Chromosomal Element e14 - NCBI

Vol. 161, No. 3

JOURNAL OF BACTERIOLOGY, Mar. 1985, p. 1112-1117

0021-9193/85/031112-06$02.00/0 Copyright © 1985, American Society for Microbiology

Excision and Reintegration of the Escherichia coli K-12 Chromosomal Element e14 HOWARD BRODY, ALAN GREENER,t AND C. W. HILL* Department of Biological Chemistry, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033 Received 24 September 1984/Accepted 3 December 1984

The genetic element e14 is a natural component of the Escherichia coli K-12 chromosome. On induction of the SOS pathways, e14 excises as a 14.4-kilobase circle. We report here on the reintegration of e14 into the chromosome of cured (e14°) E. coli K-12 derivatives. Using a Tn1O insertion mutant of e14, we found that reintegration occurred specifically at the locus originally occupied by e14 and with the same orientation. The reintegration event required neither the RecA nor the RecB functions. The attachment site of the free form was located within a 950-base-pair HindIII-AvaI fragment and shared sufficient homology with the host attachment site to form detectable DNA-DNA hybrids. Even though E. coli C and B/5 did not contain e14, they did possess a HindIll restriction fragment that hybridized to the free e14 attachment fragment. E. coli C could be transformed with e14-1272::TnlO, resulting in integration at this site of homology. The TnlO mutants were also used in mapping the point of e14 attachment. We found the following sequence: fabD purB atft4 umuC. Furthermore, analysis of a recombinant plasmid that contained both the e14 attachment site and the purB locus showed that these two loci occur within 11 kilobases of each other.

selecting Tetr; the TnJO was then eliminated by cotransduction with metE. Other mutants used were as follows: JC8947, a umuC::TnS (el4°) derivative of AB1157 obtained from A. J. Clark; PC0254, a purB51 (el4°) mutant available as CGSC 5038; CH1418, an e14-1272::TnJO derivative of PC0254 prepared by P1 transduction; L48, afabD temperature-sensitive mutant available as CGSC 5638; CH1407, an e14-1272::TnJO derivative of L48 prepared by P1 transduction. CH440 has been described previously (10). DNA isolation and analysis. Plasmid DNA was prepared by the high salt-sodium dodecyl sulfate procedure described by Maniatis et al. (14). Genomic DNA was prepared as described by Hartl et al. (7). Restriction endonuclease digestions and DNA ligation with T4 DNA ligase were done as specified by the enzyme manufacturers. Separation of DNA fragments involved the use of 0.75% horizontal agarose gels in 0.04 M Tris acetate (pH 8.0)-0.002 M EDTA, with 0.5 jig of ethidium bromide per ml. Southern transfer (16) to diazotized paper was done as described by Hill and Harnish (9). Hybridization probes were prepared by nick translation with [ot-32P]dCTP (Amersham Corp.) as described by Maniatis et al. (14). Size standards used were restriction fragments of lambda DNA or pBR325. Induction and DNA extraction of e14 variants. A culture of the recA441 (e14) strain was grown overnight at 32°C. This culture was diluted 1:10ANnto L broth previously warmed to 42°C, and incubation was continued for 4 h. Small circular DNA was extracted by the boiling procedure of Holmes and Quigley (11). Recombinant plasmids. Recombinant plasmids are shown in Fig. la. The isolation of pAG2 was described previously (6). pHB10, which contained all of e14, was prepared by using standard techniques (14) to digest e14 with EcoRI and ligating the product into the EcoRI site of pBR325 (2). The e14 used was a gift of Mike Capage (4). The ligated preparation was used to produce Tetr transformants of CH734, and a transformant was streaked once for single-colony isolation and used to prepare a liquid culture. This culture was adjusted to 8% dimethyl sulfoxide and frozen at -70°C.

