Vol. 164, No. 1JOURNAL OF BACTERIOLOGY, OCt. 1985, p. 270-2750021-9193/85/100270-06$02.00/0Copyright © 1985, American Society for Microbiology

Novel One-Step Cloning Vector with a Transposable Element:Application to the Myxococcus xanthus Genome

TEIICHI FURUICHI, MASAYORI INOUYE, AND SUMIKO INOUYE*Department ofBiochemistry, State University ofNew York at Stony Brook, Stony Brook, New York 11794-5215

Received 29 April 1985/Accepted 23 July 1985

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructeda transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycinphosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by TnSinverted repeats (ISSOL and ISSOR). TnV can transpose to many different sites of DNA in E. coli andMyxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloningof a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolatedfrom TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed intoE. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNAcan then be used as a probe for characterization of the gene and screening of clones from a genomic library.We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.

Transposons have been widely used to characterize aspecific gene by mutagenesis (14). Tn5, which has a geneencoding neomycin phosphotransferase II (NPTII; kanamy-cin resistance), provides an ideal system for isolation ofmutants and subsequent cloning of the genes (3). TnS-inserted DNA fragments from the mutants can be cloned ina vector plasmid or bacteriophage by using the Kmr pheno-type as a screening marker.To achieve TnS mutagenesis followed by cloning of the

TnS-inserted genes in one step, we constructed a newTnS-derived transposon capable of autonomous replication,designated TnV. This transposon consists of the replicationorigin derived from pSC101 and NPTII flanked by TnSinverted repeats. On transposition, mutants are isolated asKmr colonies. By ligating a restriction endonuclease digestof the mutant chromosomal DNA, TnV-inserted DNA frag-ments from the mutants can be directly cloned into Esche-richia coli host cells by transformation. We applied the newtransposon vehicle to Myxococcus xanthus, a gliding bacte-rium which undergoes sporulation coupled to primitivemulticellular development on starvation of nutrients (13), toisolate genes responsible for morphogenesis during differen-tiation.

MATERIALS AND METHODSBacterial strains, phages, and plasmids. M. xanthus strain

DZF1 (11) was used. E. coli JA221 (Ipp/F' laqIq) (21), GM33(dam) (17), and D110 (polA endI) (obtained from R.Sternglanz) were used for plasmid constructions. P1 clr-100Cm (23) and P1 clr-100 Cm::TnS (15) were used. E. coli C600(2) was used for the transposition experiment. E. coli HB101(5) was used to clone M. xanthus DNA. pBR322::TnS is apBR322 derivative containing transposon Tn5 at the mapposition of about 3.2 kilobases (kb) (10). pUC9 (25) was usedas a vector plasmid. pSC101 (7, 8) was used for isolation ofthe DNA fragment involved in the replication function (rep).Growth conditions. E. coli cells were grown in LB medium

(19) and on LB-agar (LB plus 1.5% agar). M. xanthus cellswere grown vegetatively in Casitone yeast extract (CYE)medium (6) and on CYE-agar (CYE plus 1.5% agar). CYE-

* Corresponding author.

soft agar was composed of CYE and 0.7% agar. Fruitingbody formation was induced by placing M. xanthus cells onCF-agar (9) and coli-agar (cell suspension of E. coli JA221[200 Klett units] in 0.1% MgSO4 7H20 autoclaved with1.5% agar). When necessary, the following antibiotics wereadded: (i) for E. coli, ampicillin (50 jxg/ml), tetracycline (12.5,xg/ml), chloramphenicol (25 ,ug/ml), and kanamycin sulfate(50 ,g/ml); (ii) for M. xanthus, kanamycin sulfate (40 ,ug/ml).DNA preparation. Plasmid DNA isolation was carried out

as previously described (20). For rapid analysis, DNA wasprepared by the alkaline lysis procedure (4). P1 phage DNAwas isolated from the lysates by a rapid isolation procedurefor k phage DNA (16). Chromosomal DNA of M. xanthuswas prepared by the method of Yee and Inouye (26) forcloning and by that of Avery and Kaiser (1) for rapidanalysis.

