A MECHANISM FOR ADDING SEGMENTS TO ... - Semantic Scholar

CIRCULARIZATION OF TRANSDUCED FRAGMENTS: A MECHANISM FOR ADDING SEGMENTS TO THE BACTERIAL CHROMOSOME MOLLY SCHMID

AND

JOHN R. ROTE1

Department of Biology, University of Utah, Salt Lake City, Utah 84112 Manuscript received July 2, 1979 Revised copy received October 1, 1979 ABSTRACT

Generalized transducing fragments that have redundant sequences in direct order can circularize during transduction events. The length of the required redundant sequences can be at least as short as IS10 (1.4 kb) (KLECRNER 1977). The circular transduced fragment is able to recombine with homologous sequences in the chromosome. Circularization and insertion of transduced fragments allow addition of segments to the bacterial chromosome rather than replacement of recipient segments as in a normal transductional cross. It also provides a method for translocation of bacterial genes to a variety a€ specific sites on the chromosome in either orientation. The significance of these events to bacterial eiolution is discussed.

H E transposable drug resistance element TnlO has been used to mediate Tvarious bacterial chromosome rearrangements ( CHUMLEY,MENZELand ROTH1979; CHUMLEY and ROTH1980; KLECKNER, ROTHand BOTSTEIN 1977). One of these is the directed transposition of small chromosome segments by generalized transduction ( CHUMLEYand KOTH 1979) For this type of rearrangement to occur, the donor strain in the transduction must have a chromosome segment bounded by two TnlO insertions (TnlO-chromosome segment-TnlO) . The recipient strain in the transduction must hare a TnlO element at the site of the desired transposition. Directed transposition occurs by homologous recombination between the TnZO elements in the donor transduced fragment and in the recipient chromosome. The recombination events that lead to this type of directed transposition can be described by the two different mechanisms shown in Figure 1. Genes from the histidine operon are being transposed in to the site of a pro: : TnlO insertion. By the first mechanism, the transduced fragment containing the TnlO-his-Tnl0 sequence remains a linear molecule, Crossover events between each of the two donor TnlO elements that border the his operon 2nd the recipient TnlO located in the pro region would be required to complete the transposition event. By the second mechanism, the TnlO-his-TnZO transduced fragment uses its redundant TnlO sequences to circularize. The circular transduced fragment contains a single TnZO element, which recombines with the chromosomal TnlO. A total '

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Genetics 94: 15-29 January, 1980

16

M. S C H M I D A N D J. R. R O T H

FIGURE 1.-Two recombination mechanisms for creating a directed transposition strain by generalized transduction.

of two crossover events is required to incorporate the TnlO-his-Tn10 transduced fragment by either mechanism. This paper describes genetic experiments that distinguish between these two mechanisms of directed transposition. In the crosses described here, inheritance by linear substitution occurs with approximately the same frequency as inheritance by integration of a circular transduced fragment. In the majority of these experiments, TnlO sequences provide the redundancies that allow circularization of the transduced fragment. However, experiments with a small his duplication show that other redundant sequences can permit circularization. It is also shown that redundant sequences as short as ISlO are sufficient to cause circularization of a transduced fragment. MATERIALS A N D METHODS

Bacterial strains: Bacterial strains used i n this study are listed in Table 1. All strains are derived from Salmonella typhimurium LT2. Bacterial strains which have “TT” prefixes contain a TniO insertion or were constructed €rom TnZO-containing strains. (1) his9533 (TT1704): This deletion strain was obtained in a selection for hisG mutations (JOHNSTON and ROTH1979). The deletion removes all of the his operon and a large segment of the surrounding material; it is too large to be transduced to His+ by a single P22-transduced fragment. It was probably generated by the zee-1::TnZO insertion (zee-1::TniO is located outside of the his operon, near hisE). The deletion mutant is Tets, but seems to retain some TniO homology at the endpoint of the deletion (M. SCHMIDand F. CHUMLEY, unpublished). (2) TT2361, TT2387, TT2632: These are directed transposition strains built according to the methods described by CHUMLEY and ROTH (1979). They all carry the large deletion his-9533 and TnlO- (hisOi242-H)-TnlO at a new chromosomal location. These strains were constructed by generalized transduction, using TT.2348 as the donor of the TniO-his-TniO genes. The recipient carried his-9533 and a TnZO insertion a t the site of the desired translocation. Selection for HisD+ recombinants (histidinol growers) resulted in 70-100% of the transductants inheriting the his genes at the new location. The HisD+ phenotype of these strains is unstable since recombination between the two TniO insertions bordering the his genes leads to loss of the transposed his material. (3) TT3171 (OinT-his-TniO): Construction of this strain was similar to the construction of

17

CIRCULARIZED T R A N S D U C I N G F R A G M E N T S

TABLE 1 Bacterial strains Strain

TT513

Genotype

Method

zee-2::TnlO (A)