We have described a cryptic episome like element, named e14, present in the Escherichia coli K-12 chromosome (6). This element was observed as a 14.4 kilobase (kb) DNA circle which was excised on induction of the SOS pathway by any one of several stimuli. Stimuli used for induction have included UV light, thermal shift of a recA441 (tif-1) mutant (6), and transformation with certain P1 miniplasmids (4). The element is not essential to E. coli, since naturally cured derivatives of K-12 such as Ymel are observed. In addition, strains possessing e14 have been cured by screening survivors of SOS induction (6). The curing caused no change in growth rate or growth requirements. The element is not present in either E. coli C or E. coli B/5 (6). Recently, van de Putte et al. (17) have found that E. coli cells harboring e14 can complement phage Mu gin mutants. In addition, this gin complementing function, called pin, can cause the inversion of an internal portion of e14. The induced excision of e14 strongly implied that it is an episome or defective episome which codes for a repressible, site-specific recombination system. We report here on the further characterization of both the excision and the reintegration of e14. MATERIALS AND METHODS Bacterial strains. Some of the mutants used were derivatives of E. coli K-12 strain CH734, and their relevant genotypes are listed in Table 1. The symbols (e14+) and (e14°) refer to bacterial strains with and without e14 integrated into the host chromosome, respectively. TnJO insertion mutants were prepared by infection with XNK55 (12). P1 transduction (8) and transformation (3) procedures have been described. recA56 was introduced by cotransduction with srl-300::TnJO followed by elimination of TnJO as described by Maloy and Nunn (13). CH1493 (polAl) was prepared by transducing CH1332, with CH1274 (metE zig-1274::TnlO polAJ) as donor * Corresponding author. t Present address: Department of Biology, University of California, San Diego, La Jolla, CA 92093.

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EXCISION AND REINTEGRATION OF e14

VOL. 161, 1985 TABLE 1. Bacterial strainsa Strain

CH734

CH826 CH1035

CH1246 CH1332 CH1333 CH1371 CH1480 CH1491

CH1492 CH1493

CH1494

Source or reference Genotype trpA36 lysA glySH xy14 6 i1vD130 argHI tsx (e14+) 6 CH734 Lys' recA441 (e14+) 6 CH734 Lys' recA441 (el4°) P1 transduction of CH826 CH734 Lys' recA441 umuC::TnS purBSJ (el4°) UV-induced curing of CH734 CH734 (el4°) CH734 recAS6 (el4°) P1 transduction of CH1332 See text CH734 Lys' recA441 (eI4-1272::TnlO) CH734 recA56 (e14+) P1 transduction of CH734 CH734 Lys+ recB21 Hfr conjugation with CH734 (e14+) CH734 Lys' recB21 Hfr conjugation with CH1332 (el4°) CH734 polAl (el4°) See text CH734 (e14-1272::TnlO) P1 transduction

a All strains were F- and were derived from CH734 in one or more steps as indicated.

This frozen stock was then used as an inoculum for all experiments requiring pHB10. Special care was essential since pHB10 transformed cells grew very poorly, and more rapidly growing variants were found to have rearranged plasmids. Generally, a crystal from the frozen stock was thawed and streaked on plates containing 10 ,ug of tetracycline per ml, and, while still very small, colonies were used to inoculate several small-volume liquid cultures. Those cultures which showed slow growth were pooled to inoculate a larger culture, from which pHB10 was extracted. pHB101 and pHB108 (Fig. la) were subcloned from pHB10 by standard techniques (14). RESULTS Cloning and subcloning e14. Previously published work by Greener and Hill involved the use of a recombinant plasmid, pAG2, which carried an 11.1-kb HindIII fragment of e14 as its insert (6). More thorough characterization of e14 required cloning of the entire element. This was accomplished by digesting a preparation of e14 circular DNA with EcoRI and ligating the linear DNA into the EcoRI site of pBR325 to produce the recombinant plasnmid pHB10 (see above). The pHB10 plasmid was shown to contain the entire e14 sequence, since on digestion with EcoRI a 14.4-kb fragment was produced which strongly cross-hybridized with pAG2. The restriction maps of pHB10 and subclones derived from it are shown in Fig. la. Cells carrying pHB10 grew very slowly, and so maintenance of this plasmid was a significant problem (see above). e14 excision site. To determine which segment of free e14 contained the novel joint created on excision, we took advantage of the simple expectation that each restriction fragment of free e14 should be present in integrated e14 except for the fragment containing the novel joint. To identify this fragment, two restriction fragments whose sum made up the entire free e14 were used to probe restriction endonuclease-digested DNA prepared from E. coli K-12. With the 11.1-kb HindIlI insert from pAG2 as hybridization probe to HindIII digests of genomic and free e14, Southern analysis revealed a fragment in both preparations identical in

