construction of chromosomal rearrangements in salmonella by

Copyright 0 1994 by the Genetics Society of America

Construction of Chromosomal Rearrangements in Salmonella by Transduction: Inversions of Non-Permissive Segments Are Not Lethal

Lynn Miesel, Anca Segall’ and John R. Roth

Department of Biology, University of Utah, Salt Lake City, Utah 84112 Manuscript received January 31, 1994 Accepted for publication May 4, 1994

ABSTRACT Homologous sequences placed in inverse order at particular separated sites in the bacterial chromo-

some (termed “permissive”) can recombine to form an inversion of the intervening chromosome segment. When the same repeated sequences flank other chromosome segments (“non-permissive”), recombination occurs but the expected inversion rearrangement is not found among the products. The failure to recover inversions of non-permissive chromosomal segments could be due to lethal effects of the final rearrangement. Alternatively, local chromosomal features might pose barriers to reciprocal exchanges between sequences at particular sites and could thereby prevent formation of inversions of the region between such sites. To distinguish between these two possibilities, we have constructed inversions of two non-permissive intervals by means of phage P22-mediated transduction crosses. These crosses generate inversions by simultaneous incorporation of two transduced fragments, each with a sequence that forms one join-point of the final inversion. We constructed inversions of the non-permissive intervals trp (‘34) to his (‘42) and his (’42) to cysA (’50). Strains with the constructed inversions are viable and grow normally. These results show that our previous failure to detect formation of these inversions by recombination between chromosomal sequences was not due to lethal effects of the final rearrangement. We infer that the “non-permissive” character of some chromosomal segments reflects the inability of the recombination system to perform the needed exchanges between inverse order sequences at particular sites. Apparently these mechanistic problems were circumvented by the transductional method used here to direct inversion formation.

R EARRANGEMENTS of the bacterial chromosome can arise by recombination between repeated chro-

mosomal sequences. Exchanges between direct-order repeats generate deletions and duplications (Figure 1); these rearrangements are common among spontaneous mutations (CAPAGE and HILL 1979; LEHNER and HILL 1980; ANDERSON and ROTH 1981; PETES and HILL 1988; reviewed in ANDERSON and ROTH 1977). In contrast, inversions, the product of intrachromosomal exchanges between inverse-order repeats (Figure 2) , are rarely reported. The rarity of reported inversions could be due to problems of detection, to a paucity of inverse-order chromosomal repeats, to poor viability of strains canying inversions, or to mechanistic problems in forming inversions.

The limitations to inversion formation have been ex- plored in Salmonella typhimurium and Escherichia coli by placing genetically marked, homologous sequences in inverse order at sites flanking various chromosomal segments (REBOLLO et al. 1988; SEGALL et al. 1988). Since this situation provides both recombining sequences and a detection method, it circumvents two of the possible limitations to the observation of inversions. In both Sal- monella and E. coli, these experiments demonstrated

’ Current address: Department of Biology, San Diego State University, San Diego, California 921824057

Genetics 137: 919-932 (August, 1994)

that formation of inversions depended on the chromosomal location of the recombining sequences used.

Inversions were a significant fraction of selected recombinants for sequences placed at some sites (termed “permissive”). When the same recombining sequences were placed at other paired sites (termed “non- permissive”) recombination acted to exchange markers between the repeated sequences, but no inversions of the intervening chromosomal segment were detected among the recombination products (KONRAD 1977; ZIEG and KUSHNER 1977; REBOLLO et al. 1988; SEGALL and ROTH 1989).

We have entertained two alternative explanations for these observations. One possibility is that inversions form for all chromosomal regions, but some inversions (non-permissive) are lethal and cannot be recovered as viable recombinants. The alternative explanation is that the recombination system acts differently on sequences depending on their chromosomal location; perhaps inversion is mechanistically impossible for sequences placed at sites flanking non-permissive regions. The mechanistic reasons for the failure to invert some regions would be supported if inversions of non- permissive regions could be constructed by a means other than recombination between chromosomal sequences (as diagrammed in Figure 2). The viability of strains with the final constructed inversion would argue

920 L. Miesel, A. Segall and J. R. Roth

A. Non-reciprocal exchange that forms a deletion

1 Deletion Recombinant

B. Non-reciprocal exchange that forms a duplication

1 b c d

Duplication Recombinant

FIGURE 1 .-Recombination between direct order homologous repeats on different sister chromosomes. Recombinants bearing deletions or duplications can arise by non-reciprocal exchanges between direct order repeats on sister chromosomes. Non-reciprocal recombination is defined here as the rejoining of only one pair of flanking markers by a single recombination event.

against lethality as a reason for the failure to form this inversion by intrachromosomal recombination.

We present data to demonstrate the non-permissive character of two particular chromosome intervals and we demonstrate a method for directing the formation of these inversions by a transductional cross. Since the constructed inversions are not lethal, we infer that their “non-permissivity” (judged by recombination between chromosomal sequences) is likely to reflect mechanistic problems that limit the recombinational ability of sequences at particular chromosome sites.

MATERIALS AND METHODS

Bacterial strains: The strains used in this study were all derived from S. typhimurium strain LT2. Their genotypes are listed in Table 1.

Media: Rich medium (NB) was Difco nutrient broth (8 g/liter) with NaCl (5 g/liter). To make solid medium, agar (BBL) was added to 1.5%, final concentration. Minimal medium was E medium supplemented with 0.2% glucose or a variant of E medium lacking citrate (NCE) (VOGEL and BONNER 1956 BERKOWITZ et al. 1968). NCE medium was supplemented with 0.2% lactose as the sole carbon source. Concentrations of nutritional supplements were as described in (DAVIS et al. 1979). Ampicillin (Ap) was added at final concentrations of 30 pg/ml in NB medium and 15 pg/ml in minimal medium. Tet- racycline HCI (Tc) was added at final concentrations of 20 pg/ml in NB and 10 pg/ml in minimal medium. Kanamycin SO, (Kn) was added at final concentrations of 50 pg/ml in NB and 125 pg/ml in minimal medium. Amino acid supplements and antibiotics were obtained from Sigma. The indicator dye 5-bromo-4chloro-3-indolyl-PDgalactopyranoside (X-gal; Bachem Fine Chemicals) was added at a final concen-

Reciprocal exchange that forms an inversion

Parent Strain with Inverse-Order Repeats

Full Exchange Inversion Recornbinant

FIGURE 2.-Inversion formation by intrachromosomal recombination. Inverse order homologous sequences present at separate chromosomal sites can, in theory, recombine to invert the intervening chromosomal segment. Inversion formation by intrachromosomal recombination requires a reciprocal exchange: both pairs of flanking markers are rejoined from a single recombination event.

tration of 25 pg/ml. Kligler iron agar and MacConkey agar were purchased from Difco.

Transduction methods For most crosses, the high frequency transducing mutant of phage P22 (HT105/1 in t -201) was used as described (DAVIS et al. 1979; SANDERSON and ROTH 1988). Another high frequency transducing mutant of P22 (HT12/4 Tpfr72 Ap68, phage MS2104) was used for the his- cysA inversion construction. This transducing phage, a gift from MIRIAM SUSSKIND, is deficient in lysogen formation (int) and in tail fibers due to the Tpfr72 deletion and has a relaxed specificity of DNA packaging due to the HT 12/4 allele. Tail fibers must be added in vitro to lysates of this phage to permit further infectivity. Use of this phage in transduction crosses minimizes lysogen formation and phage killing and improves transductional efficiency. Transducing lysates of phage MS2104 were prepared as described by (DAVIS et al. 1979); phage tails were added as described by YoUDERlAN et al. (1988).

MudA phage: The transpositiondefective derivative of Mu dl(Ap, lac) phage, MudA, (CASADABAN and COHEN 1979; HUGHES and ROTH 1984) was used as a repeated chromosomal sequence for recombination tests. Upon insertion in the chromosome, the MudA transposon can fuse its lac operon to a target operon and conveys resistance to ampicillin; this element is about 38 kb in length.