Plasmid constructions. All molecular cloning procedureswere performed as described by Maniatis et al. (16). pTF1was obtained as follows. The 1.5-kb (12) HindlIl-HincIlfragment containing the NPTII gene of TnS was cloned intothe HindIII-HincIl site of pUC9; thus, pUC9-kan was ob-tained. The HaeII fragment (1.8 kb) containing the repregion of pSC101 was treated with the large fragment ofDNA polymerase I to remove the protruding 3' single-stranded regions and then was inserted into the HincIl site ofpUC9-kan. Although pUC9 cannot replicate in DNA poly-merase I-defective (polA) cells, the resulting plasmid pUC9-kan-rep is capable of replication in polA- strain D110 byusing the replication origin of pSC101 which functions in it.We cut pBR322::TnS with BclI to delete the internal 2.7-kbregion of TnS and treated it with bacterial alkaline phospha-tase. This was ligated with the 3-kb BclI-BamHI fragment,spanning the kan-rep region, of pUC9-kan-rep. The finalproduct, pTF1, is composed of the entire pBR322 and thetransposon TnV (pBR322::TnV).

Transposition. To construct P1::TnV, we transposed TnVfrom pTF1 into P1. An overnight culture of E. coli C600containing pTF1 in LB containing kanamycin was diluted50-fold with LB containing kanamycin and 5 mM CaCI2 andincubated at 37°C. At late-log phase, P1 lysates were addedto the cells at a multiplicity of infection of 0.05. After mixing,the cells were left at room temperature for 30 min to allow

270

on April 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ONE-STEP CLONING VECTOR 271VOL. 164, 1985

Transposon TnV

IS5OL Kan Rep IS5OR

Transposition intoM. xonthus chromosome

RE RE RE RE RE

Digestion withrestriction enzyme ( RE)

Ligation

Transformation of E. co/i

Isolation andnick- translationof plasmid DNA

Colony- hybridization withM. xanthus library

Isolation ofa wild-type clone

FIG. 1. Experimental scheme for one-step cloning of M. xanthuschromosomal DNA (see the text for details).

phage absorption. Infected cells were spread on LB-agarplates containing kanamycin and chloramphenicol followedby incubation at 30°C for 24 h. Phage lysates were preparedfrom Kmr Cmr cells by the method of Miller (19). C600 cellswere infected with the phage lysates as described above.Infected cells were shaken at 300C for 1 h and then spread on

LB-agar plates containing kanamycin. After incubation at

30°C for 24 h, the Kmr transductants obtained were screenedfor sensitivity to ampicillin and tetracycline to distinguishPl::TnV lysogens from pTF1-containing cells generated byP1-generalized transduction. Then phage lysates were pre-

pared from Kmr Aps Tcs cells and assayed for the ability ofTnV to transpose.

Transposition of TnV from P1::TnV into the M. xanthuschromosome was carried out as follows. M. xanthus DZF1was grown in CYE at 300C. The cells were harvested at a cell

density of 109 cells per ml (about 170 Klett units) andsuspended in 1/10 of the original volume with 1% Trypticase(BBL Microbiology Systems)-4 mM MgSO4. Phage lysateswere diluted with an equal volume of P1 buffer (4% LB, 1mM CaCI2, 1mM MgSO4, 10 mM NaCI). The cell suspensionwas mixed with 2 volumes of the phage mixture and placedat room temperature for 30 min. Infected cells were mixed in0.2- to 0.4-ml portions with 2.5 ml of CYE-soft agar contain-ing 20 1Lg of kanamycin per ml and then poured ontoCYE-agar plates with 20 ,ug of kanamycin per ml (25 ml perplate). After 24 h of incubation at 30°C, 3 ml of CYE-softagar containing 533 pg of kanamycin per ml was overlaid onthe plate to give a final concentration of about 75 ,ug/ml.Incubation was continued for 4 to 5 days. Since P1 cannotreplicate in M. xanthus cells (15), the resulting Kmr cellsshould have TnV integrated into the chromosome.