T

hisOG203

T T T T T T

his3050 hisOGD646 hisOGD646 hisOGD646 hisOGD646 hisOGD646

TT628 strA1 pyrC7/F'tsll4 lac+ zzf-2l::TnlO TT1127 hisC8667: :TnlO (B) TT1151 hisC8691: :TnlO (A) TT1704. TT1770 TT1883 TT1889 TT1918 m 4 7 TT2348 TT.2361 TT2387 TT2632 TT3003 TT3010 TT3164 TT3170 TT3I 71 TT3286 TT3291 "3852 TT3853 TT3854 TI'3855 'IT3856

hisO-E9533 zee-1::TnlO his+ zee-2::TnlO trplOlO::TdO, his9533 zee-2::TnlO his01242 pro668: :TnlO, his9533 hisOGD646, hisH8698: :TnlO zee-2: : (TnlO-hisO1242-H8698-TnlO) trpl010: : (Tnl0-hisO1242-H8698-TnlO) ilvA2091: : (TnlO-his01242-H8698-TniO) pro668: :(TnlO-hisOl242-H8698-TnlO) hisOGD646, hisC8691: :TnlO (A) hisOGD646, hisC8667: :TnlO(B) zee-2: :TnlO hid129 zee-2: :TnlO (A) hisO-C8691: :TnlO (A) zee-2: :TnlO (A) hisO-C8667: :TnlO(B) Dp (hisO1242-H8698)-TnlO(his01242, ACD2236, BHAFIE), eddzee-2: :TnlO his01242-H8698::TnlO, phs+ Dp(hisOGD646, C8667) -TnlO(B)- (hisO-E+) Dp(hisOGD646, C8667) -TnlO(A) - (hid-E+) Dp(hisOGD646, C8667) -TnlO(A)- (hisO-E+) Dp( hisOGD646, C8667) -TnlO(A) - (hid-E+) Dp(hisOGD646, C8667)-TnIO(A)-(hid-E+)

Recipient

Donor

Pooled TnlO insertions (CHUMLEY, MENZEL and ROTH 1979) T LT2 Pooled TdO insertions T LT2 Pooled TnlO insertions Spontaneous HisGT TT580 TT513 T TI'17o.E 73.95 T his01242 TT513 T TTI 704 TT190 T hisOGD646 TT1158 T TT.2347 TT1889 T TT1883 TT2348 T TT1884 TT2.348 T 73'1918 TT2348 T hisOGD646 TT115I T hisOGD646 TI'l127 T hid129 TT513 T TT3003 TT3164 T TT3010 TT3164 T hisCD2236 TT2361

TT2.348 TT3171 TT3171 TT38171 TT3171 TT3171

Strains listed were used in this study and were constructed in this laboratory. The method of construction is listed. "T" indicates P2Zmediated transduction. TnlO (A) refers to a TnlO insertion in orientation A; TnlO (B) refers to a TnlO insertion, in orientation B. and ROTH1980), except that the two TniO insertions standard Tnl0-his-Tnl0 strains (CHUMLEY used are in opposite orientation in the chromosome. The TnlO insertions used to build this strain were oriented by the method of CHUMLEY, MENZELand ROTH (1979). The OlnT-his-TnfO strain is much less prone to loss of the his material between the two TnlO insertions than are Tn10-his-Tnl0 strains. The latter segregate HisD- TetR colonies at a frequency of approximately 1W to 10-3 after ten generations of nonselective growth, whereas the OlnT-his-TnlO strain yields HisD- colonies at a frequency of about 10-4 after ten generations of nonselective growth. The stability of the HisD+ phenotype is expected from a strain that has TnlO insertions in opposite orientation, since most homologous recombination events would invert the chromosomal segment, but not remove it. T h e rare exchanges between IS10 sequences bordering the his genes could cause deletion, but would be expected tc. occur only rarely due to the restricted length of these homologies (1.4 kb in IS10 compared to 9.3 kb for the entire TnlO element). (4) TT3286 (hisO1242-H)-TnlO-(hid1242, ACD2236, BHAFZE) : This duplication strain was the result of circularization and insertion of a transduced fragment carrying trp:: [TniO-

