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PLASMID 12, 149-160(1984) A Specialized Host-Vector System for the in Vivo Cloning of the trp Operon of Wild-Type and Mutant Strains of Salmonella typhimurium by Generalized Transduction THOMAS PATTERSON’ AND RONALD BAUERLE Department of Biology, University of Virginia, Charlottesville, Virginia 22903 Received February 23, 1984; revised August 8, 1984 Using in vitro methods a 14.2-kbEcoRl fragment of the Salmonella typhimurium chromosome containing the trp operon plus associated flanking sequences from deletion mutant AtrpDCB763 was cloned into the EcoRI site of plasmid pBR322 in a S. typhimurium host. An in vivo cloning vector was constructed from the recombinant plasmid by the in vitro excision of a Sal1 fragment that contains the entire trp operon. The derived plasmid (pSTP21) carries a hybrid insert made up of the 5.4-kb EcoRI-Sail upstream Banking sequence and the 3.2-kb SalI- EcoRI downstream flanking sequence. Plasmid pSTP21 has been used as a receptor plasmid to clone a variety of mutant and wild-type trp operons by RecAdependent in vivo recombination between the insert DNA of the plasmid and the homologous trp flanking sequences of transducing DNA fragments transferred into the cell by bacteriophage P22. The host-vector system developed for the in vivo cloning permits the dilferentiation of plasmid transductants from chromosomal transductants on the primary selective medium. Expression of the cloned trp operons is regulated normally by tryptophan. A substantial amplification of trp enzymes is attainable upon derepression. The recombinant plasmids are stably inherited in RecA+ and RecA- S. typhimurium hosts. However, conditions of high expression of the trp operon lead to a rapid loss of cehular viability and of plasmid stability. Q 1984 Academic Press. Inc. The correlation of the nature, location, and consequences of mutational changes in regulatory and structural genes continues to be an effective approach in the study of structure-function relationships in macro- molecules. In order to facilitate such an analysis of mutant genes of the trp operon of Salmonella typhimurium. we have devel- oped a S. typhimuriumlpBR322 host-vector system that permits the molecular cloning of essentially any mutant or wild type trp operon by in vivo recombination. The method differs from other in vivo cloning strategiesthat have been described (e.g., Stauffer et al., 1979; Horwitz et al., 1980) in that the formation of the desired recombinant plasmids is achieved by means of a simple transductional cross. Lysates of bacteriophage P22, prepared on the strains whose trp operons are to be cloned, are used to infect a recipient strain ’ Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 11724. carrying a specialized plasmid cloning vector (a derivative of pBR322 constructed by in vitro methods). This plasmid serves as a receptor molecule for the recombinational insertion of a defined segment of transducing DNA that always includes the trp operon. The resulting recombinant plasmids are structurally identical except for the muta- tional differences in the trp insert sequence. The development and characterization of the cloning system and preliminary studies of the expression of cloned trp genes in S. typhimurium hosts are described in this re- port. MATERIALS AND METHODS Bacteria, phage, and plasmids. The S. typhimurium strains used are listed in Table 1. Except for the transfer of plasmids by transformation, strains were constructed by transduction using bacteriophage P22 int-4 (Smith and Levine, 1967) or P22 HT105 int- 149 0147-619X/84 $3.00 Copyright 8 1984 by Academic Press. Inc. All rigbls of reproduction in any form rcxrval.
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PLASMID 12, 149-160(1984)

A Specialized Host-Vector System for the in Vivo Cloning of the trp Operon of Wild-Type and Mutant Strains of Salmonella typhimurium

by Generalized Transduction

THOMAS PATTERSON’ AND RONALD BAUERLE

Department of Biology, University of Virginia, Charlottesville, Virginia 22903

Received February 23, 1984; revised August 8, 1984

Using in vitro methods a 14.2-kb EcoRl fragment of the Salmonella typhimurium chromosome containing the trp operon plus associated flanking sequences from deletion mutant AtrpDCB763 was cloned into the EcoRI site of plasmid pBR322 in a S. typhimurium host. An in vivo cloning vector was constructed from the recombinant plasmid by the in vitro excision of a Sal1 fragment that contains the entire trp operon. The derived plasmid (pSTP21) carries a hybrid insert made up of the 5.4-kb EcoRI-Sail upstream Banking sequence and the 3.2-kb SalI- EcoRI downstream flanking sequence. Plasmid pSTP21 has been used as a receptor plasmid to clone a variety of mutant and wild-type trp operons by RecAdependent in vivo recombination between the insert DNA of the plasmid and the homologous trp flanking sequences of transducing DNA fragments transferred into the cell by bacteriophage P22. The host-vector system developed for the in vivo cloning permits the dilferentiation of plasmid transductants from chromosomal transductants on the primary selective medium. Expression of the cloned trp operons is regulated normally by tryptophan. A substantial amplification of trp enzymes is attainable upon derepression. The recombinant plasmids are stably inherited in RecA+ and RecA- S. typhimurium hosts. However, conditions of high expression of the trp operon lead to a rapid loss of cehular viability and of plasmid stability. Q 1984 Academic Press. Inc.