1113

size to the probe, indicating that the novel joint was not contained within this interval. In contrast, when the 3.4-kb HindIII insert from pHB101 was used to probe HindIII-AvaI digests of genomic and free e14 DNA, a 2.5-kb HindIII-AvaI fragment was present in preparations of free and genomic e14, but a 0.95-kb HindIII-AvaI fragment present in free e14 was absent in the genomic digest. Instead, two larger fragments hybridized. This showed that the ends of the integrated e14 were joined in the 0.95-kb HindIII-AvaI interval on excision of the element. This was verified by using the 0.95-kb HindIII-AvaI fragment (the pHB108 insert) to probe a HindIII-AvaI genomic digest (Fig. 2, lane 2). In this lane, the 0.95-kb fragment (seen in the digest of pHB10; Fig. 2, lane 1) is absent, but two larger fragments (4.6 and 8.5 kb) are present. These contain the ends of the integrated e14. Since they hybridized to the probe with similar intensities, the crossover point must have been near the middle of the 0.95-kb HindIII-AvaI interval. Various E. coli K-12 strains lacking e14 were analyzed by Southern hybridization to see whether homology between e14 and the host exists. A 12-kb HindIII-AvaI band from a cured mutant, CH1332 (e140), hybridized to the 0.95-kb probe (Fig. 2, lane 3). This fragment was not present if e14 was integrated (Fig. 2, lane 2), showing that it contained the site of e14 integration into the chromosome. An important inference from this result was that e14 and its chromosomal attachment site shared a region of significant homology. This chromosomal attachment site was present on a 20-kb fragment if Hindlll alone was used to digest genomic DNA. E. coli B/5 (Fig. 2, lane 7) and E. coli C (Fig. 2, lane 8) possessed an apparently identical 20-kb HindIII interval that exhibited weak homology with the 0.95 kb HindIII-AvaI probe. A second region on the E. coli K-12 chromosome showed slight homology with e14. The 11.1-kb HindlIl insert of pAG2 hybridized weakly with a 5-kb HindIII fragment in digests of either cured or e14-containing E. coli K-12 cells. Thus e14 shared limited homology with some other portion of the E. coli K-12 chromosome remote from its site of integration. Isolation of TnlO insertions in e14. Studies of e14 have been hampered by the lack of a readily selectable phenotype. Therefore, we prepared a set of TnJO insertions into the element so that tetracycline resistance could be used to indicate the presence or absence of e14. To do this, phage P1 was grown on a pool of W3102 derivatives with TnJO inserted at a variety of sites. The lysate was used to infect a purB recA441 mutant (CH1246), selecting for Tetr and scoring for Pur+. Integrated e14 is linked to purB (6). Of 33 letr Pur+ recA441 transductants induced by thermal elevation, three exhibited the excision of an enlarged e14. This is shown for one example in Fig. 3, lane 4. The positions of these three TnJO insertions are shown in Fig. lb. Site-specific reintegration. The availability of an e14 derivative with a selectable phenotype enabled us to test whether e14 would reintegrate at the same site from which it had excised. A K-12 strain cured of e14 (CH1332) was transformed with an extract from thermally induced CH1371, a recA441 e14-1272::TnJO strain. Tetr transformants were selected. Genomic DNA was extracted from ten of these transformants, digested with HindIll, and subjected to South. ern analysis. When probed with the 0.95-kb HindIII-AvaI fragment, all ten gave patterns indistinguishable from those of the original K-12 (el4+) strain. An example of one of these is shown in Fig. 2, lane 6. Thus el4-1272::TnJO had reintegrated at the same site from which it was excised.