18

NI. SCHMID A N D J. R. ROTH

(hkOl242-H)-TnlO] into the his region of hisCD2236, using his homology. The strain is His+ (smooth) and segregates 1 to 2% HisD- Tets colonies after approximately ten generations of nonselective growth. Both copies of the his region carry the regulatory mutation his01242. Genetic mapping of zee-2::TnlO: It was necessary to show that hisOGD646 deletes the site of the zee-2::TnlO insertion (the operator-proximal TnlO insertion used to build the translocation strains). Transduction crosses between donor zee-2: :Trill) (TT513). and recipient hisOGD646 were performed selecting either TetR or HisD+. Subsequent scoring of the unselected marker showed complete linkage of the TetR and His+ phenotypes (74 of 74 His+ were TetR and 100 of 100 TetR were His+). The lack of recombinant His- TetR or His+ TetS iypes demonstrates that hisOGD646 lacks the site of the zee-2::TnlO insertion. Nonzenclature: Nomenclature is generally described in CHUMLEY and ROTH (1980) and, more extensively, in CAMPBELL et al. (1977) and CHUMLEY,MENZELand ROTH (1979). The nomenclature z--::TnfO refers to a TnlO insertion in a “silent” DNA region; the ‘z--’describes the map position of the insertion. In order to describe the directed transposition strains in as concise a manner as possible, the notation trp::(TnlO-his-TnlO) is used in the text to describe a translocation of his genes into the trp region. A strain that has TnlO insertions in opposite orientation bordering the his region is denoted OfnT-his-TnlO. Media: All media used are the same as in CHUMLEY and ROTH (1980) with one exception: Complex medium supplemented with Tetracycline is 8 g/l Bacto-tryptone, 1 g/l yeast extract, 5 g/l NaCl, 15 g/l Difco agar, 0.84% glucose, 10 mg/l Tetracycline (Sigma). Kligler Iron Agar (Difco) was prepared as recommended (55 g/l) and used to distinguish Phs+ and Phs- phenotypes. Individual plates were poured immediately before use. Colonies to be tested were stabbed into the agar with a toothpick. This seemed sufficient to create the necessary anaerobic environment, The color reaction was generally quite clear after overnight incubation; however, plates were allowed to incubate for 24 to 48 h r since a delayed positive reaction was sometimes observed. mM L-histidine Minimal (E) plates were supplemented with 1 mM histidinol and 5 x to detect HisD- segregant colonies. On these plates, HisD- colonies appear small, pale and have a characteristic shape, while HiaD+ colonies have a normal appearance. General methods: General transduction methods are as described in CHUMLEYand ROTH (1980). Transductions in which HisD+ (histidinol growth) was selected were performed on plates containing histidinol and a low concentration of histidine (described above). The transductants that were directed transpositions often took 72 to 120 h r at 37” to appear. Orientaiion of TnlO inwrtions: Orientation of TnlO insertions was determined according to the method of CHUMLEY, MENZELand ROTH (1979). The general method involves integration 01 an E. coli F”(ts) Lac+ zzf-2O::TnlO into the Salmonella chromosome, using TnlO homology to direct the site and orientation of F” insertion. The F’(ts) Lacfzzf2O::TnlO episome lacks genetic homology to promote recombination events at sites other than the TnlO insertion. The Hfr strains formed in this manner transfer the chromosome in a direction that depends on the orientstion of the chromosomal TnlO inseriio’n. RESULTS

A. Circularization of transduced fragments: Directed transposition of transduced chromosome segments might occur by either of the two mechanisms diagrammed in Figure 1. The two directed transposition mechanisms differ in the state of the transduced fragment before recombination with the chromosome. Circular transduced fragments can be formed by recombination between the two TnlO elements [hat border the his genes. These two TnlO elements are in the same orientation. and a single recombination event results in a circular transduced fragment. Either circular or linear transduced fragments can recom-

19

CIRCULARIZED TRANSDUCING FRAGMENTS

bine with the chromosome and result in genetically identical directed transposirion strains. In order to distinguish between recombination events involving linear or circular transduced fragments, the transduction cross outlined in Figure 2 was performed. I n this cross, the use of a directed transposition strain as the transductional donor of his genes limits homology between donor and recipient. The donor strain (TT2632) has the first five genes of the his operon bracketed by two TnlO elements and located in the pro region (minute 7). The donor strain’s normal his region (minute 44) has been completely removed by deletion (his9533). When this strain is used as a transductional donor of his material, all donated his fragments are derived from the transposed his region. The recipient in this cross, hisOGD646, lacks the site of the “near-his” TnlO insertion, which forms one end of the donor’s transposed his region. Thus, there i:, no genetic homology between the transduced fragment and the his region of the recipient chromosome on one side of the hisOGD646 deletion (Figure 2). I n this cross, HisDf recombinants can arise in two ways: (1) Using pro homology, a linear transduced fragment can recombine into the pro region, causing a Pro- HisD+ TetR phenotype. This recombination can occur in a standard manner, between a linear transduced fragment that has pro homology on both sides of the transposed his and TnZO genes. The donor pro region[pro:: (TnlO-hisOGDCBH-TnlO) ] is substituted for the wild-type recipient pro region. The HisDf TetR Pro- transductants should segregate HisD- TetR Pro- colonies by recombination between the TnlO elements in the inserted TnlO-his-TnlU stmcture. (2) HisDf recombinants can arise by recombination events that occur in the his region. Those events in the his region that involve linear transduced fragments require homology on both sides of the recipient mutation and thus PRO REGION

HIS REGION his “lo ‘pro P

-

C

i

I

I

1 insufficient

Pro- HisD’Tet‘

.I homology

-hi$OGDCBHAflE\

Pro+HisD+Tet

FIGURE 2.-A method f o r detecting transductants arising by linear or circular transduced fragnzents. A linear transduced fragment derived from a Tni0-his-Tn10 structure in the pro region can recombine in the recipient pro region to give Pro- HisD+ TetR recombinants. This linear transduced fragment cannot repair hisOGD646 since there is no homology between donor and recipient to the left of the recipient his deletion. A circular transduced fragment can recombine with the his region to give Pro+ HisD+ TetR transductants.

