IS2-IS2 and IS3-IS3 relative recombination frequencies in F integration

PLASMID

3, 48-64 (1980)

IS2-IS2

and M-IS3 Frequencies

Relative Recombination in F Integration

RICHARD C. DEONIERAND R. G. HADLEY M&c&r

Biology,

University

of Southern Cal(forniu,

University

Park, Los Angeles,

California

90007

Received April 26, 1979 The relative integrative recombination frequencies of the F plasmid IS2 and Is3 elements were determined at two Escherichia coli K-12 chromosomal sites by hybridization analysis of Hfr DNA. The sequence organizations of the independent Hfr strains formed by F integration at a& indicate that the relative recombinational frequencies at the two F plasmid 1S.I elements olJ3, and u,& are not significantly different. A comparison of the relative recombinational frequencies of the IS2 and Is3 elements of F was provided by analysis of DNA from Hfr strains having F integrated between lac and proC (i.e., at the IS2 or at the 1% element (cy&,) located in this region). No instances of F integration at 05& were detected, indicating that integrative recombination at IS2 is significantly more frequent than at 1S.I in this chromosomal region.

during F integration. Previous studies of this process revealed two anomalies. First, investigation of six independent integration events at the chromosomal IS3 element as& yielded no instances in which the F plasmid a& element was involved (Deonier and Davidson, 1976; Hadley and Deonier, 1979). Because sequence divergence has been documented for IS2 elements (Ghosal et al., 1979), it seemed possible that cv,/3, and az& might have sequence differences that would affect their ability to mediate F integration. Second, F integration within the chromosomal region between lac and proC occurred at IS2 in both of the two cases so far examined (Hu et al., 1975a; Deonier et al., 1977) even though a neighboring IS3 (a)&,) was available. If the IS2-IS2 recombinational activity were the same as the IS3 -IS3 recombinational activity, integration at LY& should be favored, because F has two IS3 elements and one IS2 element. The number of Hfr structures so far examined is insufficient to establish whether or not either of these observations really reflects intrinsic differences in the recombinational activities of different pairs of IS elements. We have used hybridization techniques

Tandem direct repetitions of IS elements are associated with several distinct phenomena. Direct repetitions of IS1 are thought to be involved in the amplification of drug resistance determinants of certain R plasmids (Hu et al., 1975b; Ptashne and Cohen, 1975), and they also define the borders of the chloramphenicol transposon Tn9 (MacHattie and Jackowski, 1977). Either IS2 or IS3 present on the F plasmid can mediate F integration at corresponding sites on the bacterial chromosome (Davidson et al., 1975), leading to an Hfr strain in which the integrated F DNA is flanked by direct repetitions of IS2 or IS3 (Deonier and Davidson, 1976; Hu et al., 1975a). This is structurally analogous to the two cases mentioned above. Recombination at direct repetitions of IS elements may share some mechanistic steps with recombination processes characteristic of single IS elements or of IS elements present as inverted repetitions, and possible relationships between these kinds of recombination can be explored by genetical and physical analysis of the sites at which recombination occurs. The present study provides physical data on recombination between IS2 or IS3 pairs 0147-619X’80/010048-17$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

48

F INTEGRATION

to determine the integration sites on F for seven Hfr strains with points of origin located in two regions of the Escherichia coli K-12 chromosome: at the IS3 a&, (between lac and proA), and at (Y&, or IS2 (between lac and proC). The former set of Hfr strains allows a comparison of the relative recombinational activities of the two IS3 elements of F (clllpl and a&J, whereas the latter set provides a comparison between IS2 and IS3. These new data are compared with previous results to assess whether there are differences in pairwise recombinational activities of IS2 and IS3 elements at these sites. MATERIALS

AND METHODS

Strains constructed or employed in this study are indicated in Table 1. Genetic analysis. The Hfr character and transfer direction were examined by crossstreak matings (Berg and Curtiss, 1967) on appropriately supplemented minimum medium plates (Clowes and Hayes, 1968)using both recA’ (AB 1157 or PB3 14) and recA(ED1 111)recipient strains. Transfer ofproC was tested using x478 as a recipient. Sensitivity to bacteriophage f 2 was also tested by the cross-streak method, using EMB-glucose plates. Strains containing RSF2124 or derivative plasmids were scored for ampicillin resistance by plating on L-plates (Lennox, 1955) containing 20 Fg/ml ampicillin. The Cal+ phenotype was determined by the procedure of Fredericq (1957). Preparafion of DNA. Methods employed for DNA isolation and ethidium bromide extraction have been described previously (Deonier and Mirels, 1977). Restriction enzymes. Endonucleases EcoRI, HindIII, and Bum HI were obtained from Miles Laboratories. Reactions with EcoRI were conducted at pH 7.5 at 37°C using the solution components of Polisky et al. (1975). Hfr DNA was digested with at least a twofold excess of EcoRI. Half of the enzyme was added at the beginning of the reaction and the remaining half was added 1 h later. Digestion was continued for one more hour. In most instances, parallel digestions using Bacterial

strains.

49

AT IS2 AND IS3

control DNA were performed to verify the enzyme activity. Digestion conditions for Hind111 were those of Danna and Nathans (1971). Digestion with BamHI was conducted using 50 mM NaCl, 6 mM Tris-HCl, 6 mM MgC&, and 6 mM @mercaptoethanol, pH 7.4, at 37°C. Eiectrophoretic analysis. Methods employed for electrophoresis and mobility analysis for gels stained with ethidium bromide have been previously described (Hadley and Deonier, 1979). To analyze molecular weights of bands appearing in autoradiograms, densitometer traces were made after short-term exposures to determine the positions of the most intense bands relative to the origin. Positions of bands of lesser intensity were determined by densitometry after prolonged exposures. Relative mobilities were determined with reference to f,2 or f,4 bands of F in the autoradiogram. Fragment sizes were determined by using the calibration curve either constructed from the original gel (using EcoRI fragments of F and F’ plasmids) or, in some cases, constructed from the autoradiogram itself. Discrepancies sometimes appeared for fragment sizes larger than 15 kb,’ and in these cases, calibration from the autoradiogram was employed. Hybridization procedures. Procedures for labeling probe DNA with 3H by nick translation (Rigby et al., 1977), for transferring DNA to nitrocellulose filters (Southern, 1975), for hybridization (Engel and Dodgson, 1978),and for autoradiography (Laskey and Mills, 1975) have been described previously (Deonier et al., 1979). Electron microscope heteroduplex procedures. Heteroduplexes were prepared and

mounted as described previously (Ohtsubo ef al., 1974; Deonier and Davidson, 1976). FA(33-43) DNA was used as a double-strand length standard. Measurements were adjusted to correspond to a total F length of 94.5 kb. Construction

oj’plasmids

containing

fR#

of F. F fragments were separated electro1 Abbreviation

used: kb, kilobase.