Phenotype tests: The hisD gene product catalyzes the conversion of histidinol to histidine. A HisD’ phenotype is scored as the ability of a strain to grow on minimal medium or, if blocked elsewhere in the his pathway, to use histidinol (1 mM) as a histidine source on minimal medium. The trpAB gene products catalyze the conversion of indole to tryptophan. The TrpAB’ phenotype is the ability of a strain to grow on minimal medium or to use indole as a tryptophan source. The phs gene product is required for anaerobic conversion of thiosulfate to sulfide, which forms a dark brown precipitate (FeS) in Kligler iron agar; mutant strains ( p h s ) form no precipitate in this medium (Phs-). The vitamin B12dependent utilization of 1,2-propanediol, the Pdu phenotype, was tested on Difco MacConkey agar supplemented with 1 % propanediol (w/v) and cyanocobalamin (0.2 pg/ml). On this medium, Pdu’ strains form red colonies and Pdu- colonies are white (BOBIK et al. 1992). The vitamin B12dependent utilization of ethanolamine as a carbon source (the Eut phenotype) was tested on NCE medium supplemented with 0.2% ethanolamine and cyanocobalamin (0.2 pg/ml) (ROOF and ROTH 1988).

Chromosomal recombination tests: Construction of strains used for the assay of recombination between inverse-order mutant copies of the lac operon and methods for isolating and

Constructed Chromosomal Inversions 921

TABLE 1

Bacterial strains

Straina Genotype * LT2 Wild type TR59OO DEL299 (his‘ GO, phs- , pdu-, cob-) lT513 zee-2:TnlO lT1330 trpE1049:TnlO “ A lT3667 zee-729:TnlO “B” his’ lT7701 hisH9962:MudA “A” lT9241 hisB,Hl26l::MudA “B” lT10508 cysA1585:MudA “B” lT12246 trpA2485:MudA “A” lT12262 srl-201 trpD,E249O:MudA(lacZ::Tn5) hisD9533:MudA( lacZ95O:TnlO) TT13399 DEL299 hisB,Hl26l::MudA “B” TT13400 DEL299 hisB,Hl261::MudA “B” trpElO49:TnlO “A” lT13401 trpA2485:MudA “A” zee-729:TnlO “B” lT13402 trpE1049:TnlOINV1795[(trpA2485*MudA*hisB,Hl261) DEL299 (trpE1049*TnlO*zee-729)] TT13403 INV1796[(trpA2485*MudA*hisB,H1261) DEL299 (trpE1049*TnlO*zee-729)] TT13404 trpE1049:TnlO INV1797[(trpA2485*MudA*hisB,H1261) DEL299 (trpEl049*TnlO*zee-729)] lT13405 INVl798[(trpA2485*MudA*hisB,H1261) DEL299 (trpE1049*TnlO*zee-729)] lT13406 trpEl049:TnlO INV1799[(trpA2485*MudA*hisB,H1261) DEL299 (trpE1049*TnlO*zee-729)] TT13407 INV1800[(trpA2485*MudA* hisB,HI261) DEL299 (trpE1049*TnlO*zee-729)] ‘IT13408 TT13422

zee-729:TnlO srl-201 hisD9953:MudA( lacZ95O:Tnl O)pyrC2688:MudA( lacZ:Tn5)

TT13441 IT17283

zfa-3649:TnlO “B” DEL299 hisH9962:MudA “A”

IT17284 DEL299 hisH9962:MudA “A” zfa-3649:TnlO TT17285 DEL299 cysA1585:MudA “B” TT17286 serBl469:TnlOdGn his646(DELhisOGD) TT17288 srl-201 hisD9953:MudA “A” (lacZ95O:TnlO) cysA215B::MudA “B” (lacZ::Tn5) TT17289 DEL299 INVl781[( hisH9962*MudA*cysA1585)-(zee-2*TnlO?zfa-3649)] TT17290 DEL299 INV1782[(hisH9962*MudA*cysA1585)-(zee-2*Tnl~zfa-3649)] TT17291 DEL299 INV1783[(hisH9962*MudA*cysA1585)-(zee-2*Tnl~z~a-3649)] TT17292 DEL299 INV1784[(hisH9962*MudA*cysA1585)-(zee-2*Tnl~z~a-3649)] TT17293 DEL299 INV1785[( hisH9962*MudA*cysA1585)-(zee-2*TnlO?zfa-3649)] lT17294 DEL299 DUP1786[(zfa-3649)*TnlO*(zee-2)] hisH9962:MudA lT17295 DEL299 DUPl787 [ (zfa-3649) *Tn 1 O( zee-2) ]

All strains used in this study were derived from S. typhimurium strain LT2 and were constructed in this laboratory. The letters “A” and “B” denote the orientation of the preceding inserted element. The orientations are described in MATEW AND METHODS.

characterizing the resulting Lac+ recombinant clones were described previously (SEGALL and ROTH 1989).

Construction of strains used for directed inversion formation: The his-trp chromosome segment: The transductional recipient strain (TT13400) used to direct formation of the his-trp inversion was constructed as follows. The hisB,H1261::MudA insertion (from strain “T9241) was transduced into a strain (TR5900) carrying the large deletion DEL299 (hisGphs-pdu-cob) selecting ApR. The introduced hisB,H::MudAelement isin the “B”orientation (counterclockwise transcription of the lac operon); the lac operon is not fused to the his promoter (transcribed clockwise). The trpE1049:TnIO insertion (from strain TT1330) was then introduced by transduction, selecting TcR. The introduced TnlO insertion is in the “ A orientation, as defined previously (CHUMLEY et aZ. 1979; HUGHES and ROTH 1985). The final strain (TT13400) is phenotypically His- Trp- ApR TcR Lac- (see Fig- ure 5). Strain ‘IT13400 grows on indole as a tryptophan source because a secondary promoter, downstream of the trpE::Tn IO insertion site, expresses the trpBA genes (BAUERLE and W- GOLIN 1967; JACKSON and YANOFSKY 1972). This strain can use histidinol as a histidine source since it has an intact hisD gene, expressed from a foreign promoter at the opposite end of the deletion DEL299.

The transduction donor used to direct inversion formation was constructed as follows. The initial strain (TT3667) has a TnlO insertion near the his locus (zee-729:TnlO; orientation “B”). The trpA2485:MudA element was transduced from

strain TT12246 into strain ‘IT3667 selecting ApR. This trpA::MudA insertion is located downstream of the internal trp promoter; since this element is in orientation “A,” this strain expresses the ZacZgene from the trp promoter and forms blue colonies on X-gal indicator plates. The final strain (TT13401) is phenotypically His’ Trp- ApR TcR Lac’ and cannot use indole as a tryptophan source.

The his-cysA chromosomal segment: The transduction recipient used to make this inversion was constructed as follows. The hisH9962:MudA insertion (“A” orientation; from strain TT7701) was transduced into a strain (TR5900) which carries the large DEL299 deletion [ hisGphs-pdu-cob]. Transductants resistant to Ap were selected and tested for retention of the recipient deletion which confers a Phs- phenotype. A TnlO insertion clockwise of the eut operon (zfa-3649:TnlO; “B” orientation) was transduced into this strain (TT17283) selecting TcR. The final strain, TT17284, has the phenotype: TcRApR Lac’ His- Hol’ Pdu- Phs- and Cob- (see Figure 7).

Genetic tests for inversions: Inversion-bearing strains can be identified by observing linkage disruption at the site of the recombining sequences that have become inversion break- points (SCHMID and ROTH 1983). The homologous repeats used here to direct inversion, either by a transduction cross or by chromosomal recombination, are transposable elements (MudA or Tnl 0) inserted in structural genes; each insertion causes a particular auxotrophy and provides a characteristic drug resistance. If recombination events between these inserted elements leads to an inversion, the parental order of


chromosomal sequences flanking the insertion sites are disrupted and the two parts of each disrupted biosynthetic gene are moved to widely separated chromosome locations. This “linkage disruption” caused by an inversion makes it difficult (roughly 25fold lower transduction efficiency) to repair the auxotrophic requirement of an inversion strain by transduction with a wild- type donor lysate. When an inversion-bearing strain is used as a transduction recipient, the inversion break-points can be repaired, albeit at a reduced frequency, by events that return the inverted segment to i t s original orientation. (Crosses of this type will be described in more detail in the RFSULTS.)

Linkage disruption in a donor strain makes it difficult for a normal recipient strain to inherit the inserted element (drug resistance) located at an inversion break point. When an inversion-bearing strain is used as a transduction donor, a breakpoint marker can only be inherited at low frequency by a two- fragment transduction event in which both inversion break- points are inherited. This event regenerates the donor’s inversion in the recipient chromosome ( ~ : H M I D and ROTH 1983).

