEGALL, L.MIESELand J. R. ROTH, unpublished results). However, in the data setof REBOLLO,FRANCOIS and LOUARN( 1 988) and in two of the 45 intervals we have tested, inversions were recovered that showed impaired growth. One of these examples is the argAhis region reported here. The other is the ara-trp region for which slow-growing inversions arose and were recovered at high frequency (SEGALL, MAHAN and ROTH 1988, MAHAN,SEGALL and ROTH 1990). While impaired growthor lethality could be suggested to account for the failure to recover inversions of some regions, we see normal growth of strains with constructed inversions of nonpermissive intervals. These results suggest that a mechanistic barrier is more likely to represent the general explanation of nonpermissive intervals. The “terminus rule” that invertible segments must contain a terminus or origin of replication still seems interesting despite some exceptions. If the two intervals with very infrequent exchanges are classified as nonpermissive, then the terminus ruleis obeyed by all of the 1 1 intervals tested here in the altered parental background. One of the previously reported exceptions to the terminus rule (trp-pyrC) has been reevaluated and now appears to bepermissive in agreement with the rule (L. MIESEL and J. ROTH, unpublished results). Some remaining exceptionsmay be explained by the periterminal rule of REBOLLO,FRANCOISand LOUARN(1988); e.g., the 3-1’7-minute interval (aranadA) is permissive in the wild type background; this region does not include the terminus but does not disrupt the periterminal region. Regardless of whether or not some combination of these rules can be made to predict all inversion behavior, we suggest that nonpermissivity is not due to lethality of inversions, but rather to a restriction on recombination between sequences flanking particular chromosome regions. We currently entertain two models, both of which suggest that folding or packaging of the bacterial chromosomemightcontribute to limiting recombination between particular sites. (1) If the folding or positioning of portions of the chromosome restricted contact between particular sites in the same circular molecule, inversion formation would be prevented. T h e noninversion recombinants could arise by sister chromosome exchanges (SEGALLand ROTH 1989) or by excision and reintegration of circles in the case of the recombination system used here (MAHAN and ROTH 1988). (2)The second possibility is an extension of the recombination model of MAHAN and ROTH Restrictions on Inversion (1989). According to this model, reciprocal exchanges require repairof double strand breaklgaps generated in the process of recombination. Excessive degradation of these ends would prevent completion of a reciprocal exchange within the short inverse repeats and lead to a failure of inversion formation. Chromosome folding or sequestration of particular chromosomal regions might influence the extent of end degradation. Regardless of the specific mechanisms involved, the distribution of permissive and nonpermissive intervals and the effects of chromosomealterations such as those presented here should reflect aspects of chromosome structure that are physiologically relevant. We have presented data that theability to form inversions is influenced by sequences includedin the tested segment and is less sensitive to the natureof sequences that flank that segment. An important aspect may be possession of the terminus (or origin)of replication. This work was supported by U.S. Public Health Service grant GM 27068 from the National Institutes of Health. M. J. M. was supported by predoctoral training grant T32-GM 07464-1 1 from the National Institutes of Health. 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Communicating editor: E. W. JONES