The correlation of the nature, location, and consequences of mutational changes in regulatory and structural genes continues to be an effective approach in the study of structure-function relationships in macro- molecules. In order to facilitate such an analysis of mutant genes of the trp operon of Salmonella typhimurium. we have devel- oped a S. typhimuriumlpBR322 host-vector system that permits the molecular cloning of essentially any mutant or wild type trp operon by in vivo recombination. The method differs from other in vivo cloning strategies that have been described (e.g., Stauffer et al., 1979; Horwitz et al., 1980) in that the formation of the desired recombinant plasmids is achieved by means of a simple transductional cross. Lysates of bacteriophage P22, prepared on the strains whose trp operons are to be cloned, are used to infect a recipient strain

’ Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 11724.

carrying a specialized plasmid cloning vector (a derivative of pBR322 constructed by in vitro methods). This plasmid serves as a receptor molecule for the recombinational insertion of a defined segment of transducing DNA that always includes the trp operon. The resulting recombinant plasmids are structurally identical except for the muta- tional differences in the trp insert sequence. The development and characterization of the cloning system and preliminary studies of the expression of cloned trp genes in S. typhimurium hosts are described in this re- port.

MATERIALS AND METHODS

Bacteria, phage, and plasmids. The S. typhimurium strains used are listed in Table 1. Except for the transfer of plasmids by transformation, strains were constructed by transduction using bacteriophage P22 int-4 (Smith and Levine, 1967) or P22 HT105 int-

149 0147-619X/84 $3.00 Copyright 8 1984 by Academic Press. Inc. All rigbls of reproduction in any form rcxrval.

150 PATTERSON AND BAUERLE

TABLE I

BACTERIAL STRAINS USED

Genotype

Strains” TB20

l-B50 TB1409

TB1678

TB1686

TB1690

TB1700

TB1719

TB1743

Strains with plasmids b TB1207 TB1209 TB1210 TB1844 TB1862 TB1864 TBl865 TBl934 TB1935 TB1937

A(trpPLEDCBA-chr-

OPPVOl AtrpDCB763 AtrpPLEDCBAI67 ara-9

recA 1 AtrpE1676 AmetA

metE55 I ilv-452 xyl- 404 rpsLl2OfIaA66 hsolT6 hsdSA29 recA1

AtrpE1676 AtrpAlO9 ara- 9 recA1

AtrpED1679 AtrpA109 ara-9 recA1

AtrpE1676 pyrF146 ara-9 rec.4 1

AtrpE1676 cysB517 ara-9 recA 1

A(cysB-top-trpPLQ22 ara-9 leuP500

TB1409/pBR322 TB1678/pSTPI TB1678/pSTPI I TB 1690/pSTPl TB1686/pSTPOI TBZOJpBR322 TBZO/pSTPI TB 1686/pSTP2 I TB1686JpSTP26 TB20/pSTP2 1

’ Strain TB20 is from the original Demerec collection. Strains TB50 and TB1743 were isolated in previous work in the laboratory of Paul Margolin. All other strains were isolated or constructed in this study.

b The origins and physical maps of the various plasmids are presented in Fig. 2.

101, an integration-deficient derivative of a high-frequency transducing mutant (Schmie- ger, 1972), kindly supplied by J. Roth (Uni- versity of Utah). Plasmid pBR322 (Bolivar et al., 1977) was isolated from Escherichia coli strain CR63 and was transformed into a restrictionless strain of S. typhimurium (strain CL4419, kindly supplied by C. Colson,) with selection for T$. S. typhimurium-mod&d plasmid was isolated from this strain and

transformed into S. typhimurium strain TB1409 with selection for TcR. The derived strain (TB1207) was used as the source of pBR322 DNA for all subsequent work.

Media, Chemicals, and Enzymes. Com- plete medium was either NZY or LB broth and agar (Maniatis et al., 1982). Minimal salts medium and supplements were as pre- viously described (Wuesthoff and Bauerle, 1970). Enriched minimal agar (EMA) con- tained 1% (v/v) Difco nutrient broth; casa- mino acids agar (CAA) was minimal agar supplemented with 0.1% Difco casamino ac- ids. Tetracycline (Calbiochem-Behring) was used at 25 pg/ml in complete media and at 10 pg/ml in minimal media and in selective media for plasmid transformation. Ampicillin (Sigma) was used at 50 pg/ml in complete media and 25 &ml in minimal media. Restriction endonucleases and TCDNA ligase were from Bethesda Research Laboratories.

Transductions and transformations. Phage lysates were prepared and transductions per- formed essentially as described previously (Wuesthoff and Bauerle, 1970). Transforma- tion of S. typhimurium with plasmid DNA was according to the method of Lederberg and Cohen ( 1974).