Genetic tests for tandem duplication of chromosomal segments: Strains carrying duplications generate haploid segregants by recombination between the two copies of the repeated sequences (AwnERsoN and ROTH 1977; ANDERSON and ROTH 1981). These segregants arise at high frequency (1/10- 1 / 100 bacterial cells) and have lost the genetic marker located at the join-point of the original duplication.

RESULTS

The his-trp and cysA-his intervals are non-permissive for inversion by recombination between chromosomal sequences: Mutant lac% alleles were placed in inverse order at separated sites in the chromosome and Lac’ recombinants were selected and tested as described previously (SEGALI. et al. 1988; SEGALL and ROTH 1989; SE- GALL and ROTH 1994). Each lacZ sequence is part of a transpositiondefective MudA prophage and carries a transposon insertion (either Tn5 or TnlO) within the laci! gene. Thus the parental strain is phenotypically Lac-, and recombination between the two mutant l a d genes can generate a lac+ gene (see Figure 3). Each Lac’ recombinant can be tested for the presence of an inversion and for the fate of the parental lac mutations (scored as drug resistances). Three Lac+ recombinant classes can be generated by the recombination events shown in Figure 3 (SEGALL and ROTH 1994).

A single full intrachromosomal exchange in the region of the l a d gene between the Tn5 and TnlO insertion sites of the two copies will generate an inversion with one lac+ operon; the other lac operon may carry one drug resistance elements or both (depending on whether or not gene conversion is associated with the exchange). If we assume that only two copies of the lac region interacted to form the inversion, then the Iacalleles at the ends of the inverted segment are the two products of that exchange and we can score as gene conversion any event in which there was a loss of one or more of the drug resistance elements (SEGALL and ROTH 1994).

Two full exchange events between the participating lac regions in the same chromosome can generate one lac’ allele and one with both drug resistances without

Full Exchange

Lac+ Inversion Recombinant

Two Full Exchanges

: r Lac+ Double Recombinant (No Inversion)

Apparent Conversion

Lac+ Apparent Gene Conversion Recombinant (No Inversion)

FIGURE 3.-Recombination events between inverse order MudA-lac repeats in the same circular chromosome. Thick arrows represent MudA-lac sequences. Triangles represent in- sertions of drug resistance elements within the homologous sequences.

causing an inversion. Because both of the lac regions are simultaneously altered by recombination, we feel con- fident that both were involved in the exchange that formed the lac+ allele.

A non-inversion Lac+ recombinant class that has lost the drug resistance element from one of the two lac regions represents the third recombinant type. This sort of recombinant could arise in several ways, including sister-strand exchanges. Because only one lac allele is altered and no inversion has occurred to indicate involvement of both alleles, we cannot be sure that the recovered lacalleles are both products of one exchange. Therefore we cannot score this type as a true gene conversion event. We have designated this type as “apparent” gene conversions because of this uncertainty.

Table 2 includes data on the sorts of Lac+ recombinants recovered from three different strains, each with mutant lac regions placed in inverse order at a different pair of sites. In one strain the sequences flank a permissive interval (hisD-pyrC). In the other two strains, the sequences are placed flanking the non-permissive intervals hisD-trp and cysA-hisD. When recombining lac sequences flank the permissive interval (hisD-pyrC), approximately 50% of the Lac’ recombinants carry an inversion. For the strains with the lac operons flanking a nonpermissive intervals (his-trp and his-cysA), we obtain Lac’ recombinant clones of the double recombinant and apparent gene conversion types, but none of

923 Constructed Chromosomal Inversions

TABLE 2

The his-cysA and his-- intervals are %on-permissive” for inversion formation

Freq. of Lac’ recombinant types (fraction of total)

Endpoint I Endpoint I1 Lac+ Inversionsd Apparent (map (map Interval recombinants No. of Lac’ gene Double

Strain position) position) ’ typeb (X lo5) clones tested - Conv. + Conv. conversion recombinants

‘IT13422e pyrC (‘23) hisD (’42) P 7.1 120 0.1 1 0.41 0.13 0.35 ‘IT12262’ trpA (’34) hisD (’42) NP 11 1078 0 0 0.76 0.24 ‘IT17288 hi5D (’42) cysA (’50) NP 20 698 0 0 0.76 0.24

The endpoint refers to the chromosomal position of the Mu&( lac, A p ) element. The elements are present in inverse orientation. Permissive intervals (P) are those that form inversions by recombination between chromosomal repeats. Nonpermissive intervals (NP) fail to

Frequencies are expressed as the number of recombinant clones/105 bacterial cells. generate inversion-bearing recombinants.

Conv.” denotes those inversion rearrangements which lack an associated gene conversion event. “+ Conv.” denotes those inversion rearrangements which have an associated gene conversion.

e Data for this strain have been presented previously (SEGALL and ROTH 1994). /Data for this strain have been presented previously (SEGALL and ROTH 1989).

the inversion type are found. Inversion was scored by testing “linkage disruption”-the reduced ability to repair the parental auxotrophies by transduction (see MATERIALS AND METHODS).

Directing inversion formation with a transduction cross: The basic strategy for directing inversion formation is diagrammed in Figure 4. Two DNA “linkers” are simultaneously provided as fragments transduced by phage P22. Each fragment has a scorable genetic marker flanked by sequences that are identical to separated sequences in the recipient chromosome (see parts A and B of Figure 4). If either single fragment is incorporated individually, the recombining sequences are oriented such that the chromosome is broken (Figure 4B). Si- multaneous incorporation of both fragments by independent events regenerates an intact chromosome with an inversion. These events can be visualized as occurring between sister chromosomes as diagrammed (Figure 4C), but could also occur in a recipient cell with a single circular chromosome. Formation of a viable recombinant with the selective marker on fragment 1 (as diagrammed) requires co-inheritance of fragment 2. Therefore selection for one marker causes a secondary selection for inheritance of the second marker. We have used this general approach to construct inversions of two nonpermissive chromosomal segments (his-trp and

Design of the cross that constructs inversions of the his-trp interval: The requirements of the general scheme described above are met by using transposable elements as some of the recombining sequences. These elements can be placed at appropriate positions and orientations in the donor and recipient strains. The genotypes of the donor and recipient strains used in construction of the his-trp inversion are diagrammed in Figure 5A.

Both of the parental strains have a Tn 10 and a MudA element, but these elements are disposed in opposite orientations and at different ends of the his-trp interval

his-cysA).

in the two strains. The transduction donor strain (TT13401) carries a T n l 0 element inserted in the “B” orientation upstream of the his operon; the recipient has a Tn 10 in the “ A orientation within the trp operon. The donor strain (TT13401) carries a MudA element inserted in the trpA gene (in the clockwise “A” orientation). Since this MudA fusion is transcribed from the trp promoter, strain TT13401 is Lac’ and forms blue colonies on X-gal indicator medium. The recipient strain (TT13400) has a hisH::MudA insertion in the counterclockwise “B” orientation. This lac region of this MudA element is not transcribed, so the recipient strain forms white (Lac-) colonies on X-gal indicator medium.

The recipient’s his operon carries a large deletion (DEL299) which extends from hisG, removes the his promoter and extends across the nearby cob operon. This deletion is too large to be repaired by a single P22- transduced fragment (Figure 6) (SCHMID 1981; JETER et al. 1984). However, at a high multiplicity of donor phage, two transduced fragments carrying adjacent chromosomal material of the donor can recombine with each other and provide sufficient homology to repair the large deletion at a low frequency.

To construct an inversion, the above recipient is transduced at a high multiplicity of infection using a P22 lysate grown on the donor and His’ transductants are selected. All His’ recombinants require the incorporation of at least two transduced fragments. The possible recombinant types are discussed below.