1114

BRODY, GREENER, AND HILL

J. BACTERIOL.

a

b pulrB

U/BC /279

D

S

SA

-0

-5

AS

/290

/272 AR

D

0

a

5

8

10

BA

\D

'IS

D

20

e 14

pH106

pHB107-I

FIG. 1. Restriction maps of free and integrated e14. The map of free e14 is shown in (a) along with various cloned fragments. The map of integrated e14 along with the flanking chromosomal regions is shown in (b). The fragment cloned in pHB106 extends from an AvaI site in e14 to the AvaI site in the flanking chromosome. The fragment cloned in pHB107 extends from this latter AvaI to the HindIII site on the other side of the point of e14 integration but does not contain any e14 DNA. pHB107 complements a purB51 mutant, but pHB106 does not. All recombinant plasmids use pBR325 as vector except pAG2, which used pBR313. The sites of three TnlO insertions into e14 are also shown in (b). The restriction sites are designated as follows: A, AvaI; B, BamHI; D, HindIII; S, Sall; R, EcoRI. Additional EcoRI sites occur in the flanking chromosomal region but are not mapped.

To determine whether e14 could integrate into the E. coli C genome, el4-1272::TnJO was used to transform an E. coli C recipient. Tetr transformanis were obtained, and Southern analysis showed that the integration was into the 20-kb HindIll interval. An example is shown in Fig. 2, lane 9. Thus the sequence in E. coli C which had been shown to be homologous to e14 could serve as a site for integration of the element. RecA independence of e14 integration. Site-specific recombination generally does not require the RecA protein. However, in view of the significant homology between the 0.95-kb HindIII-AvaI fragment of e14 and the chromosomal attachment site, it was important to determine whether e14 could integrate in a recA mutant. Isogenic recA56 (e140) and recA56 (e140) mutants were compared with recA'+ (el4°) strains for their ability to serve as recipients for transformation by el4-1272::TnJO. The recA56 (e14) strain could -be transformed as efficiently as the recA+ (e14°) strain, showing that e14 integration does not require the RecA protein (Table 2). Furthermore, Southern analysis of DNA extracts showed that the reintegration in the recA56 mutant had occurred at the normal site (Fig. 2, lane 10). If the recA56 recipient already carried e14, transformation was severely depressed.

We have also found that e14 can transform a recB21 (el4°) strain as efficiently as a Rec+ (el4°) strain, demonstrating lack of dependence on the RecB function as well. This integration also occUrred at the normal site (Fig. 2, lane 11). Map position of e14. The isolation of TnWO insertions into e14 facilitated the mapping of the element. Previous work had shown that e14 is closely linked to purB (6). To place it relative to other purB linked markers, we carried out a series of P1 transductions between mutants with markers in e14, purB, fabD, and umuC. The crosses performed and the results obtained are described in Table 3. The order deduced was fabD purB e14 umuC. Note that two of the strains used in these crosses, PC0254 and JC8947, were cured of e14. The presence or absence of e14 in the donor greatly affected the cotransduction frequency observed (Table 4). e141272: :TnJO was eliminated from the recipient by cotransduction with purB' only 25% of the time if the donor carried e14 but 85% if the donor was cured of e14. The latter percentage is probably more useful in calculating the map distance separating purB and the attel4 site (see below). The relationship between e14 and the purB gene was further refined by complementing a purBSJ mutant with a plasmid that contained DNA from the regions flanking the