Isolation and characterization of DNA. Chromosomal DNA was isolated from S. typhimurium strain TB50 by the procedure of Saito and Muira (1963). Plasmid pBR322 DNA was isolated from chloramphenicol- amplified (250 &ml) cultures of strain TB 1207 grown in MM + CA + Trp at 37°C by the procedure of Clewell and Helinski ( 1969). Plasmid pSTP1 DNA was isolated from strain TB 1862 grown in LB + Tc + Ap by the method of Holmes and Quigley (198 1). Plasmid DNA was purified by CsCl-ethidium bromide density gradient centrifugation. Small scale plasmid preparations were by the method of Holmes and Quigley (1981). Digestions with restriction endonucleases and ligations were carried out according to the manufacturer’s specifications. DNA prepa- rations were analyzed by electrophoresis in horizontal agarose slab gels cast and run in TE buffer (Maniatis et al., 1982).

IN VW0 CLONING OF Salmonella trp GENES 151

Determination of trp enzyme activities. The preparation and assay of celhilar extracts for trp enzyme activities were as described (Ta- nemura and Bauerle, 1977). Cultures were grown in MM + CA at 37°C; tryptophan and other supplements were supplied as in- dicated.

RESULTS

Construction of Recombinant Plasmids Carrying the trp Region of S. typhimurium

Chimeric derivatives of plasmid pBR322 with inserts of the trp region of the S. typhi- murium chromosome were constructed by digesting bulk chromosomal DNA from strain TB50 with restriction endonuclease EcoRI and ligating it with EcoRI-digested pBR322 plasmid DNA purified from S. typhimurium strain TB1207. It is known that there is a single EcoRI site within the trp operon, early in the trpB gene (Crawford et al., 1980) and that the closest downstream site is around 5 kb removed (Selker et al., 1977). Strain TB50 was chosen as the source of chromosomal DNA since it carries an internal deletion of the trp operon that eliminates the EcoRI site in trpB, thereby ensuring that each trpcon- taming fragment cloned into the plasmid will include both upstream and downstream flanking sequences.

The ligated DNA was used to transform strain TB1678. Both single selection for Tc resistance using LB + Tc agar and double selection for Tc resistance and Trp prototro- phy (i.e., complementation of AtrpEl676) using CAA + Tc agar was made. The fre- quency of TcRTrp+ transformants among the total TcR transformants was about 5 X 10m4. A number of TcRTrp+ transformants of in- dependent origin were purified and their plasmids characterized by agarose gel electro- phoresis. Recombinant plasmids of at least two different sizes were detected. EcoRI digestion of the smallest plasmid type yielded two fragments, one comigrating with EcoRI- digested pBR322 and the other of larger size (about 14 kb), presumably the chromosomal

insert bearing the trp operon of TB50. Diges- tion with SalI and with Hind111 gave rise in each case to three fragments, indicating that besides the single cleavage sites in the pBR322 sequence, there are two SalI and two Hind111 sites within the insert DNA. Moreover, two distinct Safi and Hind111 fragment patterns were observed, indicating that plasmids with inserts of both orientations had been isolated.

A partial restriction map of a representative of each of the two plasmid types, designated pSTP1 and pSTPl1, was determined by the analysis of the fragments generated in a series of double digestions with endonucleases EcoRI, HindIII, and San. The results were consistent with those reported earlier for the trp region of S. typhimurium (Nichols et al., 1980; Crawford et al., 1980; Nichols and Yanofsky, 1979) and, taken together, estab- lished the counterclockwise orientation of the trp operon in plasmid pSTP1 and the clockwise orientation in pSTPl1. The phys- ical maps of the two plasmids are presented in Fig. 1.

Genetic Analysis of Plasmids pSTP1 and pSTPl1

Complement&ion analyses were performed to verify the presence of the trpE and trpA genes on plasmids pSTP1 and pSTPl1 and to test the presence of other chromosomal genes lying adjacent to trp. A series of appro- priately marked strains were transformed with purified plasmid DNA with selection or screening of transformants for the comple- mented phenotypes. It was found (Table 2) that both plasmids carry functional trpE and trpA genes, as expect&, however, neither carries intact any of the upstream (pyrF, cysB, and top) or downstream (chr) loci that are cotransducible by bacteriophage P22. This indicates that top,’ the structural gene for topoisomerase I (Trucksis et al., 198 1) and the closest known marker on the promoter

* The top locus has been referred to in earlier publi- cations as supX. The change in nomenclature is in accordance with the recommendations of Sanderson and Roth (1983).

152 PATTERSON AND BAUERLE

TB50 + pBR322

g%.%?+

FIG. 1. Origins and physical maps of plasmids pSTP 1, pSTP I I, pSTP2 1, and pSTP26. R = EcoRI; H = HindIII; S = Sun.

side of trp, is probably separated from trp by more than 7 kb and that chr, analogous to tonB in E. coli and the closest, characterized marker on the trpA side, is probably more than 3.5 kb away, in agreement with the physical mapping done in E. coli (Postle and Reznikoff, 1978).