20

M. SCHMID AND J. R. ROTH

cannot OCCLW in this cross (Figure 2). Circularization of a transduced fragment allows a single recombination event in the his region to insert the TnZO and his material found in the donor transposition. A linear TnIO-his-TnZO fragment can circularize by using its own TnlO homologies. The circular transduced fragment can recombine into the chromosome, using homology between the his region of the transduced fragment and the his region of the recipient chromosome. The result is a duplication of the his region that has a TnlO element at a join point [in the form (hisOGD646-H)TnlO-(hisOGDCB . . .)I. Such transductants will acquire not only HisD+, but also a reconstructed his operon; their phenotype is expected to be Pro+ His+ TetR.Since these transductants carry a single TnZO element bracketed by homologous segments of the his operon, they should generate HisD- TetS segregants that lose TnZO along with the added his segment. The data for this experiment are shown in Table 2. Two types of transductants were obtained. Prof His+ TetR types were found; these strains segregate HisDTetS colonies, and are inferred to be the result of circular transduced fragments that recombined in the his region. Pro- HisD+ TetRtypes were also found; these strains segregate HisD- TetR colonies and are the result of transduction events in the pro region. Colony morphology was used to identify recombinants that had inherited a complete his operon, since the translocated his region of donor strain TT2632 carries the regulatory mutation, hisO1242. When the hisH and hisF genes are expressed at a high level, as happens when the his01242 mutation controls exand HARTMAN pression, colonies have a rough or wrinkled morphology (MURRAY 1972). If the circularized TnlO- (his01242-H)-TdO segment is transduced into the his region of hisOGD646, a complete His+ region is adjacent to his01242 and a rough colony morphology will result. If the TnlO- (his01242-H)-TnZO segment is elsewhere in the chromosome, the transductants will be smooth, since only half of the his operon ( h i d - H ) is adjacent to hid1242. As seen in Table 2, all of the Pro+ His+ colonies are rough, while the Pro- HisD+ colonies are smooth. The lack of homology between the translocated his region of the donor strain and the operator side of hisOGD646 is important in concluding that circular TABLE 2 Results of a transduction cross demonstrating formation of circular transduced fragments Transductants phenotype

Pro+ HisD+ TetR Pro- Hi&+ TetR

Frequency

Segregation pattern

17/27 10/27

Pro+ HisD-TetS Pro- HisD-TetR

Colon

morphozgy

Rough Smooth

Recombination site

Linear/circular transduced fragments

His Pro

Circular Linear

~

The Pro+ HisD+ TetR transductants are the result of recombination between a circular transduced fragment and a single site in the his region. Selection was made for HisD+. Donor: 72632 [pro: : (TniO-hisOZ242-H-TnlO), his-95331. Recipient: hisOGD646.

21

CIRCULARIZED TRANSDUCING FRAGMENTS

transduced fragments lead to the formation of the prototrophic transductants in this cross. It has been shown that recombination cannot OCCUT between the operator side of hisOGD646 and the his or TnZO sequences present in the translocation strains (see MATERIALS AND METHODS). To rule out the possibility that sequences in or near the pro region have homology with sequences outside of hisOGD646, the experiments described so far with the pro:: (TnlO-his-TnZO) translocation strain were repeated, using other translocations as the donor strain. Donors carrying a TnZO-his-TnlO translocation in the trp operon (TT2361) or in the ilv region (TT2387) give the same results in this cross as the pro translocation donor. In all cases, recombinants arise that do not inherit the auxotrophy of the donor translocation and are unstable for the His+ phenotype. These unstable transductants lose TnZO along with the his region, suggesting that they are partial his duplications with TnlO at the join point (Figure 2). We feel that these experiments strongly support the conclusion that transduced fragments are circularized and then inserted by a single recombination event involving the homologous segments of the his operon in the donor fragment and the recipient chromosome. B. Frequency of circularization: In order to estimate the relative frequency of transductants that occur by linear or circular transduced fragments, crosses were performed in which either type of transduced fragment could recombine with the recipient his region. By comparing recombination events occurring in a particular region of the chromosome, estimates of the frequency of linear and circular transduced fragments are not complicated by the need to compare events occurring in different regions of the chromosome. When homology exists between transduced fragment and chromosome on both sides of a recipient his deletion, either linear or circular transduced-fragments can recombine with the chromosomal his region to give HisD+ recombinants (Figure 3 ) . In a cross between recipient hisCD2236 and donor TT2361 [trp::

-

1

x x

[phs hisOG-

1 phs his OGDCBHAFIE -/

a& /

his OGDCBH

his OGDCBH X -HAFIE\

,X

Q~&GG-BHAFIE'\ phs OGDCBH

i OG

BHAFE I I

\/

I

phs OG,SHT~@OGDCBHAFIE \

His' Tet His' Tet His' Tet' FIGURE3.-A method for determining the frequency of transductants due to circular transduced fragments. The donor strain carries T d 0 - h i s - T d 0 inserted in the trp region. 'His+ transductants arising by circular transduced fragments are TetR, while those arising by linear transduced fragments are Tets.