Two recombinant classes, diagrammed in Figure 5C, are formed by cooperation of donor fragments 1 and 2; both of these recombinants carry an inversion of the his-trp segment. These recombinants form by simultaneous inheritance of a transduced fragment carrying the donor his operon and the nearby Tn 10 element and a second transduced fragment carrying MudA and adjacent trp sequences; this fulfills the requirements of the scheme for inversion construction. Incorporation of either transducing fragment by itself results in breakage


A. Parental Strains

Recipient

Donor a d Fragments - a b c d :

-+ +

2 !L-$ selicted marker

scotred marker

B. Incorporation of Fragment 1

+ abed: +

/ - - 3 Fragment 1

a b c d e

a b c d e 4

a b c d e Inviable recombinant

C. Incorporation of Fragments 1 and 2 to generate an inversion n b c d e

a b c d e

Inverted segment .c

r "8

a d c b e Inversion recombinant

FIGURE 4.-Schematic representation of a two-fragment transduction cross which directs inversion formation. Trans- duced fragment 1 carries a selectable marker flanked by sequences homologous to two separate sites in the recipient chromosome. One of the flanking homologies (d) is in inverse orientation vis ci vis the recipient chromosome. (B) Incor- poration of fragment 1 alone generates an inviable structure due to the orientation of the homologous sequences. (C) Simulta- neous incorporation of fragments 1 and 2 generatesaviahle chre mosome carrying an inversion rearrangement. Note that inversion formation by a transduction cross does not require intrachromosomal recombination or reciprocal exchange.

of the chromosome (as in Figure 4B). The inversion recombinants inherit the selected His' marker on fragment 1, and also acquire an unselected Lac' phenotype because the recombination events that incorporate fragment 2 connect the hisH::MudA element (untran- scribed in the recipient) to the trp promoter. Since the large recipient his deletion (DEL299) is not repaired in the course of forming the inversion, all of these recombinants retain the Cob- phenotype of the recipient deletion and the ApR phenotype of the recipient MudA element. Depending on the position of the exchanges in the trp region between fragment 2 and the chromosome (2-1 or 2-11 in Figure 5B) the inversions will be either Trp' (class I ) or Trp- (class 11). The Trp' phenotype arises by complementation between the ex-

A. Parental strains (no inversion)

Fragmenl 1 Fragmenl2 r Fraqm'ent 3 I

Donor Strain his '42 I Tn IO (l-r,MOl) , MudA fp'34

A ' A B C D E 'P Phenolype: His* TCR APR Lac+ TIP- IMOV

Phenotype: HIS. ApR Lac' Trp- lndol' (TrpCBA+) TcR

6. Transduction events that direct inversion formation (select His+)

h8SEIFAH MCDG' " E - -

Fragrnenl 1

hrs+EIFAHBCDGO - IrrlABCDE Fragment 2

" -

h6EfFAH 'HBCDG'

C. Inverted His+ recombinants I Inverted Segrnenl

Class I: ~ r p + h~S'42 I I p ' M hrs '42 fp '34 -- 'ABCDE 'P Phenolype His+ TcR (TrpCBAf) ApR Lac+TrpECOE+

Trp+ by cornplernenlalm

Inverled Segment

Class II: Trp- his .42 I I fp ' 34 hn'42 fp ' 34

'EDCBA - Phenofype His+ TcR (TrpCBAf) ApR Lac+ Trp' Indot+ (TrpABC') TcR

FIGURE 5.Transduction cross used to direct inversion of the his ('42) to trp ('34) chromosome segment. (A) Structure of the parent strains. The complex feathered arrows denote Tn IO elemen&: the internal arrow (feathered) denotes the orientation of the central resistance determinant, the external black arrows refer to the flanking inverse order ISlOelements. Thick arrows (gray) denote MudA(Inc, AP) elements. The large deletion, DEL299, is not repairable by a single transduced fragment. (B) Transduction events that generate an inversion. The selection is for inheritance of a his- operon. In the transduced fragments 1 and 2, TnlO and MudA(Ap, Znc) elements provide homologous sequences which link sister chromosomes and direct inversion formation (as described in the text). (C) Structures of the inversion-bearing recombinants (classes I and 11).

pressed trp genes at one end of the inverted segment (trpC'R+A+) and the trp genes at the other end ( trpR+C+D'E').

One possible outcome of this cross generates His' recombinants without constructing an inversion; this is diagrammed in Figure 6. This His' recombinant (class 111) is derived by a transduction event in which two do- nor fragments (fragments l and 3 in Figure 5A) c o o p erate to repair the hisG-phs- cob deletion (DEL299) and remove the recipient hisHxMud4 insertion. These transductants inherit the donor hi^+ operon and the nearby Tn 10 element; they also gain a Cob' phenotype and lose the ApR phenotype that was associated with the recipient hisH::M~d4 element. These transductants remain Lac-since no Inc operon is left in the final recombinant.

Results of the transduction cross that forms the his- trp inversion: As described above and in Figure 5, cul-


A. Transduction events that do not form an inversion

- - Fragment 1 =

Phenotype: His- ApR Lac- I TIP- lndol+ (TrpABC+) TcR

6. His+ recombinant without an inversion his '42 L r p 34

"E B

Phenohlpe: Hisf Aps Lac-TcR Trp' k d d + (TrpAEC') TcR

FIGURE 6.Transduction events that yield His' recombinants without an inversion (his-trp cross). The donor and recipient strains are described in Figure 5A. (A) Transduction events. Incorporation of fragments 1 and 3 (Figure 5R) generates an intact his+ operon by replacing the his::MudA insertion and DEL299 with his' sequences. (B) Structure of the His+ recombinants that do not carry a his-trp inversion (class I l l transductants).

tures of recipient strain TT13400 were transduced with phage P22 grown on donor strain TT13401 and His' recombinants were selected. Transductants were phenotypically tested and classified according to their Trp, Indole (TrpAB'), Lac, Cob, and Ap phenotypes. The data are shown in the last line of Table 3.

Classes I and I1 are composed of presumed inversion products and accounted for approximately 6% of the His+ transductants. These recombinants are all phenotypically His' Tc' ApR Lac' Cob- and either Trp' (class I; 40% of inversions) or Trp- (class 11; 60% of inversions), depending on the location of the crossover point in trp homology (see Figure 5 , B and C).

The largest class, class 111, is composed of deletion repair transductants described in Figure 6. They are phenotypically His' ApS Lac- Tc' and Trp-. They com- prise approximately 91 % of the His' transductants. Two such isolates were examined in detail and found not to carry an inversion (see below).

From over 1100 transductants tested, 30 isolates (2.5% of the His' recombinants) had phenotypes distinct from the three predicted classes described above. None of these unpredicted recombinants had an inversion. Eight of these transductants are Aps and His', signifjmg loss of the MudA insertion and repair of the large deletion, but are also Trp'. These recombinants probably arise by repair of the his operon by adjacent transducing fragments, as well as repair of the trpE::Tn IO by a third transducing fragment. This unlikely event probably occurred due to the extremely high concentration of infecting phage used. Five recombinants become His' but are Ap' and Lac-. In this case, the recipient cell may have carried a duplication of the his region; the his operon of one copy may have been repaired by adjacent transducing fragments, while the second copy retained the his::MudA insertion.

Control crosses were performed to show that inversion formation requires the MudA and Tn I O elements in the donor. When both elements are absent from the

donor (wild-type strain LT2) transduction of the original recipient strain (TT13400) to His+ generated only class 111 recombinants (line 1 in Table 3). Absence of even one element, the tr@:TnI 0, from the recipient strain prevents recovery of transductants of the inversion class. When a strain (TT13399) lacking the trpE::TnIO insertion was transduced with phage grown on the original donor strain (TT13401) no class I or class I1 inversions were formed (line 2 in Table 3).

Evidence that transductants of class I and I1 carry a his-trp inversion: The preceding crosses yielded recombinants whose phenotypes were those predicted for strainswith a his-trp inversion. These recombinantswere tested genetically for the presence of this inversion. In strains with an inversion, the sequences that recom- bined to form the inversion (at the inversion break- points) are flanked on either side by sequences from distant sites in the wild-type chromosome. When an inversion strain is used as a transduction donor, the novel arrangement of sequences flanking the inversion join- point should impair inheritance of join-point markers by a recipient with wild-type gene order. We have tested this expectation for the constructed his-trp inversions.