50

DEONIER AND HADLEY TABLE 1 BACTERIALSTRAINS Strain No. Hfr or F+ strains W6F+ W1485 W1655F+

Genetic description

Source (reference)

F+ metBl rel-1 F+ supE42 hFA(33-43) metB1 rel-I hR X-

B. Bachmann (Bachmann, 1972) N. Davidson (Sharp et al., 1972) N. Davidson (Broda, 1967; Anthony et al., 1974) P. Broda (Broda, 1967) P. Broda (Broda, 1967) P. Broda (Broda, 1967) P. Broda (Broda, 1967) P. Broda (Broda, 1967) P. Broda (Broda, 1967) B. Bachmann (Hirota and Sneeth, 1961) R. Curtiss III (Curtiss and Stallions, 1969)

ED939 (=B2) ED940 (=B3) ED941 (=B4) ED943 (=B6) ED944 (=BS) ED946 (=B 11) Hfr6- 1

Hfr Hfr Hfr Hfr Hfr Hfr Hfr

x876

Hfr OR56 prototroph derived from K-12-112

F- strains ABllS7

C600 ED1111 PB314 W6FW1485Fx478 Strains containing recombinant or other plasmids “ColEI-Apa” M2903

metB1 rel-I metB1 rel-I metB1 rel-I metBI rel-1 metB1 rel-I metB1 rel-1 metB1 rel-I mtl-8 khRA-

AhhAm hhmet-2 mal-20

thi-1 argE his-4 proA leu-6 thr-I lacy1 galK2 ara-14 mtl-1 xyl-5 str-31 tsx-33 sup-37 thr-I leu-6 tonA thi-1 lacy1 supE44 Xlac purE thi tsx str recA lac purE thi tsx str metB1 rel-I supE42 hara leu ton 1acZ proC tsx purE trp lys str mtl xyl metE thi

C600 (RSF2124) C600 (pSH2)

XRD3 XRD6 RD22

C600 (pXRD3) C6OO(pXRD6) thi-I rel-I A (proB-lac)Xlll

RD117

supE44 recA56lORF203 metBI rel-1 A-lpRD117

RD119

metB1 rel-I

RH7

ED111 1 (pRH7); pRH7 = F

A-lpRD119

B. Bachmann (De Witt and Adelberg, 1962)

H. Shizuya (Signer et al., 1965) N. Davidson (Broda and Meacock, 1971) N. Davidson (Broda et al., 1%4) A&dine orange curing of W6F+ Acridine orange curing of W 1485 N. Davidson (Curtiss and Stallions, 1969)

T. Thomas (So et al., 1975) R. Thompson (Achtman et al., 1978) This paper This paper (Deonier and Mirels, 1977) Spontaneous F’ segregant from ED946 Spontaneous F’ segregant from ED946 (Hadley and Deonier, 1979)

lac+ proC+ purE+

phoretically on agarose gels after digestion with EcoRI. DNA corresponding to the partially resolved f,4 and fJ fragments was electroeluted from appropriate agarose gel slices, and was mixed with an amount of

EcoRI-cleaved RSF2124 DNA chosen so that the f,4/RSF2124 mole ratio would be - 1. The DNA was incubated at 4°C with 0.1 unit/pg of T4 ligase (Miles) at a DNA concentrationof-lO&mlfor24-30 h, and

F INTEGRATION

51

AT IS.2 AND IS3

cates fragments produced by EcoRI.) Plasmid pSH2 contains f,2 cloned on an RSF2124 vehicle (Achtman et al., 1978). Two new plasmids, pXRD3 and pXRD6, were constructed by inserting f,4 in both of the possible polarities into RSF2124 (So et af., 1975) as described under Materials and Methods. The f,4 was obtained from the F plasmid contained in W 1485. The structures FIG. 1. Structural maps of recombinant plasmids containing f,2 (pSH2) (Achtmann et ul., 1978) or fa4 of pXRD6 and pSH2 are summarized in (pXRD6) of the F plasmid. The Is3 elements a& and Fig. 1. LY& are indicated by solid areas, and the IS2 element Structures of pXRD3 and pXRD6 were of F (designated IS2,) is indicated by the hatched confirmed by electrophoretic analysis of area. Other portions of fk2 or fk4 are indicated by samples digested with EcoRI, HindIII, or open areas. The bottom sections of each structural map represent the RSF2124portion, with the Tn3 region BarnHI + HindIII. Digestion of both pXRD3 indicated by a heavier line. Relevant F coordinates are and pXRD6 with Hind111 produced (among indicated on the inside and IS element identifications others) fragments whose sizes were 2.6,2.4, or restriction enzyme cleavage sites are indicated on the outside of the circular maps. The pXRD3 plasmid (not shown) differs from pXRD6 only in the orientation of f,4.

then the reaction solution was diluted to -4 pg/ml DNA and incubated for another 72 h. The reaction solution contained 33 mM Tris, 0.066 mM ATP, 6.6 mM MgClz, and 10 mM dithiothreitol, pH 7.6 (Weiss et al., 1968; Modrich and Lehman, 1970). E. co/i strain C600 was transformed (Cohen et al., 1972) with ligated DNA, and ampicillinresistant clones were selected and tested for colicin production. Sodium dodecyl sulfate lysates of Cal- clones were screened electrophoretically for recombinant plasmids (Barnes, 1977), and plasmids containing f,4 were identified from mobility analysis of DNA digested with EcoRI. All experiments involving strains containing pSH2, pXRD3, and pXRD6 were conducted under Pl containment conditions as specified by the National Institutes of Health (U.S.) Guidelines for Research Involving Recombinant DNA Molecules. RESULTS Construction and Structure Hybridization Probes

of

For hybridization studies of F integration, probes containing EcoRI fragments f,2 and f,4 of F were used. (The subscript R indi-

8

C

932F

“i” \\ /\ pSH2 pXRD6

-m--l ‘A, 6.9F

,’ “QO;eF ,‘L ” ‘,7 / 8, ’ 13.3F ) 14.6F

WF

FIG. 2. (A) Heteroduplex structure formed from pSH2 and pXRD6. (B) Interpretive drawing of structure shown in panel (A). Single-strand regions are represented by the thinner lines, and double-strand regions are represented by the thicker lines. (C) A diagram of the structure shown in (A), with the F coordinates of the various features indicated. The long duplex segment composed of RSF2124 sequences has been arbitrarily separated into two portions to permit linear representation.