Construction of Receptor Plasmid pSTP21

Since there are two Safl restriction sites within the cloned EcoRI insert of plasmid

pSTP1, one in each of the trp flanking se- quences (Fig. l), the sequence between the two sites can be removed by Sall digestion, creating a plasmid that is devoid of all trp DNA and has a hybrid insert made up of the 5.4 kb upstream and 3.2 kb downstream flanking sequences. It was reasoned that such a plasmid might be utilizable as a specialized in vivo cloning vector into which the trp region carried on another DNA molecule, such as a transducing DNA fragment, could be inserted by homologous recombination

IN VW0 CLONING OF Salmonella trp GENES 153

TABLE 2 COMPLEMENTATION ANALYSIS FOR CHROM~KBMAL MARKERS ON RECOMBINANT PLAWIDS pSTP1 AND pSTPl1

Transformation’ by plasmid Recipient Selected

strain Recipient genotype phenotype” pBR322 pSTP1 pSTPl1

TB1686 AtrpE AtrpA recA Tcx + + + TcR TrpA+ - + + TcR TrpA+ TrpE+ - + +

TB1700 AtrpE pyrF recA TcR + + + TcR TrpE+ - + + TcR TrpE+ PyrF+ - - -

TB1719 AtrpE cysB recA TcR + + + TcR TrpE+ - + + TcR TrpE+ CysB+ - - -

TB1743 A(cysB-top-trpPLE) TcR + + + TcR Top+ - - -

TB20 A(trpPLEDCBA-chr-opp) Tc’ + + + TcR Chr+ - - -

0 Transformations were performed as described under Materials and Methods. ‘Tests for drug resistance and for complementation of nutritional markers were made by direct selection of

transformants using the following agar media: Tc R, LB agar + Tc; TcR TrpA+, EMA + Anth + Tc; TcR TrpA+ TrpE+, TcR TrpE+ Pyrp, and TcR TrpE+ CysB+, EMA + Tc. Because of the unique phenotypes of Top- and Chr- strains, the tests for these two markers were carried out by secondary screening of TcR transformants selected on LB agar + Tc. Deletion mutations of the top locus act as weak suppressors of a leu promoter mutation, leuP500 (Mukai and Matgolin, 1963). and also prevent the induction of the SOS repair system of the cell (Gverbye and Margolin, 1981). Therefore, Top- deletion strain TBl743, which also carries the leuP500 mutation (Table l), is phenotypically Leu+ and UV sensitive. Since in partial diploids, Top+ is dominant to Top-, the test for the presence of the top gene on plasmids pSTP1 and pSTP1 I entailed the screening of TcR transformants of the TB1743 crosses for the loss of leucine prototrophy, using EMA + Cys + Anth + Tc, with and without Leu, and for the loss of UV sensitivity. The phenotype of deletion mutants of the chr locus, such as TB20, is manifest as an extreme reduction in the growth of colonies on minimal salts agar lacking citrate, due to the presence of high levels of chromium in commercial agar (Corwin et al., 1966). Since chr+ is dominant to chr-, the TcR transformants of the TB20 crosses were screened for their Chr phenotype by comparing colony formation on standard and citrate-free EMA + Trp + Tc.

between the two flanking sequences of the plasmid and donor DNA. If so, it would be possible to clone the trp operon of any wild- type or mutant strain of interest into this vector, so long as the chromosome of the donor strain retains adequate homology in both flanking regions.

This “receptor” plasmid was constructed as follows. Purified pSTP1 DNA was partially digested with San, religated and used to transform strain TB 1686, which carries dele- tions of both trpB and trpA (Table l), the two intact genes on pSTP 1. Selection was on LB + Tc agar and a TcRApRTrp- transfor- mant (TB1934) was isolated. Its plasmid was designated pSTP21. Alternate selection was

made using EMA + Ap and a TcSApRTrp+ transformant (TB1935), expected to arise by the excision of the SalI fragment extending from the site inside the upstream flanking sequence to the site in the Tc gene of pBR322, was isolated. Its plasmid was designated pSTP26. Plasmids pSTP2 1 and pSTP26 were purified and their structures verified by re- striction analysis (Fig. 1).

Cloning of a Variety of trp Operons into Plasmid pSTP21 by in vivo Recombination

It has been found that plasmid pSTP21 can be successfully used as a receptor mole-

154 PATTERSON AND BAUERLE

cule for the in vivo cloning of trp genes transferred into the cell by generalized trans- duction. A critical feature of the cloning procedure is the use of phenotypic differences between the wild-type and mutant alleles of the nearby chr locus (see legend of Table 2). The recipient strain for the transductional cloning (TB1937) was constructed by trans- forming plasmid pSTP21 into strain TB20, which carries a large deletion removing all of trp and chr (A(trp-chr-opp)lOl) (Higgins et al., 1983). Since plasmid pSTPl does not carry chr (Table 2), it follows that pSTP21 also does not. Thus, strain TB1937 is phe- notypically Trp-Chr-ApRTcR. Since the strain is devoid of all trp genes, it is unable to utilize the pathway intermediate indole, to satisfy the Trp auxotrophy (Ind- phenotype).