22

M. S C H M I D A N D J. R. ROTH

(TnlU-his-TnZO)1, HisD+ transductants were Felected. The transduced fragment has homology on both sides of recipient deletion hisCD2236. Thus, linear transduced fragments carrying the transposed his region can replace the recipient his mutation. These transductants will be Tets. Alternatively, transduced fragments can circularize using the TnlG elements bordering the transposed his region. These circular transduced fragments can also recombine with the recipient his region to give HisDf transductants. These transductants will be TetR. Transductants occun-ing by recombination events involving a linear transduced fragment in the t r p region can be eliminated by selecting simultaneously for Trp+ and HisDf recombinants Under these conditions, the frequency of TetR and TetS transductants can be compared directly as indications of circular or linear transduced fragments. The data for this experiment are shown in Table 3. Approximately 55% of the HisDf transductants are TetR, indicating that about half of the transduction events involved a circular transduced fragment. Purified TetR transductants segregate HisD- TetS colonies. as expected from strains carrying the his duplication formed by addition of a circular transduced fragment. Another estimate of the fraction of circularized fragments can be made using the phs locus, which is linked to the his operon (VOLL,SHILLER and CASTRILLI 1974). The phs genes are necessary for the production of hydrogen sulfide, and phs mutants have an easily scorable phenotype on Kligler Iron Agar (see MATERIALS AND METHODS). Deletion mapping has ordered the markers in this region as phs- (zee-2::TnlO) -hisOGD . . . (F. CHUMLEY,unpublished data). A transduced fragment that has Tn10-his-TnlO derived from the normal his region (as in TT3291) will lose the phs marker if it circularizes, since phs is outside of the region bounded by the two TnZO insertions. In transduction crosses between a recipient that has a his deletion extending through the phs locus and donor TABLE 3 Transduction crosses showing the frequency ofcircularization using various Tn10-his-Tn10 strains % tfansductants Recipient

a. hisCD2236

b. hisCD2236 C. hisOGD646 d. hisOGD646 e. his3GD646 f. hisOGD646 g. hisOGD646

Donor

TT2361 trp: : (Tnl0-his0-H-Tnl0) TT2632 p r o : :(Tnl0-hid-H-Tn10) TT513 zee-2: :TnlO TT3171 OlnT-his0-C-Tnl0 TT3 170 Tnl0-hi&-C-TnZ0 TT3291 Tn10-his0-H-Tni0 TT1770 Tnl0-hisO-E-Td0

Select

HisDf Trpf HisDf Prof HisD+ HisDf HisD+ HisD+ HisDf

Score

TetRlS TetRlS Phs+/PhsflPhsflPhsflPhs+/-

by circularization

58% (51/88) H% (21/39) 0% (0/200) 4.8% (4/83) 463% (46/99) 52.0% (104/200) 42.5% (34/80)

(a,b) Two different directed transposition strains were used to show the frequency of circularization. In these crosses, TetR transductants arose from a circular transduced fragment, while T e P transductants arose from a linear transduced fragment; (c-g) in these crosses, P h s transductants result from circular transduced fragments, while Phs + transductants result from recombination events using linear transduced fragments; (c) a control cross in which the donor strain has only one TnlO element; (d) circularization can occur in a strain with TnlO insertions in opposite orientation; (e-g) a comparison of the frequency of circularization in strains carrying various lengths of the his region.

23

CIRCULARIZED T R A N S D U C I N G F R A G M E N T S

i +hsi HisD+Tet" Phs+

OGDCBHAFIE HirD+TeP Phs-

FIGURE 4.-A second method for determining the frequency of transductants due to insertion of circular transduced fragments. The donor strain carries TnI0-his-TnZ0 at its normal chromosomal site. The recipient carries deletion hisOGD646, which removes p h s and the his operon. His+ transductants arising by linear transduced fragments are Phs+, while those arising 1 ) ~ circular transduced fragments are Phs-.

37'3291 (Phs+ Tn1O-hisOGDC-TnZO),two types of HisD+ transductants should be found. A transductant that has been formed by a linear transduced fragment will be Phs+ HisD+, while a transductant formed by a circular transduced fragment will be Phs- HisDf (Figure 4 ) . The results of this experiment show that 50% of the transductants are PhsHisD+ (Table 3). Thus, in two independent tests, half of all transductants arise by circular transduced fragments. The frequency of circular transduced fragments has been found to be approximately the same when varying lengths of his material are located between the two TnlO elements (Table 3, lines e, f and g) . The frequency of circular transduced fragments does decrease when less homology is provided for circularization (see Section E and Table 3, lines d and e) * C. Circularization does not depend on TnlO sequences: I n the experiments described so far, TnZO sequences have provided the homology for circularization of the transduced fragment. To show that circularization is dependent only on repeated sequences carried by the transduced fragment and not on sequences unique to TnlO, a his-TnZO-his duplication was constructed (see MATERIALS AND METHODS). Directed transposition of his material can occur only if the his-TnZOhis transduced fragment circularizes using his homology. This cross is diagrammed in Figure 5 . The recipient in these crosses (TT1918) has a TnZO insertion in the pro region and a large deletion of the his operon for which the corresponding wild-type sequence cannot be carried by a single transduced fragment. Therefore, the proper homology does not exist for recombination between the recipient chromosome and a linear HisD+ transduced fragment. If the hisTnZO-his transduced fragment circularizes using redundant his homology, directed transposition of the his segment can occur by recombination between