52

DEONIER AND HADLEY

G)W

P WI L-J

*

-m fR4

fR2A

GG- 0-2 fR4*

IfR2B

al4 =z,e, I ‘I?2 fR48

FIG. 3. Possible Hfr structures resulting from integration of standard F at the chromosomal a& The IS2, Is3, fs2 and fa4 sequences are indicated as in Fig. 1. Bacterial DNA is represented by a sawtooth line, and F DNA is represented by a straight line. Open triangles locate the EcoRI cleavage sites immediately clockwise and immediately counterclockwise of a&. The amount of bacterial DNA between each EcoRI site and a& is also indicated. The Hfr structure resulting from integration at aI/3, of F is shown in (i). Mixed subscripts on cupelements flanking the integrated F DNA indicate presumed recombinant elements. F sequences originally present in fa2 now appear in the junction fragments, fa2A and fa2B. The junction fragment designated by A is the junction that is more closely linked to proximal Hfr markers, and the one designated by B is closer to distal markers. Structure (ii) represents the result of F integration at (Y&. Nomenclature for junction fragments involving fa4 DNA is similar to that used above.

0.86, and 0.41 kb, which are in reasonable correspondence with fragments f,6, f,9, fi, 12, and fi, 13 that are produced by Hind111 digestion of F and that are completely contained within f,4 (Childs et al., 1977). RSF2124 contains one BarnHI site (Fig. 1; unpublished results) but no Hind111 sites. Fragment f,4 contains no BumHI sites. Digestion of pXRD3 or pXRD6 with both BumHI and Hind111 produces the interior set of Hind111 fragments listed above and two additional fragments, which contain the EcoRI sites that define the junctions between f,4 and RSF2124. The molecular weights of these junction fragments differed for pXRD3 and pXRD6, indicating that the polarity of fR4differed in these two plasmids.

The presence and polarities of the f,4 fragments in pXRD3 and pXRD6 were confirmed by forming heteroduplex structures from each with pSH2. A heteroduplex structure formed from pSH2 and pXRD6 is shown in Fig. 2. The long duplex region is formed from the homologous RSF2124 sequences. The short duplex region (whose length is 1.3 kb) is formed by hybridization of c& to a&, indicating that the polarities of f,2 and f,4 (as defined by the standard F map) are the same in pSH2 and pXRD6. Heteroduplex structures formed from pSH2 and pXRD3 contained a large uninterrupted substitution loop whose branches corresponded in size to f,2 and fR4(data not shown). The absence of the short (Y$&& duplex

F INTEGRATION

AT IS2 AND IS

53

FIG. 4. Autoradiogram/fluorograms of DNA from Hfr strains digested with EcoRI and hybridized to 3H-labeled f,2 DNA (A) or f,4 DNA (B). F is integrated at c& in all Hfr strains shown. The pRH7 control plasmid was derived from Hfr OR1 1, whose F structure is identical to structure (i) of Fig. 3. In (A), the f,2A and f,2B junctions correspond in size to those expected from structure (i) of Fig. 3. The f,4A and f,4B junctions have sizes expected for structure (ii) of Fig. 3. Fragments fR4,f,4A and f,4B, which contain cr.&, a&, and QL&, respectively, appear at lower intensity as a result of crosshybridization with the a,& sequence present on the f,2 probe. Other low-intensity bands correspond to bacterial fragments containing IS3 elements, and the one containing IS.?.7 is labeled f,(IS.7). In panel (B), hybridization to f,4 is seen for strains B3 and B4 at positions expected for the f,4A and f,4B junctions identified in (A). No intense bands of these sizes appear with B2. The numerous other bands correspond to IS2 and IS elements located in the bacterial DNA of these strains (Deonier et al., 1979; M. Hu, personal communication).

region confirms that the pXRD3 polarity is opposite to that of pXRD6. The pXRD6/pSH2 heteroduplex structure provides a basis for a more precise mapping of a& and IS2 with respect to the EcoRI sites defining f,4, which is important for interpreting the subsequent hybridization data. We take the counterclockwise EcoRI site defining f,4 to be 8.9 F on the F map (Guyer, 1978). Given the 9.3-kb size of the f,4 (Childs et al., 1977), the other EcoRI site defining this fragment can be placed at 18.2 F. The longer branch of the smaller substitution loop in the pXRD6/pSH2 heteroduplex structure corresponds to the distance from the EcoRI site at 18.2 F to Q&, and its length is 3.67 kb. This, together with the observed duplex length of 1.32 kb for the

cy,pl/c& hybrid duplex, locates the ends of az& at 13.3and 14.6 F, some 0.4 kb counterclockwise of the published coordinates (Davidson et al., 1975). We use the previous value of 1.3 for the distance separating IS2 from (Y&, and obtain corrected IS2 coordinates of 15.9 to 17.2 F. Hybridization Analyses of Hfr Strains Integrated at a&

F integration at as& causes the alteration of F DNA sequences as depicted in Fig. 3. The F restriction fragment at which F integrates (either f,2 or f,4 in this case) will be divided by the integration event into two components (f,2A and f,2B or f,4A and f,4B). Each of these new junction fragments

54

DEONIER AND HADLEY TABLE 2 OF EcoRI FRAGMENTS HYBRIDIZING TO f,2 FROMHfr STRAINSFORWHICH F IS INTEGRATED AT &Is

SIZES AND IDENTIFICATION

Fragment (kb) Strain or plasmid pRH7 B2 B3 B4 B6

f,2

f,2A

f,2B

f,4

f,4A

f,4B

12.9 12.2 (12.7)’