When a P22 lysate grown on a Chr+Ind+ strain (this would include essentially all wild- type and mutant trp strains with the exception of those that are TrpB-) is used to transduce TB1937, two types of recombination events can give rise to Ind+ transductants (Fig. 2): (1) recombination between the transducing DNA and the host chromosome, thereby replacing the trp-chr-opp deletion with the trp-chr-opp region of the donor strain; and (2) recombination between the transducing DNA and plasmid pSTP2 1, thereby inserting the region of the donor strain lying between the two components of the hybrid insert, a region that includes trp but not chr. Because recombination is limited to different se- quences in the two cases, all chromosomal recombinants are Ind+Chr+ while all plasmid recombinants are Ind+Chr-. Since Chr- col- onies are smaller than Cbr’ colonies and also have a distinct morphology, the transductants with recombinant plasmids are readily distin- guishable on the transduction plate (Fig. 3A).

The cloning cross has been performed successfully with a large variety of wild-type and mutant strains. In most cases the number of Chr+ (large colony) and Chr- (small col- ony) transductants are nearly equivalent, as exemplified in Fig. 3A, in spite of the fact that there are more targets for plasmid re- combination than for chromosomal recom-

Chmmcamrl Recombination in TB1937

Plasmid Recombination in TB1937

FIG. 2. A comparison of the recombinational events leading to the formation of Chr- plasmid transductants and Chr+ chromosomal transductants in strain TB1937. The terms “up” and “dn” indicate the upstream and downstream flanking sequences of the trp operon. The figure is meant to be illustrative and is not strictly to scale.

bination due to the high copy number of the plasmid. When strain TB1864 (TB20/ pBR322) is used as recipient, only Chr+ transductants are observed (Fig. 3B). This is as expected since the absence of the insert DNA in pBR322 precludes the formation of plasmid, but not chromosomal recombinants.

Analysis of Transductants Carrying Recombinant Plasmids

When small scale plasmid preparations from purikd Chr- transductants are analyzed by agarose gel electrophoresis, two different plasmid banding patterns are observed (Fig. 4). Either the supercoiled plasmid band co- migrates with plasmid pSTP21 or it migrates with a reduced mobility. This indicates that the major plasmid species in a particular strain is either of the nonrecombinant

IN VIVO CLONING OF Salmonella trp GENES 155

FIG. 3. Colonial morphology of plasmid (Chr-) and chromosomal (Chr+) transductants arising in crosses of plasmid-bearing strains with P22 phage grown on strain TB78 (LT2 wild type). Recombinants were selected by plating the infected cells on CAA + Ind + Tc. (A) TB1937 (TB20/pSTP21) as recipient; (B) TB I864 (TB20/pBR322) as recipient.

(pSTP21) type or of the larger, recombinant type. Patterns suggesting a mixture of recom- binant and nonrecombinant plasmids were not observed. One plasmid pattern does not appear to be more favored than the other, since of the initial 18 transductants analyzed, 8 had nonrecombinant-type and 10 had re- combinant-type patterns. Subsequent exper- iments have shown that this is merely a segregation phenomenon. Purified transduc- tants possessing the recombinant-type plas- mid pattern can be recovered from every cross by screening a number of clones (usually no more than 10 is required). Unfortunately, there are no obvious differences in colony size or morphology between the two types of segregants when grown on media selective for the Ind+ phenotype.

The plasmids of the Chr- transductants were analyzed further in transformations with strain TB1409 (AtrpEDCBAZ67 recAZ). Both single selection for TcR using EMA + Trp + Tc and double selection for Ind+TcR using EMA + Ind + Tc were carried out. The former provides a measure of both recombi- nant and nonrecombinant type plasmids in each preparation, while the latter of only recombinant type plasmids. It was found (Table 3) that the relative abundance of Ind+TcR transformants is directly correlated with the plasmid banding pattern of each preparation. Preparations with recombinant type patterns yield equivalent numbers of TcR and Ind+Tc? transformants, while those with nonrecombinant type patterns give rise to Ind+Tc? transformants at a greatly reduced

156 PATTERSON AND BAUERLE

TM937

TP5

TP13

TP21

TP28

TP20

TPlO

TPll

FIG. 4. Agarose gel electrophoresis of plasmids isolated from various Chr- Ind+ transductants of strain TB1937. Small scale plasmid preparations were made from 1.5 ml of cultures grown in LB + Ap. Electrophoresis was performed in a 0.7% agarose slab gel. The origin and characteristics of each TP transductant am presented in Table 3. The supercoikd and relaxed forms of the major plasmid species of each strain are apparent in the prep arations. The minor, more slowly migrating bands in TB1937 and in the nonrecombinant-type TP strains (TP5, TPI I, and TP28) are presumably the dimeric form of receptor plasmid pSTP2 I.

( 1 O-’ to 10P3) frequency. This is further evidence that in each Ind+Chr- transductant of TB 1937 the plasmid population is either predominately (possibly completely) recom- binant or predominately nonrecombinant.

Ind+TcR isolates from each transformation were purified and analyzed for phenotypic and plasmid characteristics. It was found that all now exhibit the recombinant-type plasmid pattern, regardless of the nature of the pattern of the parental transductant (data not shown). In those cases where the cloned trp operons carry large deletion mutations, the recombi- nant plasmids are smaller, consistent with the extent of the deletion as determined by genetic mapping. These results indicate that each isolate has been transformed by a single plasmid molecule and now carries a homo- geneous population of its particular recom- binant plasmid differing only by the nature of the cloned crp insert.