According to the design of the crosses, a constructed inversion should have a Tn I Oelement at one breakpoint and MudA element at the other (see Figure 5C). R e p resentatives of the various recombinant classes (classes I and I1 inversions; and class 111 non-inversion) were tested for their ability to transfer the TcR and ApR phenotypes (encoded at the inversion break points) into a wild-type recipient. The data for the TcR transductions are shown in Table 4. The parental strains without an inversion (strains TT13400 and TT13401) gave rise to high numbers of TcR transductants and Ap' transductants. The two putative inversioncarrying classes differ in their ability to donate Tc'. The class I inversion strains (Trp') are expected to have a Tn IO element located at the inversion break-point and this is the only TnlO element in their genotype. These strains transduce TcR to a normal recipientwith low efficiency, and the few transductants are all prototrophic, suggesting that they arise by transposition of the donor element or by multiple- fragment transduction events that involve inheritance of both break-points and regenerate the donor inversion in the recipient. The class I1 inversions (Trp-) are expected to carry two Tn I O elements, one at the inversion break-point and the other within the trp operon just outside the inverted segment. These Trp- inversions donate Tc' to a wild-type recipient at a high frequency and the TcR transductants obtained are all Trp-, as expected since they inherit the trp::Tn IO element that is not at an inversion breakpoint. The non-inversion class 111 recombinants also donate TcR at a high frequency.

All the putative inversion strains donate ApR at a lower frequency than the donor with wild-type gene order (data not shown), as expected if the MudA insertion


TABLE 3

A transduction cross that forms the his-trp inversion

Number of transductant typesb

Putative inversionsC

Class I Class I1 repair Other DEL299

Donor' Donor genotype Recipient Recipient genotype Trp' Trp- class IIId recombinantse

LT2 wild type 'IT13400 hisBH1261::MudA DEL299 0 0 120 0

'IT13401 his' tee-729:TnIO 'IT13399 hisBH1261::MudA DEL299 0 0 120 0

trpElO49:TnIO

trpA2485:MudA

'IT13401 his' tee-729:TnIO 'IT13400 hisB,H1261::MudA DEL299 44 26 1099 30 trpA2485::MudA trpEIO49:TnlO

The donor phage were used at a moi of 50. 'His' transductants were selected on minimal medium supplemented with tryptophan. The different transductant classes are described in the

The putative inversion class are phenotypically His' TcRApR Lac' Cob- Indol'. The inversions can be either Trp- or Trp'. These numbers

Recombinants that occur by repair of DEL299 are phenotypically His' Lac- ApS Cob' Indol' Trp- TcR.

text.

reflect the products of ten crosses each using independent cultures.

'These recombinants are described in the text.

TABLE 4

Analysis of putative inversion clones: his-trp interval

T C ~ selection '

Donor strain Donor description No. of Trp auxotrophs

Tc' transductants (% of Tc' transductants)

Controls 'IT13401 His' paternal strain >2000 0 TT13400 His- maternal strain >2000 100

Class I TT13403 Trp' putative inversion 2 0 IT13405 Trp' putative inversion 15 0

Class I1 IT13402 Trp- putative inversion 656 100 'IT13404 Trp- putative inversion 1088 100 'IT13406 Trp- putative inversion 840 100

Class 111 TT13408 Deletion repair >2000 100

'The recipient in these crosses, strain LT2, was infected at a moi between 2 and 5. Transductants were selected on NB medium with tetracycline. The transducing lysates prepared from the class I , 11, and I11 recombinants had similar titers (approximately 1 X 10" plaque-forming units per ml).

resides at an inversion join-point. The rare ApR transductants that do arise are prototrophic and were probably formed by multiple fragment transduction events that involve inheritance of both break-points and regenerate the donor inversion in the recipient (see MATERIALS

AND METHODS).

We have tested the class I, class I1 and class I11 recombinants for possession of the his-cob deletion (DEL299). We predicted that only strains in which this large deletion is repaired (class 111) would possess this material and thus be able to inherit a cob::TnIUdCm element. We found that the maternal recipient strain (TT13400) and all of the inversions tested (class I and 11) carry the deletion and inherit the cob::TnlUdCm element very rarely. The paternal parent (TIl3401) and the class I11 (deletion repair) recombinants all inherit the cob::TnIUdCm element with high efficiency as expected for strains that lack the large deletion (data not shown).

Strains with a his-trp inversion grow normally R e p resentative class I, class I1 and class I11 clones were single colony isolated on both rich (NB) and on appropriately supplemented minimal medium. We observed no dif- ferences in the growth of strains with and without the inversion. Furthermore, strains with the inversion were passaged several times in liquid culture and retested for maintenance of the inversion; no obvious secondary rearrangements were found in that the strains retained their characteristic phenotype and linkage disruption.

Design of the cross that constructs an inversion of the his-eysA interval: To construct an inversion of the his- cysA interval, the two linker fragments were provided by lysates grown on two different donor strains (see Figure 7). One donor strain (TT513) carries a TnlU element counterclockwise of the his' operon ( " A orientation). The other donor strain (TT17285) has a cysA::MudA insertion in the "B" orientation which forms a lac operon fusion to the cysA promoter. This MudA donor


A. Parent strains (no inversion) Fraomenl 1

Phenolype. Eul+ Cys+

Donor 2 Fraqmenl2

(lT17285)

Phenolps Eul' CyS /\pH Lac' His' PhS- Pdu' Cob

Donor 2 Fraqmenl2

(lT17285)

Phenolps Eul' CyS /\pH Lac' His' PhS- Pdu' Cob

B. Transduction events that form His+ inversion recombinants

I C. Inverted His+ recombinant

0 ' 5 0 '42 ' 50 h!s '42 "

Phenotype: TcR Hisf Eul' CysA ApR Lac+ Phs' Pdu' Cob

FIGURE 7.Transduction cross used to direct inversion of the his ('42) to c3'5A ('50) chromosome segment. ( A ) Struc- ture of the parent strains. Thick arrows (gray) denote MudA- (lac, A p ) elements. The complex feathered arrows denote TnlO elements: the internal arrow (feathered) denotes the orientation of the central resistance determinant, the external black arrows refer to the flanking inverse order IS1 0 elements. (R) Transduction events that form a his-cysA inversion. Se- lection is for inheritance of a his+ operon. By inheriting transduced fragments 1 and 2, TnlO and MudA(Ap , lac) elements provide homologous sequences which link sister chromosomes and direct inversion formation (as described in the text). Note that the chromosomal region between c ~ s A and the rJa::Tn 10 (including the PUI operon) is deleted in these transduction events. (C) Structure of the inversion bearing recombinant chromosome (class I ) .

strain also has the large his-cob deletion, DEL299. The recipient strain described below was transduced to His' with a mixture of these two donor lysates.

The recipient strain (TT17284) carries the large his- cob deletion (DEL299) and a his::MudA insertion; a foreign promoter counterclockwise of the deletion drives expression of the kis-lac operon fusion. The Tn IO and M d A elements in this recipient strain are in chromosomal positions and orientations that are reversed relative to the location of the same elements in the in the donor strains. The recipient element zfa::TnIO "B" is locatedjust clockwise of the put operon and the adjacent cysA locus (see Figure 7A). The recipient hisH::MudA sequence is inserted in the "A" orientation.

Transduction of the recipient strain (TT17284) with a mixture of both donor lysates (TT513 and TT17285) can yield three types of His+ transductants (see Figure 7). The first class, diagrammed in Figure 7, carries an inversion of the his-cysA chromosome segment. Incor-

poration of fragment 1 alone (from donor TT513) by the exchanges diagrammed in Figure 7B would break the chromosome. This problem is avoided by simultaneous incorporation of fragment 2 (from donor TT17285) using cpA sequences near the cys::MudA element to recombine with one sister chromosome and MudA sequence itself as a region of homology to recombine with the identical element at the his locus of the other sister chromosome (see Figure 7B). These paired events invert the chromosomal segment between the his and cysA loci. These events also delete the material between rysA and zfu::Tn IO (clockwise of the Put locus) leading to a deletion and a novel Eut- phenotype that is not present in either the donor or recipient strain. Appearance of this unselected Eut- phenotype among the selected His' recombinants is striking evidence that the cross proceeded as diagrammed in Figure 7.

The His+ recombinants which have a his-cysA inversion were identified by the following phenotypic char- acteristics. Only inversion recombinants carry a deletion removing the region behveen the cysA locus and zfa- 3649:TnIO and are therefore Cys- and Eu- . The inversion class also retains the large recipient his-cob deletion (DEL299) and is thus Phs- Pdu- Cob-. Due to a foreign promoter at the distal end of deletion DEL299, the inversion class can still express i t s hisD gene and the lac operon of the MudA element at one of the inversion breakpoints; a wild-type his operon is present near the other break point. Thus the inversion recombinants are phenotypically His+ Lac' and ApR. In summary, the inversion class is expected to be phenotypically TcR His' Eut- Cys- ApR Lac' Phs- Pdu- Cob- (see Figure 7C).