VOL. 161, 1985

1 23 12-

8.5

4

5

6

7

8 9

1115

10 11

-

No

t

*

-

4.6-

20 14 10

;w

0.955 FIG. 2. Digests of E. coli genomic DNA probed with the 0.95-kb HindIll-Aval insert from pHB108. A combination of HindllI and AvaI used to digest samples in lanes 1 through 3, whereas only HindIII was used for samples in lanes 4 through 11. Conditions for agarose gel electrophoresis, Southern transfer, and hybridization were as specified in the text. Lane 1, control digest of pHB1O; lanes 2 and 4, CH734 (e14+); lanes 3 and 5, CH1332 (el4°); lane 6, CH1332 transformed with el4-1272::TnIO; lane 7, E. coli B/5; lane 8, E. coli C; lane 9, E. coli C transformed with el4-1272::TnJO; lane 10, CH1333 (recA56) transformed with el4-1272::TnlO; lane 11, CH1492 (recB21) transformed with el4-1272: :TnlO).

was

point of e14 attachment. In this connection, we prepared two plasmids, pHB106 and pHB107, by the strategy shown in Fig. 4. In the first step, pHB105 containing the e14 attachment region (a 1.3-kb fragment subcloned from a IindlIISall digest of pHB10) was used to transform a polAl (el4°) strain (CH1493). pBR325-derived plasmids cannot transform a polAl recipient unless they integrate into the host chromosome (6). In this case, we anticipated that pHB105 would integrate by a recombination between the cl}ped e14 attach-

12

3 4

_. -el4::TnlO chomosomal

ment site and the chromosomal attachment site, since the sites seemed to share significant homology. Transformants were obtained, and the fact that they had integrated at the chromosomal attachment site was verified by Southern analysis of genomic DNA isolated from the transformant. Digestion of the genomic DNA with AvaI followed by ligation to produce a DNA circle resulted in pHB106. pHB106 contained 4.5 kb of chromosomal DNA to one flank of integrated e14 as well as a small segment of e14 (Fig. 4). In the next step, pHB106 was in turn forced to integrate into the polAl (e140) recipient, and on HindIII digestion and ligation of this genomic DNA, pHB107 was obtained. pHB107 contained the same 4.5 kb of chromosomal DNA that pHB106 had, as well as an additional 11 kb to the other side of the chromosomal e14 attachment site. We found that pHB107, but not pHB1O6, could complement a purBSJ mutant. Thus the restriction map of the e14 region is oriented with respect to purB as indicated in Fig. lb. DISCUSSION We have shown that the element e14 reintegrates into the chromosome of an e140 recipient on transformation and that TABLE 2. RecA independence of e14 integration

-e14

No. of transformants'

Recipient

FIG. 3. Thermally induced e14 excision. All strains contained the recA441 allele. Thermal induction and extraction of plasmid DNA was done as described in the text. After Southern transfer, the papers were probed with the pAG2 insert. The positions of e14 monomeric supercoiled DNA is indicated. Lane 1, CH826 (e14+) uninduced; lane 2, CHi826 (e14+) induced; lane 3, CH1371 (e141272::TnlO) uninduced; lane 4, CH1371 (el4-1272::TnlO) induced.

Relevant type geno-

from: trial 1

trial 2 CH1332 32 recA+ (el4°) 40 CH1333 17 recAS6 (el4°) 52 CH1480 recAS6 (e14+) 0 0 The recipients were transformed with a preparation of e14-1272::TnlO as described in the text. Each of the three recipients was transformed with equivalent amounts of the el4-1272::TnIO extracts. Different extracts were used in the two trials, and we estimate that less than 1 ng of DNA was added to each transformation preparation. In each trial, the transformation efficiency of each of the three recipients by pBR325 was the same.

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BRODY, GREENER, AND HILL TABLE 3.