Southern blot hybridization. Restriction enzyme-digestedDNA was electrophoresed in 0.7% agarose gels, transferredonto nitrocellulose filters (24), and hybridized with nick-translated (22) DNA (5 x 108 cpm/,g) as described byManiatis et al. (16).

Cloning of DNA mutagenized by transposon insertion.Chromosomal DNA (1 to 2 ,ug) cut with an appropriaterestriction enzyme was self-ligated in 140 [lI of ligation bufferwith 1.4 U of T4 DNA ligase at 12.5°C for 18 h. The ligationmixture (40 ,ul) was mixed with 10 [Ii of 5 x Tf buffer (50 mMCaCI2, 50 mM MgCI2, 50 mM Tris hydrochloride [pH 7.5])and was used to transform CaCl2-treated E. coli HB101 cellsto Kmr.

RESULTSExperimental design. The scheme for the isolation of the

gene of interest using the transposon TnV is outlined in Fig.1. We first constructed the transposon called TnV, which isderived from Tn5 and contains the replication origin derivedfrom pSC101 flanked by the inverted repeats (ISSOL andISSOR). TnV was transposed into the M. xanthus chromo-some, and TnV insertion mutants were isolated as Kmrcolonies. After a TnV mutation library was established, theindividual colonies were screened for a specific phenotypesuch as motility or development. Chromosomal DNA wasisolated from these cells and digested with an appropriaterestriction enzyme, such as Sall, BamHI, EcoRI, or KpnI,which do not cleave within the TnV sequence. Each digestwas then treated with ligase to produce self-ligated circularDNAs. E. coli cells were transformed with the ligated DNA,and Kmr transformants were isolated. The plasmid DNAconferring kanamycin resistance consisted of TnV DNA andregions of the gene of interest (Fig. 1). This cloned DNA canbe used directly as a probe for Southern or Northern blothybridization. Since the DNA sequence of TnV has nohomology to the DNA from pBR322, cosmid, or A phagevectors, the cloned DNA can be used directly to screen agene library constructed with these vectors. This allowsisolation of surrounding regions or the remainder of the geneof interest without further manipulation.

Construction and properties of pTF1. To carry out thisexperimental design, we constructed a derivative of pBR322designated pTF1 that has an additional DNA fragment, TnV,consisting of a transposon as well as a replication origin (Fig.2). TnV differs from transposon Tn5 by having the 1.8-kbHaeII fragment from pSC101 containing the rep gene (18) inplace of the 1.5-kb BclI-HincII fragment downstream of thegene for NPTII. Details of the construction are describedabove. The resulting pTF1 was able. to replicate in andconfer kanamycin resistance to E. coli polA cells. The TnV

on April 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

272 FURUICHI ET AL.

in pTF1 includes the two inverted repeats (IS50L and ISSOR)containing the genes responsible for transposition.

Isolation of P1::TnV. P1::TnV was isolated to introduceTnV into M. xanthus cells via P1-mediated transduction. E.coli C600 harboring pTF1 (Kmr Apr Tcr) was infected withP1 clr-100 (CMr) and tested for P1 lysogeny. Phage lysateswere prepared from two independent Kmr Cmr lysogens byheat induction, and then C600 lysogens were reestablishedunder the selective pressure of Kmr. In lysogens derivedfrom both phages, 60 Kmr colonies were tested for sensitiv-ity to ampicillin and tetracycline to eliminate lysogens re-sulting from generalized transduction of the original plasmidpTF1 itself. A total of46% of Kmr colonies were sensitive toboth antibiotics, and it was concluded that these resultedfrom lysogeny of P1 carrying TnV. To confirm the construc-tion of P1::TnV, we isolated phage DNA from lysatesprepared from three independent Kmr Aps Tcs lysogens,digested it with restriction enzymes, and characterized it byagarose gel electrophoresis. Three independent P1 deriva-tives capable of transducing Kmr had an insertion in differentfragments (data not shown).