The second transductant class, diagrammed in Figure 8, A and B, carries a duplication of the his-Put chromosome segment. The duplication can be formed by recombination events involving a single his+-Tn IO transduced fragment from donor strain TTFil3. Recombination events between sequences clockwise of the hzs operon and between one pair of the IS10 sequences (which are at either end of a TnlO element) can generate a His+ recombinant with a duplication of the at-hissegment (see Figure 8, A and B). Since a single fragment is involved in this event, the duplication transductants arise much more fre- quently than the inversion transductants despite the limited size of the recombining IS10 elements.

The duplication class is easily distinguished from the inversion recombinants by its Cys' and Eut' phenotype and by the instability of the His+ phenotype which is lost when the duplication segregates. The duplication recombinants retain the large deletion (DEL299) that extends from hisG to cob, so they are phenotypically Phs- Pdu- Cob-. Recombinants of this class also retain the parental his::MudA insertion and thus are ApR Lac+. In summary, the duplication class is characterized by the following phenotype: His' (unstable) TcR Eut+ Cys' ApR Lac+ Pdu- Phs- Cob- (see Figure 8B).


A. Transduction events that form His+ duplication recornbinants

Fragmenl 1 (iT513)

Phmtype: TcA EuIfCys+

I His' ApR Lac+ Phs- Pdu' Cob'

6. His+ duplication recombinants (no inversion)

' 5 0 '42 '50 '42

Phenotype: His+ TcR Eut*C# ApR Lac' Phs' Pdu'ccb'

C. Transduction events that form His+ deletion-repair recombinants

-Fragment - 1 (iT513) - - Ffagment 3 m513) -

=- -

WDEMPI; I

-

D. His+ deletion-repair recombinants

M s p ju mb

Phenotype: T ~ R EuI+CP* His+ Aps Lac' Phs+ Pdu+ cob+

FIGURE 8.Transduction events that form His+ recombinants without an inversion (his-cysA cross). The donor and recipient strains are described in Figure 7A. (A) Incorporation of fragment 1 alone generates a his+ operon and directs duplication of the his-cysA chromosome segment (as described in the text). (R) Structure of the chromosome of His' strains with a his-rut duplication (class I1 transductants). (C) Incor- poration of fragments 1 and 3 generates a his+ operon by replacing the his::MudA insertion and DEL299 with wild-type his' sequences. (D) Structure of the chromosome of transductants with a repaired deletion (class 111).

The third expected class of His' recombinants forms by repair of the large deletion (DEL299) at his and removal of the recipient his::MudA insertion, as shown in Figure 8, C and D. This repair event requires inheritance of two adjacent P22-transduced donor fragments. The His' transductants of this type lose the hk:MudA insertion and b e come Lac- and Aps. Since the large deletion (His- Phs- Pdu- Cob-) is repaired using two fragments that together span the en tire deletion, these His' transductants become Phs' Pdu' and Cob', unlike all the other recombinant classes. In summary, the class 111 His+ transductants, arising by deletion repair, are expected to be stably TcR Eut+ Cys' His+ Aps Lac- Phs' Pdu+ Cob' (see Figure 8D).

Results of the transduction cross that inverts the his- cys interval: A culture of the recipient strain, TT17284, was transduced with a mixture of P22 phage lysates prepared on both donors, strains TT513 and TT17285, using moi values of 0.55 and 25, respectively. The multiplicity of phage grown on strain TT513 was reduced to minimize formation of the duplication recombinant class. Transductants (His') were selected on minimal medium containing cysteine.

One hundred fifty-five transductants were tested and divided into three phenotypic classes (last line of Table

5). Three percent of the transductants had phenotypes predicted for inversions (Table 5). The duplication types comprised 97% of the His' transductants. Reduc- ing the multiplicity of phage from strain 'IT513 elimi- nated deletion repair (class HI). Most members of the duplication class (class 11; 145/150) were ApR and Lac' as expected, five were ApS and Lac-. The exceptional Aps Lac- duplications probably experienced repair of the hisHxMudA element by gene conversion using his+ sequences from the duplication join-point. As can be seen in Table 5, the inversion class (class I ) was seen only in crosses in which both donor lysates were used simultaneously. The deletion repair transductants were o b served only in crosses with a high multiplicity of the his' donor (lT513) (data not shown).

Evidence that his-cys inversions were formed The His+ recombinants that were inferred by nutritional phenotypes to carry an inversion were tested for linkage disruption at the inversion break-points to confirm the presence of an inversion. Putative inversion recombinants were tested for their ability to donate TcR (Tn 10) and His' phenotypes to a strain with a wild-type gene order. Since these markers are all present at the inversion join-point they should be transduced very ineffi- ciently into a strain with wild-type gene order compared to other markers. The putative inversion recombinants were also tested for their ability to be transduced to Cys' by phage grown on a wild-type donor.

The putative his-qd inversions show reduced ability to donate TnlO and his' join-point markers to a recipient with wild-type gene order (Table 6). In contrast, the d e nors with wild-type gene order (lT17284 and 'IT51 3) gave rise to high numbers of His+ and TcR transductants, as did the His' duplication donors. These data are consistent with the idea that the TnlGhis' join-point is flanked by sequences derived from two distant chromosome sites as would be expected for strains canying the putative inversions.

Strains with the his-cysA inversion have suffered a deletion that extends from cysA to the insertion site of the zfa-3649:Tn I O clockwise of the eut locus. Since this deletion is thought to be present at the inversion join- point, strains with this inversion should inherit a cysA' gene from a wild-type donor at a very low frequency. However, this event can occur, rarely, when two fragments return the inversion to the wild-type gene order (diagrammed in Figure 9).

When the inversion strain is transduced to cysteine- independent growth; the rare Cys' transductants should all inherit an unselected Eut' phenotype. These events are also expected to remove the elements (TnlO and MudA) from both of the inversion break points. Thus Cys' transductants should be rare and all that are recovered should be TcS Eut' and ApS. Moreover, the transductants are expected to lose the his' promoter sequences at thejoint pointwhile retaining the recipient deletion DEL299 to show a Hol' His- Phs- Pdu- Cob-


TABLE 5

Crosses that construct an inversion of the his-cysA interval

Multiplicity of infection Distribution of transductant types

Donor 1 ('IT513) Donor 2 ('IT17285) Class I a Class II Class 111 No. zee2::TnlO his' DEL299 cysA1585::MudA inversion duplication DEL299 repair tested

0 25.0 0 0 0 0 0.55 0 0 1 (0.04) 0 91 0.55 25.0 0.03 0.97 (0.03) 0 155

Strain IT17284 (diagrammed in Figure 7A) was the recipient in all of the crosses. His' transductants were selected on minimal medium

a Putative inversions were classified as those recombinants that are phenotypically His+ Eut- Cys- ApR Lac' TcR Phs- Pdu- Cob-. supplemented with cysteine.

The duplication-bearing recombinants were classified as those that are phenotypically His' Eut+ Cys' Phs- Pdu- Cob- TcR and generate His- segregants. Most of the duplications were ApR Lac' due to the hisH::MudA element. A minor class had lost this MudA element and was ApS Lac-. The frequency of this minor class is represented by the number in parenthesis.

'Recombinants that arise by repair of DEL299 are characterized by a His' Aps Lac- Phs' Pdu' Cob' Cys' Eut' phenotype. These recombinants were detected only under conditions where donor 1 (strain 'IT513) was used with a moi of 10 or greater.

TABLE 6

Linkage disruption: his-cys recombinants as transduction donors

Donor Ratio TcR/Ser+ Ratio His'/Ser' strain' Donor description transductants X lo4 transductants X 10'

'IT1 7284 Maternal strain (control) 62,000 Ob 'IT513 Paternal strain (control) 59,000 410

'IT17289 Putative inversion 'IT17290 Putative inversion IT17291 Putative inversion 'IT17292 Putative inversion TT17293 Putative inversion

<o. 1 4

10 8

<0.1

1.6 0.8 1.5 0.4 1.2

TT17294 Duplication recombinant (ApR, Lac') 56,000 2.10 TT17295

'The recipient in all these crosses is strain TT17286 (serB1469:Tn1OdCm his646 (DEL hisOGD). In these experiments, strain TT17286 is transduced separately to His', TcR and Ser'. Transduction to Ser' is used as a transduction control to adjust for variation in the transducing lysates.