6.1 6.0 6.0 (6.0)

24.7 26.2 24.2 (24.1)

9.3 9.3 9.5 (9.3)

8.4” 8.4n (8.2)

17.5 16.9 (18.6)

fdd%) 12.8 13.2 *b * 12.6 (12.8)

f&d%)

fdIS3.6)

fdI~.7)

11.4 11.8 11.6 11.1 11.2 (11.2)

11.8 * * 11.6 (11.7)

5.6 5.6 5.6 5.8 (5.8)

? 7.3 -

n B3 and B4 generated bands whose sizes were 8.43 and 8.38 kb, and which hybridized extensively to fR4probe. * Asterisks denote bands obscured by intense hybridization in nearby regions. r Values in parentheses are the actual or predicted sizes based on other mobility and hybridization studies (Deonier et al., i979; Hadley and Deonier, 1979).

will contain bacterial DNA from one side or the other of the bacterial integration site. If, for example, integration had occurred within f,2 and EcoRI-digested Hfr DNA were hybridized to f,2, two new areas of relatively intense hybridization would appear. The F fragment not involved in integration (fR4in this case) would be unaffected, and it would migrate at its normal position. If integration occurred within f,4, fR2would migrate at its normal position and sequences from f,4 would migrate in two bands. Since the EcoRI restriction sites immediately adjacent to CY&are known (Hadley and Deonier, 1979), it is possible to determine whether integration within either of these fragments occurred exactly at the IS3 elements a$1 or CX& from the sizes of the junction fragments. In an attempt to find instances of F integration within CC&&,we examined the independent Hfr strains B2, B3, B4, and B6 (Broda, 1%7) using restriction enzyme digestion and hybridization techniques. All of these strains have transfer properties similar to those of other Hfr strains with F integrated at n&. Autoradiograms resulting from hybridization of Hfr DNA to 3H-labeled fR2 or f,4 are shown in Figs. 4A and B. The simplest hybridization pattern (Fig. 4A) resulted from use of f,2 probe, which contains IS3 but not IS2. The results of this

hybridization are summarized in Table 2. B2 and B6 each contain two bands of elevated intensity that comigrate with the two junction fragments of pRH7, a Type II F’ plasmid derived from an Hfr (OR1 1) in which F had integrated at (Y& by recombination at its c& sequence (Hadley and Deonier, 1979; see Appendix). This indicates that B2 and B6 were produced by integration of F at a,&, whereas B3 and B4 were not. Lowerintensity bands in B2 and B6 have molecular lengths that correspond in size to fR4 and to the normal bacterial EcoRI fragments fR(c@& and f,(IS3.7) (Table 2). The normal 16.8-kb band containing asp3 is absent in both cases, as expected. Presence of a band corresponding to f,4 (which should crosshybridize with f,2 since it also contains an IS3 element) is expected for B2 and B6 because recombination within fR2would not affect f,4. B3 and B4 contain an intense band with the mobility of unaltered f,2, indicating that the integration event did not affect this fragment of F (or a& in particular). Bands corresponding to f,4 and to the fragment normally containing (Y& are not detected with the f,2 probe in B3 and B4, indicating that F integration to form these strains occurred within f,4 at (Y&. Two additional low-intensity bands (17.2 and 8.4 kb) present in both B3 and B4 but absent in B2 and B6 are candidates for the junction

F INTEGRATION

Cm-16.9hby , ---189kb

:7.9kb’ ’ ‘I ’ , II, ---t-IWkb ’

(iI

01

-

lac -V

%F

I ’

: (ii)

/@a

Is2FBv

8242

)

I

55

AT IS2 AND 1s

v (ffiA)i

fR2 (fR4B)i

(fR4B)ii

fR2

(fR4Atki

FIG. 5. Three F integration possibilities in the region betweenluc andproc. Symbols and designations are similar to those in Fig. 3. Distances between relevant positions on the bacterial DNA are in kilobases. The IS2 present on the bacterial DNA is designated IS2B, and products of recombination between IS2, and IS?, are designated as shown in structure (i). The three different structures shown at the bottom would all be genetically similar. Junction fragments from each of the three Hfr structures are distinguished by appropriate subscripts.

fragments f,4B and f,4A, since integration DNA to 3H-labeled f,4 probe (Fig. 4B) proat a& by recombination at a& would duced numerous bands as described previgenerate junction fragments which both ously (Deonier et al., 1979), and some are contain an IS?. An additional band whose sufficiently elevated in intensity to make size is 7.3 kb appears in B3 but is absent in discrimination between hybridization to f,4 the other Hfr strains obtained from Broda. junction sequences and hybridization to Its size is similar to that of the altered multiple equivalents of IS2 or IS? difficult. IS3-containing band identified in W6F- In the case of B2 (Fig. 4B), fR4 appears in (Deonier et al., 1979). its normal position, as expected from the Hybridization of EcoRI-digested Hfr result using f,2 probe. The patterns for B3

56

DEONIER AND HADLEY

fR2 -

fR093.7)-

61

FIG. 6. Autoradiogramifluorograms of DNA from Hfr strains having F integrated between /UC and DNA was digested with EcoRI and hybridized to RH-labeled f,4 DNA (A) or f,,Z DNA (B). The control ORF203 plasmid contains the junctions f,4A and f,4B expected for structure (i) of Fig. 5. It is seen in panel (A) that B8, Hfr6- 1, and OR56 contain both of these junctions, whereas B 11contains only f,4B. Low-intensity bands corresponding to bacterial IS2 and IS3 elements are seen, and some can be identified by comparison with the patterns obtained from W6F- and W1485F-. Panel (B) presents the results of hybridization with f,2. The two intense bands seen with B I 1 DNA correspond to f&B and f,2A junctions expected from structure (iii) of Fig. 5.

proC.