Recipient

Donor

PC0254

CH1407

MappingfiabD, purB, el4,

J. BACTERIOL. and umuC No. observed (%)

Donor markers"

Selection

fabD purB+ el4::TnlO

PC0254

CH1407

140 (45) 167 (53) 6 (2) 0 (0) 114 (44) 128 (49) 12 (5) 5 (2)

Tetr

Pur+

purB+ e14" urnuC::TnS

CH1418

JC8947

Kanr

CH1418

JC8947

Pur

96 (48) 6 (3) 98 (49) 0 (0) 15 (15) 54 (54) 31 (31) 0 (0)

_

'3ar under the loqus designation indicates that the donor allele was present in the transdtictant class.

the crossover occurs between the 0.95-kb HindIII-AvaI interval of e14 and a 20-kb HindIlI interval of the recipient chromosome. Furthermore, the reintegration produced the identical configuration to that of the original e14 in E. coli K-12 (Fig. 2, compare lanes 4 and 6). The attachment site on the chromosome showed significant homology to the attachment site on e14 (Fig. 2, lane 3). Nevertheless, the integration was independent of the RecA and RecB functions. It seems clear, therefore, that a site-specific recombination system was used. Van de Putte et al. (17), in their studies of the Pin system, found that a recombinant plasmid containing

TABLE 4. Cotransduction of e14 markers with purB+a Cotransduction Cross RcpetDnr frequency (%) Donor no. Recipient 51 1 purB+ (el4::TnJO) purB- (e140) 25 purB+ (e14+) purB- (el4::TnJO) 2 85 purB+ (el4°) purB- (el4::TnJO) 3 a Recipients and donors in the crosses were as follows: cross 1, CH1407; cross 2, CH1418 x CH440; cross 3, CH1418 x JC8947.

e14 could integrate near purB in a polA pin mutant. From this, they concluded that e14 integrated at a specific site. E. coli C and B/S do not possess e14 (6). However, they do have a 20-kb HindlIl interval that appears identical to the segment of K-12 (e140) bearing the attel4 site. We found that el4-1272::TnJO can stably integrate at this site in E. coli C. The implication is that the site specific recombination system and its control operates in E. coli C. A key recombinant plasmid in this work was pHB10, which contained e14 opened at its unique EcoRI site and ligated into the EcoRI site of pBR325. This plasmid proved exceedingly difficult to work with since it retarded growth of the host culture, and cells with altered plasmids overgrew the cultures unless great care was taken (see above). We do not understand the reason for this deleterious effect, but it was exhibited regardless of whether the host carried a chromosomal copy of e14. Because of this problem, we were concerned that pHB10 might not contain a faithful copy of e14. However, extensive comparisons of the restriction maps of pHB10, pAG2, and integrated e14 have revealed no discrepancies. None of the other subclones of e14 described in Fig. 1 had similar detrimental effects. The results of our mapping placed the site of integration of e14 in the sequence offabD purB attel4 umuC (Table 3). The fact that e140 in the donor was cotransducible with purB at a B

A

pHB106

pHBIOS D

(A D

DS

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I

DS II

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BA

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pur8

purS D S

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PC0254 x

S ASA

LOW1 JA

D

BA

L 1i Amp

IHB1I0

PHB106

DS

II

I.

D

BA

'E1_ Amp

pHB107

FIG. 4. Scheme for cloning the chromosomal regions flanking the point of e14 attachment. (A) A polAl (el4°) strain was transformed with pHB105, Since pHB105 cannot independently replicate in the polAl background, it was forced to integrate into the recipient chromosome. Judging from Southern analysis, this must have occurred by recombination between the respective attachment sites. The attachment sites are shown schematically as a filled box, vector DNA as the open bar, e14 DNA as the hatched bar, and chromosomal DNA as the thin line. Genomic DNA was extracted, digested with AvaI and ligated to produce pHB106, whose insert included DNA to one side of the attachment site. (B) pHB106 was in turn forced to integrate into the chromosome of Et polAl (e14°) recipient. On cutting the DNA with HindIII and ligating it, pHB107 was created. pHB107 included DNA to the other side of the chromosomal attachment site.