Expression of the Cloned trp Operon

It was of interest to characterize the expression and regulation of the trp operon

TABLE 3 CHARACTERIZATION OF PLASMIDS OF VARIOUS IND+ CHR- TRANSDUCTANTS OF STRAIN TB1937

Transductant 0

Parental donor TransformantsJplate c

Mutant Plasmid Genotype type pattern ’ Ind+ TcR Tea

TP5 LT2 TP23 LT7 TP13 LT2-7 TP21 trpEl724 TP28 trpEl726 TPl6 trpEl732 TP14 trpD16 TP15 trpD479 TP18 AtrpE512 TP20 AtrpE167S TP9 AtrpE1676 TP17 AtrpLE1698 TPlO AtrpED1679 TPll AtrpLEDC1682

Wild type Wild type Wild type Amber Amber Missense Amber O&e Deletion Deletion Deletion Deletion Deletion Deletion

NR 2 903 R 75 83 R 378 388 R 55 51 NR 2 219 R 206 230 R 126 145 R 137 136 NR 0 217 R 46 45 R 502 517 NR 0 109 R 405 399 NR 1 965

’ Transductants were isolated from the crosses of strain TB 1937 with P22 phage grown on the indicated parental donor as described in the text.

* Plasmid patterns were determined by agarose gel electrophoresis as described in Fig. 4. ‘Transformation of strain TB1409 was carried out as described in the text and under Materials and Methods.

IN VIVO CLONING OF Salmonella trp GENES 157

cloned in S. typhimurium. It is convenient to demonstrate trp operon regulation by monitoring the specific activity of the trp enzymes during growth of a trpRi auxotroph in minimal salts medium (MM) containing a limiting supplement of tryptophan. In the early stages of growth the enzymes are main- tained at the fully repressed level; as the tryptophan supplement becomes exhausted the rate of growth diminishes and a dramatic derepression of enzyme synthesis ensues due to the release of repression and attenuation.

The results of such an experiment carried out with strain TB1865, whose plasmid con- tains the trp promoter, operator, leader, trpE and trpA genes intact (Fig. l), are presented in Fig. 5. Upon exhaustion of the limiting tryptophan supplement, a net 40-fold de- repression of trpE activity over that of the repressed level is rapidly achieved. At this

FIG. 5. Kinetics of growth and of trp operon expression in plasmid-bearing strain TBl865 grown under conditions of tryptophan limitation. Cells were grown in MM + CA (0.2%) + Trp (4 @ml) at 37°C. Viable cell titer and plasmid stabiity were detetined by plating appropriate dilutions onto NZY agar and NZY + Ap (50 &ml) agar plates, respectively. AS-NH, activity was assayed in cellular extracts as described under Materials and Meth- ods.

point, the trpE polypeptide constitutes about 11% of the total cellular protein, as calculated from the specific activity of the homogeneous protein ((2 10 units/mg), Bauerle, unpub- lished). The activity of trpA increases in a similar fashion (data not presented). The maximal derepressed activity achieved in TB1865 under these conditions is about 10 times that found in control experiments with strain TB50 which has only a single, chro- mosomal copy of the same operon. In con- trast, the repressed level of activity of TB 1865 is about 50-fold higher than that of TB50, suggesting that, besides the dosage effect of the multicopy state of the cloned trp genes, some derepression of expression also occurs, probably as a result of repressor titration (Kelley and Yanofsky, 1982).

The high differential rate of trp expression upon exhaustion of tryptophan is apparently toxic to the cell, since there is a concomitant loss of cell viability and, in later stages, of the plasmid itself (Fig. 5). The condition of tryptophan starvation is not in itself respon- sible for cell death and plasmid instability, nor is it limiting to the maximum level of trp expression, since similar results were ob- tained when derepression was effected by partial limitation for tryptophan in a plasmid- bearing strain (data not shown). This was accomplished by using a host with a chro- mosomal trpED deletion which causes the production of a partially active, truncated trpD polypeptide which, in turn, limits the growth of the strain in tryptophan-free me- dium. These results are consistent with the finding that cell viability and plasmid stability are also poor in TrpR- plasmid-bearing strains under all conditions of growth (data not shown).

DISCUSSION

The in vivo cloning system described here is unique in that recombinant plasmids car- rying the trp operons of strains of interest are generated by a simple transductional cross. The recombinant plasmids contain a large insert from which the intact trp operon

158 PATTERSON AND BAUERLE

can be conveniently recovered as a single fragment by &/I digestion. Specific segments of the operon can then be subcloned when manipulation or detailed analysis of a partic- ular gene or sequence is desired.

The method is versatile; any wild type or mutant trp operon, with the exception of those bestowing an Ind- phenotype, can be cloned directly. This includes the operons of promoter, operator and leader region mutants as well as mutants of the trpE, trpD, trpC, and trpA structural genes. Recently we have expanded the method so that the cloning of mutant trpB operons is also possible (Hess and Bauerle, unpublished). In such cases, the TrpB- operon is transduced into receptor plasmid pSTP2 1 as before, but using a recip ient strain which also carries a second, helper plasmid bearing a trp operon deleted for trpE. Selection is made for TrpChr- trans- ductants (i.e., for complementation between the newly recombinant TrpB- plasmid and the TrpE- helper plasmid). The TrpB- plas- mid is then separated from the helper and receptor plasmids by independent transfer to an appropriate host.