Duplication recombinant (ApS, Lac-) 79,000 220

Strain 'IT17284 has a hisOG deletion, DEL299, so it cannot transduce strain IT17286 to His'.

phenotype. As shown in Table 7, all the putative inversions constructed by the cross are transduced to Cy,' at a reduced frequency relative to the paternal Cys- strain (TT17285). The phenotype of the rare transductants that were recovered is consistent with that expected for a "reversed" inversion, as diagrammed in Figure 9.

Merodiploidy of the hisD locus for his-cys rearrangements: The putative inversion transductants are predicted to be diploid for the hisD locus, whereas all three of the parental strains are haploid (Figure 7, A and C). To test this prediction, a hisD::TnIOdCm element was transduced into the inversion strains; in each case the chloramphenicol resistant transductants remained phenotypically HisD' after inheriting the hisD::Tn lOdCm insertion (data not shown), demonstrating the presence of two expressed copies of the hisD+ gene.

The his-cysA inversion recombinants grow normally: Strains carrying the putative cys-his inversion were tested for their growth rate in nutrient broth supplemented with cysteine. Table 8 shows the doubling times for the inversion and parental strains. The strains carrying the

inversions have very similar growth properties to those of the Cys- parental strain.

DISCUSSION

The experiments presented here show that two chromosomal intervals, his-tr$ and cysA-his, that are non- permissive for inversion by intrachromosomal recombination events, can both be directed to invert by a two- fragment P22 transduction cross. Strains carrying the constructed inversions grew well and could not be dis tinguished from wild-type strains by colony size on solid medium or growth rate in liquid culture. Thus, lethality of the final inversion product cannot explain the failure to obtain inversions of these intervals by recombination between inverse-order chromosomal sequences. We propose that the bacterial recombination system is unable to form these particular inversions by intrachromosomal exchanges but permits their formation by transductional crosses.

These results raise several questions. Why are the same sequences treated differently by the recombina-


A. Parental strains

Donor (no inversion) Fraamenl 1 Fraamenl2

'50 '42 e u f m .. hisEfFAHBCDG0 P

Phenotype: €ut+ Cys+ His+

Inverted His+ Cys' recipient Inverted seqmenl

I

'50 '42 '50 '42

'HBCDG

Phenotype' TcR His* Eut- CysA- ApR Lac* Phd Pdu- Cob-

B. Transduction events that form Cys+ recombinants

I C. Cys+ recombinants ( no inversion)

'50 '42 eUrcvSn . hisElFAHBCDG' -

Phs' Pdu- Cob- Phenolype: Tcs €ut+ Cys+

FIGURE 9.Transduction events that return a his-cy5 inversion to the wild-type gene order. The transduction donor is the wild-type strain, LT2, and the recipient carries the his-cysA inversion made by events depicted in Figure 7B. A Cys' recombinant can (infrequently) arise by a two fragment transduction event that returns the inverted segment of the recipient to the wild-type gene order. The Cys' transductants also regain a P U P operon and lose the Tn IO and MudA elements which were located at the two widely separated inversion join points.

tion system when they are located at different pairs of sites in the chromosome? If sequences at permissive sites recombine such that roughly one half of the selected recombinants carry an inversion, why do the same sequences placed at non-permissive sites contact each other and exchange genetic information without ever producing an inversion? The directed formation of "non-permissive" inversions by transduction demon- strates that the limitation on inversion of non-permissive intervals is not viability of the final product. Apparently some aspect of the structure or DNA sequence of the chromosomal regions surrounding repeated sequences influences and restricts the ability of these sequences to recombine. What aspect of the recombination process is limited at non-permissive sites? How does the transductional procedure described here circumvent this limitation and allow inversion of a non-permissive interval?

Formation of an inversion by intrachromosomal recombination requires that two relatively short homologous sequences, inserted at distant sites in the chromosome, interact and form a complete (or conservative) exchange that rejoins both sets of flanking sequences (see Figure 2). This requirement distinguishes inversion formation from both conjugational and transductional

TABLE 7

Linkage disruption: his-cys inversions as transduction recipients

Percent of cy^' transductants

Ratio of with phenotype Cys'/CmR expected for

Recipient transductantsb reversed Recipient" description (X 10') inversions'

1T17285 Paternal strain

'IT17289 Putative inversion 3.7 93 'IT17290 Putative inversion 5.1 100 'IT17291 Putative inversion 1.3 95 'IT17292 Putative inversion 2.2 100 'IT17293 Putative inversion 3.2 100

(control) 300 -

"The donor in all crosses is strain 'IT14940 (sprBZ469:Tnl(kiCm). 'These experiments measure the number of Cys' and CmK trans-

ductants. For the inversion bearing strains, inheritance of a cysA+ gene requires repair of an inversion breakpoint and should occur rarely. The serR::TnZOdCm element is unlinked to the inversion and should be inherited at a wild type frequency. Transduction to CmK is used as a transduction control to adjust for recipient variations in transduction efficiency.

'For the inversion bearing strains, inheritance of a cys' gene occurs primarily by a rare two fragment transduction event that re- verts the inversion to a wild-type gene order. Cys' transductants can form by events that remove both join-point markers, MudA (ApR) and TnlO (TcR) to generate an Aps Tcs Hol' His- Eut+ phenotype (Figure 9). The phenotypes of at least 20 transductants were determined for each cross.

TABLE 8

Growth rate of strains with a hiscys inversion

Strain Phenotype and class

lT513 ' I T 1 7285 'IT17284 "1 7289 l T 1 7290 1T17291 ' I T 1 7292 T T I 7293

Paternal strain (control) Paternal strain (control) Maternal strain (control) Inversion Inversion Inversion Inversion Inversion

Doubling time (min)

28 34 28 27 32 26 27 26

"Growth rates were determined from cultures at exponential growth phase in liquid nutrient broth medium supplemented with cysteine.

recombination events that replace a recipient mutation. The requirement for a full exchange also distinguishes inversion formation from the process by which chromosomal duplications and deletions are formed. In all of these other recombination events, exchanges are not necessarily conservative; recombination events on both sides of the inherited information must join only one pair of flanking sequences. The recombination enzymes may be unable to form complete (or conservative) exchanges at a non-permissive pair of sites. We have shown previously that inversions of permissive intervals form primarily by the RecBCD recombination pathway (Mi - HAN and ROTH 1989; SECALL and ROTH 1994). Thus, in considering reasons why inverse repeats at some chro-


mosomal sites fail to generate an inversion and how a trans ductional method might help circumvent the problem, one might reasonably start by considering the substrates needed by the RecBCD enzyme and how these might be absent from certain regions of the chromosome.

The RecBCD enzyme is thought to act at double- strand breaks, degrading the 3"ended strand until a properly oriented Chi stimulatory sequence is encoun- tered. After encountering Chi, the enzyme turns off its exonuclease activity and proceeds as a helicase that re- leases a 3'ended single strand (including Chi) as it un- winds the duplex substrate (STAHL et aE. 1990; DIXON and Kow~~czv~ows~l1991). If the RecBCD enzyme is respon- sible for inversion formation, then we presume that a double- strand break and a properly oriented Chi sequence might be required to stimulate its activity. Since some pairs of chromosomal sites (permissive) support activity of RecBCD in inversion formation and others do not, we presume that chromosomal regions differ in their ability to provide substrates. At least one of the recombining sequences of each permissive pair must be located near a chromosomal site of frequent double- strand breakage and a properly oriented Chi sequence must lie between that breakage site and the recombining sequence. By this hypothesis, both members of a non- permissive pair of sites would lack one or both of these features required for RecBCD activity. The transductional method of directing inversion formation could circumvent this problem by providing extrachromosomal fragments with long homologous sequences and highly recombinogenic double-stranded ends. Trans- duction also circumvents the need for a complete (conservative) exchange in that the inversion can be formed by a series of four halfexchanges (nonconservative) as diagrammed in Figures 4, 5 and 7.