24

M. SCHMID A N D J. R. R O T H

transduced fragment:

his OGDCBH

i's A

recipient:

r

his

%BHAFIE

pro'

pro'

TnlO

'pro

O G D C B H 'pro ~ his

1

1

FICURE 5.-The mechanism by which a transduced fragment carrying his-TnlO-hiscan cause directed transposition of the his and TnlO genes to the site o€ a recipient TnlO insertion.

the TnlO element on the circularized fragment and the TnlO element in the chromosomal pro region. HisD+ transductants are recovered when his-Tnl0-his (TT3286) is used as a donor, but not when his-TnZO (73313) is used as a donor. The genotypes of 30 of these transductants were checked to determine whether directed transposition had actually occurred. In strains that have the his genes located in the pro region, the his genes should be completely linked to the pro: :TnlO insertion. The 30 strains obtained from the cross were transduced to Pro+. In 23 of the 30 strains, transduction to Pro+ caused loss of both the TetRand HisD+ phenotypes. These strains are concluded to be directed transpositions, since linkage is complete between pro and TnlO-his-TnlO. The genotypes of the other seven strains were not further determined, but one explanation for the strains that are not directed transpositions is given in the next section. From these data, it is concluded that duplicated sequences are required for the circularization events, and that no unique properties of TnlO are necessary for this circularization. D. Transposition of his directed by inuerse order TnlO elements: In construction of the TnlO-his-TnZO strains described thus far, TnlO insertions were chosen that had the same orientation in the chromosome. This section describes experiments designed to determine whether TnlG insertions in opposite orientation can permit directed transposition of the his genes. Each TnlO element includes tetracycline resistance genes flanked by inverse repeats of a 1400-base sequence, ISlO. Since there are two TnlO elements, direct repeat ISlO sequences enclose the his genes and a single TnlO element (Figure 6). It seemed likely that these IS10 sequences could provide the homology required to circularize a transduced fra-ment. The notation OlnT-his-TnlO will be used to refer to TnlO insertions in opposite orientation that border the his genes. Recombination events using IS10 homology can cause circularization of the transduced fragment; two types of transduced fragments result. In these two

25

CIRCULARIZED TRANSDUCING FRAGMENTS

\

\

1

I

I

recombination between IS sequences:

J two types of circular transduced fragments

FIGURE6.-A transduced fragment carrying OZnT-his-TnZ0 sequences can circularize uskg IS10 sequences. In this figure, the orientations of the 1,910 sequences that border the TnZO element are shown as heavy arrows. The Orientations of the tetracycline resistance genes are shown by the intervening lighter arrow. Recombination events can occur using either of two pairs of similarly oriented IS20 sequences (indicated by brackets). Two types of circular transduced fragments result, differing in the relative orientation of his and Tnl0 genes.

types, the orientation of TnlO relative to the orientation of the his genes depends on which pair of IS10 sequences is used to circularize the transduced fragment (Figure 6). For this reason, circularized transduced fragments carrying the OlnT-his-TnlU segment should cause directed transposition of the his genes in either orientation into a new site on the chromosome, depending on which recombination event closed the circular fragment. Transduction crosses between donor strain TT3171 (OlnT-his-TnlO) and recipient strain TT1918 (his-9533, pro: :Tido) demonstrate that a transduced fragment containing OlnT-his-TnlO does permit directed transposition of the his genes. This cross yielded HisD+ transductants. Three of five recombinants tested inherited the his genes in the pro region. These three recombinants, when transduced to Pro+, lost both HisD+ and TetR phenotypes. The remaining two recombinants inherited the his genes at another site, since their Pro+ transductants remained HisD+ and TetR. The strains that are not directed transpositions may result from addition of a circularized OlnT-his-Tn10 segment to the end of the his deletion. The recipient’s his deletion (his-9533) was probably generated by TnlO (M. JOHNSTON, unpublished), and probably has part or all of an ISlO, or part of the TnlO element, at one endpoint of the deletion. Recombination events can occur between the TnlO element of a circular transduced fragment and the ISlB endpoint; of ;he chromosomal his deletion. The result will be an effective shortening of the his deletion by addition of his genes at the normal site of the his operon in the relatively stable configuration of (ISlO)-his-TnlO. The transductants that are not directed transpositions are stably HisD+. It should be noted that the his genes in the above crosses were originally bracketed by TnlO elements in the opposite orientation (OlnT-his-TnlO). After cir-