It is noteworthy that in the transductional clonings it was not found that the plasmid recombinants greatly outnumber the chro- mosomal recombinants (Fig. 3), as might be expected in view of the multicopy nature of the receptor plasmid. Apparently other fac- tors, perhaps differences in the length of homologous DNA available for pairing or differences in superhelical density between the plasmid and chromosome, influence the probability of each type of recombinational event. Also of interest is the finding that purified transductants either carry the non- recombinant, parental plasmid in vast excess of the recombinant plasmid, or contain the recombinant plasmid as the predominant (possibly the sole) species (Fig. 4). Similar results were obtained in experiments where aru deletions were cloned into a pBR322- derived vector by in vivo recombination (Horwitz et al., 1980). This suggests that the Ind+ selection used here is not so stringent as to ensure the enrichment of the recombi-

nant plasmid in the cell at the exclusion of the parental plasmid and that plasmid seg- regation at cell division may not be entirely random in cells carrying a mixture of plas- mids.

Even though we have exploited some unique characteristics of the trp operon in developing our cloning system, it is obvious that the general design can be applied to the cloning of other loci for which a specialized plasmid vector analogous in structure to pSTP21 can be constructed. In each case it would be desirable to eliminate the possibility of integration of the plasmid into the chro- mosome of the Ret+ cloning host by using a strain with a chromosomal deletion that ex- tends beyond the limits of the flanking se- quence segments of the receptor plasmid. It would also be helpful, but not essential, if this chromosomal deletion removes a storable locus located upstream or downstream of the flanking sequences of the plasmid. For ex- ample, even though we used a downstream trp-chr deletion strain as host, we could have used instead an upstream cysB-trp deletion strain, distinguishing Ind+ plasmid transduc- tants from Ind+ chromosomal transductants by secondary screening for their Cys- phe- notype.

Our analysis of the expression of the trpE operon of plasmid pSTP1 in cultures grown under conditions of full and partial starvation for tryptophan demonstrate that normal reg- ulation of the cloned operon occurs in S. typhimurium. However, the increase in trp enzyme levels in derepressed cultures is less than that expected from full expression of the multiple operon copies in the cell. This may be a result of the dramatic decrease in cell viability that follows shortly after the onset of trp derepression. The stress causing these effects might be either the toxicity of one of the trp polypeptides or the extreme taxation of the cell’s metabolism caused by the turn-on of the highly efficient promoter of the cloned trp operon. The possible toxic overproduction of the product of an uniden- titied gene present on one of the flanking sequences that is subject to regulation by

IN VW0 CLONING OF Salmonella trp GENES 159

tryptophan (for example aroT, a downstream locus controlling aromatic amino acid trans- port, located between trp and chr (Thorne and Cot-win, 1975)) can be excluded since strain TB1937, which carries plasmid pSTP21, does not experience the dramatic loss in cell viability upon starvation for tryp- tophan.

Other investigators have observed the ac- cumulation of plasmid-free cells in growing cultures of E. coli in which trp enzymes are overproduced from cloned genes (Imanaka et al., 1980; Hallewell and Emtage, 1980; and Hershfield et al., 1979). This has been attributed to the toxic effect of the overpro- duction of trp enzymes, which leads to the death or reduction in growth rate of plasmid carrying cells and thereby enriches for plas- mid-free segregants. This cannot be the un- derlying mechanism of instability in the ex- periments reported here, since plasmid loss occurs over a short period of time and in the absence of net cell division (Fig. 5). Perhaps under conditions of high derepression of the trp operon, the normal process of random segregation of the plasmid pool in those cells that do divide is displaced by one that is nonrandom and asymmetric. Alternatively, the plasmids may become subject to intra- cellular degradation during the period of trp derepression.

ACKNOWLEDGMENTS

We thank John Roth and Charles Colson for providing bacterial and phnge strains. This work was supported by Public Health Service Grant GM26293 and by Training Grant GM07082 from the National Institutes of Health.

REFERENCES

BOLIVAR, F., RODRIGUU, R. L., GREENE, P. J., BETLACH, M. C., HEYNEKER, H. L., BOYER, H. W., CROSA, J. H., AND FALKOW, S. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2,95-I 13.

CLEWELL, D., AND HELINSKI, D. (1969). Supercoiled circular DNA-protein complex in Escherichia coli: Pm&cation and induced conversion to an open cimular DNA form. Proc. Natl. Acad. Sci. USA 62, 1159- 1166.

CORWIN, L. M., FANNING, G. R., FELDMAN, F., AND MARGOLIN, P. (1966). Mutation leading to increased sensitivity to chromium in Salmonella typhimurium. J. Bacterial. 91, 1508-1515.