and B4 differ from that of B2 by the absence of the intense f,4 band and the presence of elevated intensity in the 8.4-kb size range. This new band corresponds to one of the two fragments tentatively identified as a junction by hybridization with f,2. In addition to these differences, B3 and B4 possess additional hybridization intensity in the 18.6to 18.7-kb size range, which corresponds within experimental error to the other putative junction fragment previously identified. The recombination site for B3 and B4 is identified as (Y&, based on the alteration in linkage of fR4 and the comparison of the observed junction fragment sizes for f,4A and f,4B (8.4 and 17.9 kb, respectively) with the predicted values of 8.2 kb for f,4A and 18.6 kbforf,4B (see Appendix). The 17.9-kb value taken for f,4B in B3 and B4 is an

average of values obtained from hybridization with f,2 and f,4 probes. Hybridization Analysis *fHfr Strains Whose Points of Origin Are Located between lac and proC The principles described in the previous section can be applied to Hfr strains in which F integrated within either the IS2a (the bacterial IS2) or the IS3 ((Y&J mapping between fat and proC. Three possibilities are diagrammed in Fig. 5. Integration of F at a& by recombination at one of its two IS3 elements could produce alterations either in f,2 or in f,4, whereas integration of F at its IS2 element (hereafter designated IS2,) would affect f,4 but not fR2. The distinction between integration within IS2, and

F INTEGRATION TABLE 3 SIZES

AND

IDENTIFICATION

OF

EcoRI FRAGMENTS OR

FROM Hfr DNA OF BS, Bll, Hfr6-1, ORS6, WHICH HYBRIDIZE EXTENSIVELY WITH EtTHER f,2 OR f,‘%='b

DERIVED

Identity of restriction fragment (kb) Source of DNA

f,2

f,2A

f,2B

f,4

f,4A

f,4B

B8 Bll Hfr6- 1 OR56 ORF203

12.8 12.8 12.8 12.7

19.8 -

4.41 -

-

2.90 ? 2.93 2.7oC 2.88

25.0 24.4 25.2 25.Y 25.0

a Data from autoradiograms shown in Fig. 5 and others not shown are presented here. * Sizes of f,2, f,2A, and f,2B are from experiments using f,2 probe. Sizes of f,4, f,4A, and f,4B are from experiments using f,4 probe. Some fragments could be detected with either probe by cross-hybridization at 1%. c Results of one experiment from fR4 probe.

a& can be made on the basis of junction fragment size. Subscripts i, ii, or iii indicate junction fragments from the corresponding structures in Fig. 5. The 25.0-kb (f,4B)i fragment and the 3.0-kb (fR4A)i fragment of the Type II F’ plasmid ORF203 are characteristic of integration of F at IS2, in this region of the chromosome (structure (i), Fig. 5) (Hadley and Deonier, 1979). The predicted sizes for the junction fragments resulting from F integration at c+,& via its CQ& element are 7.0 and 13.6 kb for ( f .4A)ii and ( f K4B)ii, respectively (see Appendix). These values differ sufficiently from the observed values for integration at IS2 (3.0 and 25.0 kb) to provide clear discrimination between these two integration possibilities. Recombination between the F plasmid cQ1 element and (Y&, would produce junction fragments with sizes of 4.9 kb ((fR2A)iii) and 19.1 kb ((fR2B)iii). The bases for both of these estimates are outlined in the Appendix. Autoradiograms resulting from hybridization of EcoRI-digested Hfr and ORF203 DNA to 3H-labeled f,4 probe DNA are shown in Fig. 6. The most intense ORF203 bands are (fR4A)i or (f,4B)i (see Fig. 5). The ORF203 bands of lesser intensity in order

AT IS2 AND ISI

57

of decreasing molecular size are the novel joint of ORF203 (18.4 kb), f,2 (12.9 kb), f R(c&) (11.3 kb), f R4 (from putative F segregants present in the sample (Deonier et al., 1977)), and fRIO (possibly crosshybridizing with T& sequences present in contaminating RSF2124 DNA from the probe). Because the concentration of ORF203 DNA is low, its bands migrated somewhat faster than those of the Hfr strains. B8, Hfr6-1, and OR56 each contain fragments that have nearly the same mobility as ( f R4B)i and ( f R4A)i from ORF203. They lack the normal f,4 fragment. The average lengths of ( f,4A)i and (f R4B)i determined from three independent autoradiograms are given in Table 3. The agreement between sizes obtained for both ( f R4A)i and (f R4B)i in the Hfr strains B8 and Hfr6- 1 with the sizes measured for ORF203 indicates that for these two Hfr strains and the Hfr progenitor of ORF203 (OR2 l), F integrated at the same site, i.e., the IS2, between lac and proC. Because OR56 is a derivative of K-12-1 12, the (fR4B)i fragment is expected to be larger than the others by 1.3 kb because of the additional Is3 (a&) contained in f R4(Hadley and Deonier, 1979). The observed size is within 0.8 kb of the expected value, and within experimental error for this size range. This agreement, together with the size agreement for ( f ,4A)i, indicates that IS2, was the F integration site for OR56 also. The sizes deviate markedly from the values predicted for integration at c+&. These three Hfr strains did not contain a low-intensity band corresponding to the bacterial fragment containing IS2B (18.9 kb), which supports the conclusions reached from the junction data. Autoradiograms resulting from hybridization of Z&RI-digested Hfr DNA to 3Hlabeled f R2 probe are shown in Fig. 6B. It is seen that B8, OR56, and Hfr6-1 DNA samples each contain an f,2 band, which is expected since the data in Fig. 6A had indicated altered linkage within f,4. The low-intensity bands correspond to bacterial fragments containing IS3 elements. The 23to 25-kb length of the topmost band is ap-