26

M . SCHMID A N D J. R. ROTH

cularization and directed transposition to a new TnlO site, the his genes should be flanked by TnlO elements in the same orientation (pro::(TnlO-his-TnZO) ). This expectation is borne out by the instability of the directed transposition strains recovered. These strains are unstable HisD+ and give rise to HisD- TetR segregants that have lost the inserted segment, presumably by recombination between the flanking, direct order TnlO elements. Recombinants that occur in the pro region might also occur by a linear OlnThis-TnlO transduced fragment. This could occur by one crossover between one IS sequence of TnlO elements that are in opposite orientation, and one crossover between TnlO elements in similar orientation. This would lead to a stable translocation in which the TnlO insertions are in opposite orientation in the new chromosomal location. This type of stable directed transposition has also been detected. E. Circularization of transduced fragments by ZSlO Homology: The frequency of circularization of OlnT-his-TnlO-containing transduced fragments was estimated by the method involving the phs locus described in section B. TT3171 (OlnT-his-TnlO) was used as a donor to transduce the HisD+ phenotype into recipient strain hisOGD646. The general moss is described in Figure 4. The linked phs locus was used as an unselected marker. Transductants arising from circular transduced fragments remain Phs-, while those arising from linear transduced fragments become Phs+. The results of this experiment are shown in Table 3, line d. Approximately 5% of the HisD+ transductants are Phs- and are concluded to have arisen from circular transduced fragments. The control cross (Table 3, line c ) uses donor TT513 (zee-2::’hlO)which has only one TnlO element in the his region and therefore cannot circularize; it can be seen that no Phs- transductants are recovered from this control cross. Therefore, the Phs- transductants from the cross between donor TT3171 and recipient hisOGD646 result from circularization of the transduced fragment. The transduced fragments that circularize must use direct repeat IS10 homology to circu1ar;ze. Thus, sequences as short as ISlO (1.4 kb) can be used to cause circularization. Transductants arising from circularization of a OlnT-his-TnlO transduced fragment in the above crosses should carry a duplication of the his region with a single TnlO element at the join point of the duplication. As shown in Figure 6, there should be two types of circular transduced fragments that differ only in the relative orientation of the his and TnlO genes. Since the circular transduced fragment recombines with the recipient chromosome using his homology, the TnlO element should be present in either orientation in different transductants. Orientation of the TnlO element in these transductants was determined by the method of CHUMLEY,MENZELand ROTH (1979). This method requires formation of an Hfr by recombination between a chromosomal TnlO and a n F’ TnlO. The relative orientation of the TnZC element in the chromosome determines the orientation of Hfr transfer. The data for this experiment are shown in Table 4. I n four of five strains, the Hfr directed clockwise transfer, while one clearly showed the opposite direction of transfer. This gives strong evidence that two

27

CIRCULARIZED T R A N S D U C I N G FRAGMENTS

types of circular transduced fragments can be formed by recombination between different pairs of direct-repeat IS sequences. DISCUSSION

We have shown that transduced fragments containing redundant sequences are able to recombine with a recipient chromosome in two different ways. First, linear transduced fragments can recombine with the chromosome in a standard manner to replace the homologous recipient segment. Two crossover events between the recipient chromosome and transduced fragment are required to complete the recombination events. Second, a transduced fragment containing redundant sequences can circularize and then recombine with the chromosome; this adds a segment to the chromosome without replacing recipient material. This second process also requires two recombination events; however, one recombination event involves only transduced fragment sequences. The recombinant resulting from these events is unstable since homologous sequences flank the added DNA segment. Two objections might be raised to the conclusion that a circular transduced fragment exists inside the recipient cell. The first objection is that the order of the recombination events involved in creation of a circular transduced fragment may be reversed. The first recombination event might be between the chromosome and a linear transduced fragment. This recombination would break the circular chromosome, leaving a linear structure with terminal repeats. The second recombination event would use these terminal repeats to reform the circular chromosome. Thus, it is possible that some transductants that have been attributed to circular transduced fragments may actually occur by this alternative mechanism. A second objection to fragment circularization is that all of the transductant types that have been attributed to circular transduced fragments could be TABLE 4 Recombination between the two different pairs of IS sequences in OlnT-his-Tn10 gives rise to two relative orientations of Tnl0 and his genes Recipient

ccw transfer

Hfr donor

TT628 X TT628 x TT628 x TT628 x TT628 x

pyrF146

cw transfer purF145

B

TT3852 TT3853 TT3854 TT3855 TT3856

4.00 25 33 45

300

loa

A A A A

TT628 X TT513 TT628 X TT1127

100 200

600 10

A B

7

23 80

Orientation

110

Controls

The different orientations of TniO in transductants arise from circularizationof OinT-his-TnlO. The first column lists the origin of the Hfr strains that oriented the TnlO insertions.