CRAWFORD, 1. P., NICHOLS, B. P., AND YANOFSKY, C. (1980). Nucleotide sequence of the trpB gene in Esch- erichia coli and Salmonella typhimurium. J. Mol. Biol. 142.489-502.

HALLEWELL, R. A., AND EMTAGE, S. (1980). Plasmid vectors containing the tryptophan operon promoter suitable for efficient regulated expression of foreign genes. Gene 9,27-47.

HERSHF~ELD, V., BOYER, H. W., YANOFSKY, C., LOVETT, M. A., AND HELINSKI, D. R. (1974). Plnsmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proe. Natl. Acad. Sci. USA 71, 3455-3459.

HIGGINS, C. F., HARDIE, M. M., JAMIESON, D., AND POWELL, L. M. (1983). Genetic map of the opp (oliopeptide pernxase) locus of Salmonella typhimu- rium. J. Bacferiol. 153, 830-836.

HOLMES, D. S., AND QUIGLEY, M. (1981). A rapid boiling method for the preparation of bacterial plas- mids. Anal. B&hem. 114, 193-197.

HORWITZ, A. H., HEFFERNAN, L., CASS, L., MIYADA, C. G., AND WILCOX, G. (1980). Construction of pBR322ara hybrid plasmids by in vivo recombination. Mol. Gen. Genet. 179, 6 15-625.

IMANAKA, T., TSUNEKAWA, H., AND AIBA, S. (1980). Phenotypic stability of trp operon recombinant plas- mids in Escherichia coli. J. Gen. Microbial. 118, 253- 261.

KELLEY, R. L., AND YANOFSKY, C. (1982). frp Apore- pressor production is controlled by autogenous regu- lation and inefficient translation. Proc. Natl. Acad. Sci. USA 79, 3 120-3 124.

LEDERBERG, E. M., AND COHEN, S. N. (1974). Trans- formation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J. Bacterial. 119, 1072-1074.

MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). In “Molecular Cloning A Laboratory Manuel’ p. 440. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.

MUKAI, F. H., AND MARCOLIN, P. (1963). Analysis of unlinked suppressors of an 0” mutation in Salmonella. Proc. Natl. Acad Sci. USA 50, 140-148.

NICHOLS, B. P., M~ozz~ru, G. F., VANCLEEMPUT, M., BENNETT, G. N., AND YANOFSKY, C. (1980). Nucleo- tide sequences of the trpG regions of Escherichia coli, Shigella dysenteriae, Salmonella typhimurium and Serratia marcescens. J. Mol. Biol. 142, 503-517.

NICHOLZ., B., AND YANOFSKY, C. (1979). Nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli: An evolutionary comparison. Proc. Natl. Acad. Sci. USA 76, 5244-5248.

OVERBYE, K. M., AND MARCOLIN, P. (1981). Role of the supX gene in ultraviolet light induced mutagenesis in Salmonella typhimurium. J. Bacterial. 146, 170- 178.

POSTLE, K., AND REZNIKOFF, W. S. (1978). Hind11 and

160 PATTERSON AND BAUERLE

Hind111 restriction maps of the att @t&or&trp region of the Escherichia coli genome, and location of the tonE gene. J Bacterial. 136, 1165-I 173.

SAITO, H., AND MIURA, K. I. (1963). Preparation of transforming deoxyribonucleic acid by phenol treat- ment. Biochim. Biophys. Acta 72, 619-629.

SANDERSON, K. E., AND ROTH, J. R. (1983). Linkage map of Salmonella tyohimurium, edition VI. Microbial. Rev. 47,410-453.

SCHMIEGER, H. (1972). Phage P22 mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 119, 75-88.

SELKER, E., BROWN, K., AND YANOFSKY, C. (1977). Mitomycin C-induced expression of trpA of Salmonella typhimurium inserted into the plssmid ColE 1. J. Bac- teriol. 129, 388-394.

SMITH, H. O., AND LEVINE, M. (1967). A phage P22 gene controlling integration of phage. Virology 31, 207-216.

STAUFFER, C. V., ZURAWSKI, G., AND BENNETT, G. N.

(1979). In vivo cloning of DNA regions carrying mutations linked to se&table genes: Application to mutations in the regulatory region of the Excherichia coli tryptophan operon. Plasmid 2, 498-502.

TANEMURA, S., AND BAUERLE, R. (1977). Internal reini- tiation of translation in polar mutants of the trpB gene in Salmonella typhimurium. Mol. Gen. Genet. 153, 135-143.

THORNE, G. M., AND CORWIN, L. M. (1975). Mutations atfecting aromatic amino acid transport in Escherichia coli and Salmonella typhimurium. J. Gen. Microbial. 90,203-2 16.

TRUCK~IS, M., GOLUB, E. I., ZABEL, D. J., AND DEPEW, R. E. (1981). Escherichia cob and Salmonella typhi- murium supX genes specify deoxyribonuoleic acid topoisomerase 1. J. Bacterial. 147, 679-68 1.

WUESTHOFF, G., AND BAUERLE, R. (1970). Mutations creating internal promoter elements in the tryptophan operon of Salmonella typhimurium. J. Mol. Biol. 49, 171-196.


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