58

DEONIER

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B 11had lost the Hfr character after a month. Some of these clones that had lost the Hfr character were f2 sensitive, and were unable to transfer luc+, proC+, or put-E+. DNA from Hfr Bll also revealed differences from the other strains by hybridization both to f,4 and to f,2. With the f,4 probe, Bll DNA showed a region of relatively intense hybridization in the expected position for ( f R4B)iand this band comigrated with the ( f R4B)i bands seen with ORF203, B8, and Hfr6- 1 DNA. However, B 11 did not produce a band comigrating with (fR4A)i (Fig. 6A), even though the experiments described below indicate that some type of f ,4A band should have been present. There are several additional bands present in B 11 but not in B8, Hfr6-1, or OR56 (Fig. 6). One of these could represent an altered (fR4A)i. The pattern resulting from hybridization off R2to B 11 DNA also differed from those of B8, Hfrci-1, and OR56. The usual f,2 band was absent, and it was replaced by two intense bands (19.8 and 4.4 kb) (Fig. 6B). The sizes of the low-intensity bands derived from B 11 correspond to the sizes of the fragments containing a& (17.2 kb), a& (13.0 kb), IS3.6 (12.0 kb), and IS3.7 (5.7 kb). There is also an additional high molecular weight band with low intensity that comigrates with the (fR4B)i fragments identified for B8, HfrB-1, and OR56-a result that agrees with the identification of this as ( f a4B)i. A band corresponding to the fragment containing CX& was clearly missing from B 11 DNA. The sizes of the two putative f,2 junction bands are 19.8 and 4.4 kb, which are in fair agreement with the predicted sizes for (fR2B)iii (19.1 Hfr Bll and Derived F’ Plasmids kb) and (fR2A)iii (4.9 kb) arising from F Hfr B 11 differed from B8, Hfr6- 1, and integration at c& by recombination at OR56 in the character and stability of its CX& (see Appendix). A band whose size donor properties. Transfer of proC by B 11 corresponds within experimental error to was low as compared to transfer from BS, the ( f R2B)iii band identified above (19.0 kb) OR56, or Hfr6-1, but it was higher than also appeared in B 11 but not in B8, Hfr6- 1, transfer from F+ strains. Unlike the other or OR56 when f R4probe was used (Fig. 6A). Hfr strains, which were very stable when Thus, Hfr B 11displays fragments characteristic of F integration both at IS2a (i.e., maintained on nutrient agar slopes, Bll gave rise to clones that were not Hfr. Ap- (fR4B)Jand at CX&(i.e., (fR2A)iii and (fB2B)iii). To assist in resolving the ambiguity in proximately 13% of the clones derived from proximately the same for DNA from B8, Hfrb-1, and OR56, within experimental error. It is present in the Hfr DNA samples and ORF203 DNA, but it is absent in the Fcontrol DNA samples. This size corresponds approximately to the size of (f,4B)i obtained previously, and its presence confirms the presence of ( f a4B)i identified from hybridization data for f,4 probe, because (f,4B), is expected to contain the IS3 CY&. The next low-intensity band corresponds to the previously identified q&containing fragment (Deonier et al., 1979), and it is seen in all cases. The next series of faint bands is obscured for Hfr B8, OR56, and Hfr6-1 because of the intense hybridization of f,2. The next band visible in all four Hfr strains has a size of 5.7 kb-the size expected for the band containing IS3.7 (Deonier et al., 1979). Because we wish to compare integration efficiencies at IS2a and cq&, it is important to establish that a& was indeed present in these strains. Its presence is clearly indicated in OR56 and Hfr6- 1 by low-intensity peaks of hybridization that are immediately below f,2 and that comigrate with bands containing cq& from W1485F- and from W6F-. A shoulder on the peak corresponding to f,2 hybridization appeared in the same region with BB, but an actual peak was not resolved. We infer that this corresponds to (Y& from its position and from the presence of cQ5 in the B2, B3, B4, and B6 Hfr strains, all of which were isolated at the same time from the same F+ strain from which B8 was isolated.

F INTEGRATION

AT IS2 AND IS

FIG. 7. Heteroduplex structures formed from FA(33-43) and plasmids derived from Hfr B 11. Relevant positions are marked with F map coordinates. For the interpretive drawings shown at the right, doublestrand segments are indicated by thick lines and single-strand segments are represented by thin lines. Panel (A) shows the structure involving pRDl19. The double-strand region containing F DNA is interrupted by a single-strand gap in this particular structure. The y8 inverted repeat loop (labeled 2.8F/8.5F) is present in the shorter branch of the substitution loop. The other branch is presumably bacterial DNA. Panel (B) presents the corresponding structure involving pRDll7. The size of the substitution loop branch containing the y8 inverted repeat loop is the same as in the pRDl19 structure. As in panel (A), the

nthcsr

hron,-b

;n

ot+r;h.,tnrl

tn

hort.m.Ll

n&T,,

59

60

DEONIER

AND HADLEY

structure of B 11, the plasmids contained in two non-Hfr segregants derived from Hfr Bll were examined. These two plasmids, pRDl17 and pRD! 19, differed from each other and from standard F. The size of pRDll7 is 78 kb (electrophoretic determination), and the size of pRDl19 is 164kb (from electron microscopy). Heteroduplex structures formed between pRDl17 or pRDl19 and standard F (Figs. 7A and B) contained the A(33-43) deletion loop expected from strains descended from W1655F+ (Anthony rt ul., 1974). This loop provided a reference point on the heteroduplex structures, and its positions on these structures were confirmed by the presence of the f R5/f,J3fusion EcoRI fragment of FA(33-43). Both structures contained substitution loops, one branch of which displayed an internal loop in 35 to 45% of the structures. The reproducible position and size of this loop (5.4 to 5.7 kb) suggest that it is the y6 loop/stem structure (Broker et al., 1977). The singlestrand distances from this loop to the forks (2.5 and 6.9 kb) and the double-strand distances from the A(33-43) loop to the forks (17.2 and 52.7 kb) indicated that the F sequences between a,fil and IS2, were deleted from both pRD117 and pRD119 (data not shown). Nevertheless, the structures indicate that the portion of F DNA that would be present in the usual ( fR4A)i fragment (Fig. 5) should be found in the Hfr DNA of B 11. This segment of F DNA presumably appears in one of the intermediate-intensity, low molecular weight bands detected by hybridization with f,4 probe (Fig. 6A, Column 4). The shorter branch of the pRDl17 substitution loop is similar in size (8.0-8.2 kb) to the distance separating IS2, and c& in ORF203 (8.3-8.6 kb). Incomplete structures formed from F and a broken pRDl19 strand (data not shown) displayed a substitution loop having branches similar to those seen for pRDl17 but terminating in a short 1.3-kb duplex region bounded on the other side by another fork. We interpret this as an indication that an IS2 is present in the bacterial segment of pRD119, and the size of the