28

M. SCHMID A N D J. R. ROTH

fraqment

\ m

f

C

6

H

his 0 G DCBHAFIE His' Trp' TetR (unstable)

FIGURE 7.-A mechanism by which a linear transduced fragment could lead to His+ transductants in a limited homology cross. This event would require the transduced fragment to carry a duplication of the transposed TnlO and his genes.

attributed to transduction events involving rare donor cells with a duplication of the transposed segment. This is a serious concern, since direct-repeat TnlO homology flanks the transposed his genes in the donor and could provide homology to create a TnlO-his-TnlO-his-TnlO duplication. A linear transduced fragment carrying this duplication could recombine as in Figure 7 to yield transductants identical to those formed by a circular transduced fragment. Transduction events such as these have been described in ANDERSON and ROTH (1977). One experiment suggests that this alternative explanation can represent only a minor fraction of the transductants. Duplication between homologous sequences is known to be dependent on a functional recA gene (ANDERSON and ROTH1977). A Rec- donor strain would not be expected to form duplications, using the TnlO insertions for homology. Isogenic Rec+ and Rec- transposition donor strains were used to transduce recipient strain hisOGD646 to His+. Homology exists between donor and recipient on only one side of the recipient his deletion in these crosses. The results of this experiment show that Rec- transposition donors give rise to the circular fragment transductant class just as well as Rec+ transposition donors (approximately one His+ transductant per IO7 pfu in Rec+ transposition donors, and 0.8 His+ transductants per IO7 pfu in Rec- transposition donors). Thus, very few of the transductants thal have been attributed to circular transduced fragments are the result of duplications in the transductional donor strain. For genetic manipulation of bacteria, the OlnT-chromosome segment-TnlO configuration offers several advantages. It allows use of a stable donor strain for directed transposition. It also results in two types of circular transduced fragments that differ only in the relative orientation of the TnlO and intervening genes. With this donor, creation of isogenic directed transposition strains differing only in the orientation of the transposed genes is possible.

CIRCULARIZED TRANSDUCING FRAGMENTS

29

The circularization events described here are probably not specific to transduction. Although it has not been determined, it seems likely that any foreign DNA segment containing redundant sequences would be capable of circularization inside a recipient cell. The circularization mechanism may be important in limited homology crosses, since only one region of homology between the donor DNA fragment and the recipient chromosome is required. The redundant sequences required to cause circularization of the donor fragment might be naturally occurring homologous regions (including IS sequences) or a duplicated region of the donor strain that is small enough i o bc carried by the donor DNA fragment; either situation would permit fragment circularization. In genetic crosses between two different species, homology for recombination may be severely limited. It is interesting to note that when such crosses are performed, unstable recombinants arise often, as would be expected if circularized fragments were being inserted (ANDERSON and ROTH1977). The unstable recombinants that result from the circularization mechanism are interesting from an evolutionary standpoint, since recipient DNA is not lost in the recombination events. Thus, the original recipient sequence can be recovered by recombination between the points initially used to integrate the donor DNA fragment. New gene combinations and hybrid genes are possible by segregation of the duplicated region, using other points of homology or by accumulation of deletions in one of the two copies.

.

This work was supported in part by Public Health Service Grant GM23408 (to J. R. ROTH)

LITERATURE CITED

ANDERSON, R. P. and J. R. ROTH, 1977 Tandem genetic duplications in phage and bacteria. Ann. Rev. Micro. 31: 473-506. CAMPBELL, A., D. BERG,E. LEDERBERC, P. STARLINGER, D. BOTSTEIN, R. NOVICKand W. SZYBALSKI, 1977 Nomenclature of transposable elements on prokaryotes. pp. 15-22. In: DNA Insertion Elements, Plasmids and Episomes. Edited by I. A. BUKHARI,J. A. SHAPIROand S. L. ADHYA.Cold Spring Harbor Laboratory, New York. CHUMLEY,F., R. MENZELand J. ROTH, 1979 Hfr formation directed by TnlO. Genetics 91: 629-655. CHUMLEY,F. and J. ROTH, 1980 Rearrangement of the bacterial chromosome using TnlO as a region of homology. Genetics 94: 1-14. JOHNSTON, H.M. and J. ROTH,1979 Histidine mutants requiring adenine: selection of mutants with reduced hisG expression in Salmonella typhimurium. Genetics 92 : 1-15. KLECKNER, N., 1977 Translocatable elements in prokaryotes. Cell 11: 11-23. KLECKER, N., J. ROTHand D. BOTSTEIN,1977 Genetic engineering in vivo using translocatable drug-resistance elements: new methods in bacterial genetics. J. Mol. Biol. 116: 125-159. 1972 Overproduction of hisH and hisF gene products MURRAY,M. L. and P. E. HARTMAN, leads to inhibition of cell division in Salmonella. Can. J. Micro. 18: 671-681. VOLL,M. J., M. SHILLFB and J. CASTRILLI,1974 His-linked hydrogen sulfide locus in Salmonella typhimurium. J. Bact. 120: 902-905. Corresponding editor: H. ECHOLS