While we find the above model attractive, other possibilities have not been excluded: (1) Perhaps sequences at all sites recombine to form Holliday structures but the resolution of these structures in some regions is biased against the exchange of flanking sequences. (2) Perhaps the folded nucleoid prevents the recombining sequences at certain sites in the same circular chromosome from contacting each other; this would prevent inversion formation. The exchange of information between such sequences that occurs in the absence of inversions could be accomplished by sister chromosome exchanges. In the transductional construction method, the extrachromosomal transduced fragments, might not be subject to the topological constraints that limit contact between genomic sequences. (3) Mechanistic re- strictions may include a viability component. To wit, recombination events that lead to inversion formation may, in some chromosomal regions, generate "toxic" intermediates. Such intermediates may cause cell death if they are not resolved appropriately. This possibility re-

mains even if the final inversion has no deleterious effects on growth.

Other reasons may explain why the transduction pro- tocol circumvented the difficulty in forming inversions. One is that the transducing phage genomes might provide functions that assist with the recombination event. We have tried to isolate inversions by intrachromosomal recombination following phage infection and were un- successful (SEGALL 1987). It should be noted that all the strains used in constructing inversions carry the his-cob deletion DEL299. This deletion also removes the sbcB gene, which encodes the recombination enzyme exonuclease I (a single-stranded 3' exonuclease). It might be argued that this deficiency helped either the process of inversion formation or the viability of the final product. We have tested this possibility and found that non-permissive intervals remain so in sbcB deletion mutants ( S E W 1987; L. MIESEL and J. R. ROTH, unpublished results).

In each interval, rearrangements other than inversions comprised the majority of transductants obtained. In the his-trp interval, the predominant transductant type arose by repair of the large his-cob deletion by a two-fragment transduction event. In the his-cysA interval, the predominant transductant type carried a duplication generated by a single transduced fragment. However, both of these non-inversion transductant classes require fewer recombination events than the inversion class: three events for deletion repair and two events for duplication formation vs. four events for inversion formation.

In summary, we provide evidence that Salmonella cells carrying inversions of the his-trp or the his-cysA intervals can be constructed by transductional crosses and that cells with these inversions grow normally. We infer from these results that mechanistic aspects of the recombination process are likely to prevent formation of these inversions by intrachromosomal exchange. We suggest that certain regions of the chromosome may fail to provide sequence features (double-strand breaks or Chi sequences) needed for the formation of inversions by the RecBCD enzyme.

This work was supported by National Institute of Health grant GM27068 to J.R.R.; predoctoral training grant T32-GM0746415 supported L.M.

LITERATURE CITED ANDERSON, R. P., and J. R. ROTH, 1977 Tandem genetic duplications

in phage and bacteria. Annu. Rev. Microbiol. 31: 473-505. ANDERSON, R P., and J. R ROTH, 1981 Spontaneous tandem genetic

duplications in Salmonella typhimuriunz arisc by unequal recombination between rRNA (rrn) cistrons. Proc. Natl. Acad. Sci. USA 78: 3113-3117.

BAUERLE, R. H., and P. ~ ~ A R G O L I N , 1967 Evidence for two sites of ini- tiation ofgene expression in the tryptophan operon of Salmonella typhamurium. J. Mol. Biol. 26: 423-426.

BERKOWTZ, D., J. HUSHON, H. WHITFIELD, J. R. ROTH and B. N. AMES,

riol. 9 6 215-220. 1968 Procedure for identifjmg nonsense mutations. J. Bacte-

BOBIK, T. A., M. A L I ~ N and J. R. ROTH, 1992 A single regulatory gene


integrates control of vitamin B,, synthesis and propanediol deg- radation. J. Bacteriol. 174 2253-2266.

CAPAGE, M., and C. W. HILL, 1979 Preferential unequal recombination in the glyS region of the Escherichia coli chromosome. J. Mol. Biol. 127: 73-87.

CASADABAN, M . J., and S. N. COHEN, 1979 Lactose genes fused to ex- ogenous promoters in one step using a Mu-lac bacteriophage: i n vivo probe for transcriptional control sequences. Proc. Natl. Acad. Sci. USA 76: 4530-4533.

CHUMLEY, F. G., R. MENZEL and J. R. ROTH, 1979 Hfr formation directed by TnlO. Genetics 91: 639-655.

DAVIS, R. W., D. BOTSTEIN and J. R. ROTH, 1979 Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

DIXON, D. A,, and S. C. KOWAL.CZ~OWSKI, 1991 Homologous pairing in vitro stimulated by the recombination hotspot, Chi. Cell 66:

HUGHES, K T., and J. R. ROTH, 1984 Conditionally transpositiondefective derivative of M u dl (Amp lac). J. Bacteriol. 159 130-137.

HUGHES, K. T., and J. R. ROTH, 1985 Directed formation of deletions and duplications using Mu d (Ap, lac). Genetics 109: 263-282.

JACKSON, E. N., and C. YANOFSKY, 1972 Internal promoter of the t r y p

J. Mol. Biol. 69: 307-313. tophan operon of Escherichia coli is located in a structural gene.

JETER, R. M., B. M. OLIVERA and J. R. ROTH, 1984 Salmonella typhimurium synthesizes cobalamin (vitamin BIZ) d e novo under anaerobic growth conditions. J. Bacteriol. 159: 206-213.

KONRAD, E. B., 1977 Method for the isolation ofEscherichia coli mutants with enhanced recombination between chromosomal duplications. J. Bacteriol. 130: 167-172.

LEHNER, A. F., and C. W. HILL, 1980 Involvement of ribosomal ribo- nucleic acid operons in Salmonella typhimurium chromosomal rearrangements. J. Bacteriol. 143: 492-498.

I M A H A N , M. J., and J. R. ROTH, 1989 Role of recBC function in formation of chromosomal rearrangements: a two-step model for recombination. Genetics 121: 433-443.

PETES, T. D., and C. W. HILL, 1988 Recombination between repeated genes in microorganisms. Annu. Rev. Genet. 2 2 147-168.

REBOUO, J.-E., V. FRANCOIS and J.". L o u r n , 1988 Detection and

361-371.

possible role of two large nondivisible zones on the Escherichia coli chromosome. Proc. Natl. Acad. Sci. USA 85: 9391-9395.

ROOF, D. M., and J. R. ROTH, 1988 Ethanolamine utilization in Sal- monella typhimurium. J. Bacteriol. 170: 3855-3863.

SANDERSON, K. E., and J. R. ROTH, 1988 Linkage map of Salmonella typhimurium, Edition VII. Microbiol. Rev. 52: 485-532.

SCHMID, M. B., 1981 Chromosome rearrangements in Salmonella typhimurium. Ph.D. Thesis, UniversityofUtah, Salt Lake City, Utah.

SCHMID, M. B., and J. R. ROTH, 1983 Genetic methods for analysis and

517-537. manipulation of inversion mutations in bacteria. Genetics 105:

SEGALL, A. M., 1987 Studies on genome rearrangements and intrachromosomal recombination in Salmonella typhimurium. Ph.D. Thesis, University of Utah, Salt Lake City, Utah.

SEGALL, A. M., and J. R. ROTH, 1989 Recombination between homologies in direct and inverse orientation in the chromosome of Salmonella: intervals which are nonpermissive for inversion formation. Genetics 122: 737-747.

SEGALL, A. M., and J. R. ROTH, 1994 Approaches to half-tetrad analysis in bacteria: recombination between repeated, inverse-order chromosomal sequences. Genetics 136: 27-39.

SEcxAl., A,, M . J. MAHAN and J. R. ROTH, 1988 Rearrangement o f the bacterial chromosome: forbidden inversions. Science 241: 1314-1318.

STAHL, F. W., L. C. THOMASON, 1. SIDDIQI and M. M. STAHL, 1990 Fur- ther tests of a recombination model in which x removes the RecD subunit from the RecBCD enzyme of Escherichia coli. Genetics 1990: 519-533.

VOGEL, H., and D. BONNER, 1956 Acetylomithase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218 97-106.

1988 Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages. Genetics 118:

ZIEG, J., and S. R. KUSHNER, 1977 Analysis of genetic recombination between two partially deleted lactose operons of Escherichia coli K-12. J. Bacteriol. 131: 123-132.

YOUDERIAN, P., P. SUCIONO, K. L. BREWER, N. P. HIGGINS and T. ELLIOT,

581-592.

Communicating editor: E. W. JONES

construction of chromosomal rearrangements in salmonella by

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