branch suggests (but does not prove) that the same 8.2-kb DNA sequence is present in both pRDl17 and pRDl19. This suggests that pRD117 and pRD119 both contain the bacterial DNA segment extending from IS2, to cy5p5(Fig. 5). The following information on F sequence organization in B 11 has been provided by the hybridization and heteroduplex experiments discussed above: (a) The characteristic bacterial EcoRI fragment containing CX$~is missing, (b) the ( f ,2A)iii and ( f i<2B)iii fragments predicted for integration at a&, appear to be present, (c) the ( f ,4B)i fragment predicted for integration of F at IS2, is apparently present, and (d) and f,4A fragment containing at least as much F DNA as appears on (fR4A)i probably is present, though not with the size expected from integration (i) in Fig. 5. The alteration of both f a2 and f,4 indicates that steps in addition to a single integration step must have occurred. The simplest hypothesis that we have been able to formulate postulates that Bll may have been formed by integration of an F’ plasmid like pRD117, which itself is an excision product derived from a previous Hfr strain. Since B 11appears not to have been formed by a single, one-step integration of F, B 11 must be excluded from statistical consideration of F integration in the IS2, CY& region. DISCUSSION

The present and previous results on F integration are summarized in Table 4 both for the chromosomal IS3 cy& and for the chromosomal a,/3,-IS2B set between lac and proC. When the probabilities (P values) for the observed distributions are calculated from the binomial (or trinomial) distribution with the hypothesis that (Y&, cy&, and ~1&,, when present (Hadley and Deonier, 1979), have equal probabilities of mediating F integration at the IS3 c&, values larger than 0.1 are obtained for F plasmids from W1485, W6F+, or W1655 and from the F insertion variant present in K-12- 112. There is thus no statistical basis for rejecting the

F INTEGRATION

61

AT IS2 AND IS3

TABLE 4 DISTRIBUTION OF INTEGRATIVE RECOMBINATION EVENTS AMONG IS2 AND Is3 ELEMENTSOF THE F PLASMID

Integrations at each site Chromosomal site

Source of F W6, W1655, or W1485 K-12-112 W6, W1655, K-12-112, or W1485

41

42

%P8

IS2

P for observed distribution”

6*

2’

-(I

-

0.109

1’ 0

0 0

2f w

5’

0.111 0.004

o Calculated from the binomial or trinomial distribution, assuming that the probabilities for integration at all IS2 and IS? elements are identical. * Hfr strains P4X (Deonier and Davidson, 1976), P804 (Deonier and Davidson, 1976), OR11 (Hadley and Deonier, 1979), B2 (this paper), B5 (Hadley and Deonier, 1979), and B6 (this paper). c Hfr strains B3 and B4 (this paper). ri Not available for recombination. e Hfr strain OR66 (Hadley and Deonier, 1979). ’ Hfr strains OR6 and OR72 (Hadley and Deonier, 1979). DBacterial IS2 element located between lac and proC. h Only available to OR56. Its presence would make integration at a& more probable than for cases involving normal F. The P value stated was calculated by ignoring the presence of q& in OR56, and thus it represents an upper bound. ’ Hfr strains Hfr 13 (Hu et al., 1975a), Hfr6-1 (this paper), OR21 (Deonier er al., 1977), OR56 (this paper), and B8 (this paper).

hypothesis that all IS3 elements in these F plasmids mediate integration of F with equal probabilities. In contrast, of five Hfr strains whose structures could be assigned unambiguously to integration events between lac andproC, all were formed by integration at IS2B. The c& element was not involved in F integration. This distribution is significantly different from what would be expected if IS2 and IS3 elements mediated F integration with equal probability (P = 0.004). Even if it is assumed that IS2 is twice as active as IS3, the P value (0.03) still indicates a significant bias toward IS2-IS2 recombination. This result agrees with the observations on F excision from the same IS2 site in a Type II F’ plasmid (ORF203) derived from one of the Hfr strains (OR21) included in this tabulation (Deonier and Mirels, 1977). In that case, 15/15 of the F excision events were mediated by IS2, whereas none of the events were mediated by either of the two other possibilities involving IS3 pairs.

The excision data from this previous study indicate that at 95% confidence (using the trinomial formula), IS2 is at least eight times as active as IS3 in mediating F excision. The present experiments indicate that in this region of the E. co/i chromosome, IS2-IS2 recombination frequencies are greater than IS3 -IS3 recombination frequencies in the integration process as well. Explanation of the preference for F integration and excision at IS2 must take into account the dependence on recA of both of these processes (Curtiss and Renshaw, quoted in Deonier and Mirels, 1977; Cullum and Broda, 1979; Deonier and Mirels, 1977). Heteroduplex data (Hu et al., 1975a; Deonier et al., 1977)indicate that the degree of homology of a& to either o& or a& is high and is similar to the degree of homology of IS2r to IS2a. Because there are no gross differences in the degree of homology, it may be that integration preference at IS2 is associated with specific sites or sequences present on IS2 but absent from IS3. This could mean that elements of both the gener-