VOL. 161, 1985

level of 85% (Table 4) suggested that atte14 lay within 0.1 min of purB (18), and the fact that pHB107 could complement a purBSJ mutant placed the loci within 11 kb of each other. Greener and Hill (6) showed that e14 was linked to purB, since the Ampr gene of pAG2, forced to integrate into a polAl (el4+) mutant, could be cotransduced with purB at a frequency of 30 to 40%. In addition, they found that Ampr and pyrC could be cotransduced at a very low level (about 1%). Since we could not cotransduce purB and pyrC, we concluded that attel4 must be counter-clockwise from purB instead of clockwise as shown unequivocally by the threefactor crosses described in Table 3. In retrospect, some artifact must have affected our test for purB pyrC cotransduction. Bachmann (1) and Semple and Silbert (15) have commented on difficulties in cotransducing these markers. Finally, our mapping results are in general agreement with the findings of Enomoto et al. (5), who mapped the locus for Pin activity as being clockwise from purB. Pin activity is now understood to be provided by e14 (17). ACKNOWLEDGMENTS We thank R.-J. Lin for providing the pool of TnJO insertion

mutants and for preparing the zig-1274::TnlO mutant CH1274, M. Capage for a sample of e14, and B. Bachmann and A. J. Clark for providing mutant strains. We also thank Tim Torchia for insightful discussions and A. Hopper for critically reading the manuscript. This work was supported by Public Health Service grant no. GM16329 from the National Institutes of Health. LITERATURE CITED 1. Bachmann, B. J. 1983. Linkage map of Escherichia coli K-12, edition 7. Microbiol. Rev. 47:180-230. 2. Bolivar, F. 1978. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique EcoRI sites for selection of EcoRI generated recombinant DNA molecules. Gene 4:121-136. 3. Capage, M., and C. W. Hill. 1979. Preferential unequal recom-

bination in the glyS region of the Escherichia coli chromosome.

J. Mol. Biol. 127:73-87. 4. Capage, M., and J. R. Scott. 1983. SOS induction by P1 Km miniplasmids. J. Bacteriol. 155:473-480.


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5. Enomoto, M., K. Oosawa, and H. Momota. 1983. Mapping of the pin locus coding for a site-specific recombinase that causes flagellar-phase variation in Escherichia coli K-12. J. Bacteriol.

156:663-668. 6. Greener, A., and C. W. Hill. 1980. Identification of a novel genetic element in Escherichia coli K-12. J. Bacteriol. 144: 312-321. 7. Hartl, D. L., D. E. Dykhuizen, R. D. Miller, L. Green, and J. de Framond. 1983. Transposable element IS50 improves growth rate of E. coli cells without transposition. Cell 35:503-510. 8. Hill, C. W., J. Foulds, L. Soil, and P. Berg. 1969. Instability of a missense suppressor resulting from a duplication of genetic material. J. Mol. Biol. 39:563-581. 9. Hill, C. W., and B. W. Harnish. 1981. Inversions between ribosomal RNA genes of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 78:7069-7072. 10. Hill, C. W., C. Squires, and J. Carbon. 1970. Glycine transfer RNA of Escherichia coli. 1. Structural genes for two glycine tRNA species. J. Mol. Biol. 52:557-569. 11. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193-197. 12. Kleckner, N., D. F. Barker, D. G. Ross, and D. Botstein. 1978. Properties of the translocatable tetracycline-resistant element TnOO in Escherichia coli and the bacteriophage lambda. Genetics 90:427-461. 13. Maloy, S. R., and W. D. Nunn. 1981. Selection for loss of tetracycline resistance by Escherichia coli. J. Bacteriol. 145: 1110-1112. 14. Maniatis, T., E. Fritsch, and J. Sambrook. 1982. Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Semple, K. S., and D. F. Silbert. 1975. Mapping of the fabD locus for fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 121:1036-1046. 16. Southern, E. M. 1975. Detecting specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 17. van de Putte, P., R. Plasterk, and A. Kuijpers. 1984. A Mu gin complementing function and an invertible DNA region in Escherichia coli K-12 are situated on the genetic element e14. J. Bacteriol. 158:517-522. 18. Wu, T. T. 1966. A model for three-point analysis of random general transduction. Genetics 54:405-410.