62

DEONIER

AND HADLEY

alized recombination system of E. coli c& from the nearest clockwise bacterial (e.g., recA ) and a specialized recombination EcoRI site (Hadley and Deonier, 1979). system are operating concertedly in F Since there are no previously confirmed integration and excision. We know of no instances of integration at cu,/&,the sizes for precedent for “mixing” of generalized and the junction fragments in this case are estispecialized recombination pathways. How- mated from the positions of the EcoRI sites ever, because recA mutations are highly flanking (Y& and the revised values for pleiotropic, such “mixing” may be possible. coordinates of azP2determined in this study. An alternative explanation for enhanced If F were integrated at its CX$~IS3, the IS2 recombination in a recA-dependent f,4A fragment (Fig. 3) would contain bacprocess is the involvement of x sites similar terial DNA extending counterclockwise from to those found in mutants of bacteriophage a3p3 to the first EcoRI site (3.3 kb), an IS3 A (Stahl and Stahl, 1977). Because x sites element (1.3 kb), and the F DNA lying beare known to act at a distance and to stim- tween c-wZpz and the clockwise EcoRI site ulate recombination to a greater extent on terminating f,4 (3.6 kb). The size of the f,4A one side than on the other, it is possible fragment is thus calculated to be 8.2 kb. A that the elevated recombinational activity similar calculation using the counterclockat IS?, compared to a& is the result of a wise terminus of f,4 (8.9 F), the revised combination of polarity and positional coordinates for a&& (13.3 to 14.6 F), and the effects controlled by x. A x site located length of bacterial DNA extending to the within or very close to IS2* on the bacterial first EcoRI site clockwise of a& indicates DNA could selectively stimulate IS2-IS2 that for this case the size of f,4B should recombination for both F integration and be approximately 18.6 kb. excision. These two hypotheses can be tested by Sizes of Junction Fragments when F is studying excisional recombination in various Integrated at IS28 or c+,& mutants. If recombination at IS2 persists The junction fragments generated when F in recA+ recB- mutants, then a x site is probably not the basis for the elevated integrates via IS2, at the IS2B located berecombinational activity at IS2 (Stahl and tween fat andproC are typified by the (f,4A), and (f,4B)i junctions of ORF203 (Deonier Stahl, 1977). The recombinational activity at IS2 can also be checked in him mutants et al., 1977; Hadley and Deonier, 1979). The (Miller and Friedman, 1977), which affect a sizes of these fragments are 3.0 and 25.0 kb, respectively. number of specialized recombinational Because there is no previous documentaprocesses. tion of F integration at o,&,, the sizes of the junction fragments expected for such an ocAPPENDIX currence must be estimated. This requires Sizes of Junction Fragments When F is knowledge of the nearest EcoRI sites flankIntegrated at a& ing (Y&. The cy&, element is located on the The junction fragments generated by bacterial EcoRI fragment immediately clockintegration of F at asPavia its crlpl sequence wise of the fragment containing ISZ8, a conhave been determined experimentally to be clusion based on restriction analysis of F’ 6.0 and 24.1 kb for f,2A and f,2B, respec- plasmids that contain bacterial DNA spantively (e.g., junctions for pRH7, Fig. 4A). ning this region (Hadley and Deonier, unThis information, together with knowledge published results). The location of the EcoRI of the amount of F DNA on each fragment, cleavage site mapping between IS2B and indicated that 3.3 kb separates (Y& from the (~$3~is determined by subtracting from the nearest counterclockwise EcoRI site on the size of the (fR4A)i fragment (3.0 kb) the size bacterial DNA and that 12.8 kb separates of the F DNA present on (fR4A)i (15.9 to

F INTEGRATION

18.2 F), which places the EcoRI site 0.7 kb clockwise of IS2B. Since (Y& is 8.6 kb clockwise of IS2 (Hu et al., 1975a, b), the length of bacterial DNA from a& to the nearest counterclockwise EcoRI site can be calculated to be 7.9 kb (8.6 - 0.7 kb). The size of the fragment containing (Y& is 11.3 kb (Deonier et al., 1979); consequently, the distance of the first bacterial EcoRI site clockwise of a& can be calculated to be 2.1 kb (11.3 - 1.3 (c&) - 7.9 kb). These results, together with the coordinates of a& (13.3 to 14.6 F) and of the f,4 termini (8.9 and 18.2 F), allow size predictions of 7.0 kb for the (fR4A)ii junction fragment and 13.6 kb for the (fR4B)ii junction fragment resulting from F integration at cy&, via its (Y@~element. Similarly, the sizes of the junction fragments predicted for integration of F at (Ye& via the a$1 element are 4.9 kb for (fR2A)iii and 19.1 kb for (fR2B)iiiT based upon the above locations for the EcoRI sites immediately flanking a,&,, the map positions of alP1 of F (Davidson et al., 1975), and the positions of the EcoRI sites defining f,2 (83.2 and 1.4 F) (Guyer, 1978; Childs et al., 1977). ACKNOWLEDGMENTS We thank P. Broda and R. Curtiss III for sharing their Hfr strains with us, Mark Guyer for helpful comments on this work, and Karin Fouts for her expert technical assistance. This research was supported by Public Health Service Research Grant GM24589 from the National Institutes of Health, by Grant BMS7520512from the National Science Foundation, and by Grant 7216 from the Research Corporation.

AT IS2 AND IS3

63

W. M. (1977). Plasmid detection and sizing in single colony isolates. Science 195, 393-394. BERG, C. M., AND CURTISS, R., III (1967). Transposition derivatives of an Hfr strain of Escherichia co/i K-12. Grnefics 56, 503-525. BRODA, P. (1967). The formation of Hfr strains in Eschrrichia coli K-12. Gene;. Res. 9, 35-47. BRODA, P., BECKWITH, J. R., AND SCALFE,J. (1964). The characterization of a new type of F-prime in Escherichicr co/i K-12. Genet. Rex. 5, 489-494. BRODA, P., AND MEACOCK, P. (1971). Isolation and characterization of Hfr strains from a recombination deficient strain ofE.scherichiu co/i. Mol. Gen. Genet. 113, 166-173. BROKER,T. R., CHOW, L. T., AND SOLL, L. (1977). The E. co/i gamma-delta recombination sequence is flanked by inverted duplication. In “DNA Insertion Elements, Plasmids, and Episomes” (A. I. Bukhari, J. Shapiro, and S. Adhya, eds.), pp. 575-580. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. CHILDS,G. J., OH~SUBO,H., OHTSUBO,E., SONNENBERG,F., AND FRE~J~DLICH,M. (1977). Restriction endonuclease and mapping of the Escherichiu co/i chromosome in the vicinity of the i/v genes. J. Mol. Bid. 117, 175- 193. CLOWES,R. C., AND HAYES, W. (1968). “Experiments in Molecular Genetics.” Wiley, New York. COHEN, S. N., CHANG, A. C. Y., AND Hsu, L. (1972). Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichiu co/i by Rfactor DNA. Proc. Nrrr. Acad. Sci. USA 69, 2110-2114. CULLUM, J., AND BRODA, P. (1979). Chromosome transfer and Hfr formation by F in ret+ and recA strains ofEscherichia co/i K-12. Plasmid 2,358-365. CUR~ISS, R., III, ANI) STALLIONS, D. R. (1969). Probability of F integration and frequency of stable Hfr donors in F+ populations of Escherichicr co/i K-12. Genetics 63, 27-38. DANNA, K., AND NATHANS,D. (1971).Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influrnzae. Proc. Nut. Accrd. Sci. BARNES,

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