Transposition of TnV into M. xanthus. We introduced TnVinto M. xanthus by using P1::TnV. It has been shown that P1can inject its DNA into M. xanthus. However, it does notmultiply in M. xanthus (15), and the P1 genome is lost duringcell growth. Therefore, the Kmr phenotype of M. xanthuscells can result only from insertion ofTnV into chromosomalDNA. Transposition frequencies ofTnV from three indepen-dent Pl::TnV phages into M. xanthus DZF1 are summarizedin Table 1. DZF1 was infected with phage P1::TnV at amultiplicity of infection of approximately 1.5. A wild-typeTnS transposed from Pl::Tn5 into M. xanthus at a frequencyof 9 x 10-8 (Kmr colonies per PFU). The efficiency oftransposition of TnV was 3 to 63% of the wild-type TnSfrequency (Table 1). Among the three Pl::TnV phages, B2-4

EcoRI HindiM \

PstII SamH

AvaI / 'P\ AvaI

Ps>//5ia 4

(Hincl[/HaeI[) PtHind

0< HindiAvaIBgpTI

HHaeII/HincIl\BamHI/BcII

FIG. 2. Structure of pTF1. pTF1 consists of pBR322 and the6-kb transposon, TnV, that has the gene coding NPTII and pSC101replication origin flanked by the inverted repeats (ISSOL and IS50R).TnV is located at the position 3.2 kb on the pBR322 map.

TABLE 1. Transposition activity of TnV

TranspositionPhage

Frequency" % of P1::TnS

P1::TnS 8.9 x 10-8 100P1::TnV Bl-8 2.9 x 10-8 33Pl::TnV B2-4 5.6 x 10-8 63Pl::TnV B2-12 2.4 x 10-9 3

aNumber of Kanr M. xanthus colonies per PFU when cells were infectedwith the phage at a multiplicity of infection of approximately 1.5.

gave the highest transposition frequency (6 x 10-8 Kmrcolonies per PFU) and was used to construct a mutationlibrary of M. xanthus.Of 855 Kmr mutants, 20 were first selected by their

abnormal morphologies during the vegetative or develop-mental stages. To confirm the presence of a TnV insertion inthe chromosome, we digested DNA isolated from theseindependent Kmr mutants with SalI and tested it by South-ern blot hybridization with nick-translated pTF1 DNA as aprobe. Since there is no Sall site within TnV, a positive bandin Southern blot hybridization consists of a target Sallfragment from M. xanthus chromosomal DNA plus the 6-kbTnV DNA. TnV was transposed into different SalI fragmentsin all cases except for strains 221, 328, and 530 (Fig. 3).Southern blot hybridization with BamHI digests of chromo-somal DNA also revealed positive bands appearing in dif-ferent positions in all cases except for strains 221, 328, and530 (data not shown). These results indicate that TnV caninsert into many sites in the M. xanthus genome. The size ofthe fragments hybridizing with pTF1 of mutants 221, 328,and 530 were the same for both SalI and BamHI digestions(15-kb Sall and 7.9-kb BamHI fragments; see the cloningsection), indicating that these mutants are probably identi-cal.

Isolation of TnV insertion mutants defective in development.Among 855 independent Kmr mutants, 6 were unable to form

o cm M t U) t o0)Xocm co.4 U) 0aP-OD0 r _ v-r cm r-v - MC

23.1 -

9.4 -

6.6 -

~_

VI*I.N

_t' -_#040-4.4 -

FIG. 3. Southern blot analysis of Kmr M. xanthus. Sall digestsof chromosomal DNA were probed with nick-translated pTF1.Lanes: 1, wild-type DZF1; 2, mutant 221; 3, mutant 238; 4, mutant338; 5, mutant 386; 6, mutant 408; 7, mutant 414; 8, mutant 530; 9,mutant 545; 10, mutant 761; 11, mutant 773; 12, mutant 786; 13,mutant 794; 14, mutant 366; 15, mutant 469; 16, mutant 554; 17,mutant 588; 18, mutant 591; 19, mutant 685; 20, mutant 769; and 21,mutant 818. The numbers on the left indicate molecular sizes inkilobases.

J. BACTERIOL.

on April 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ONE-STEP CLONING VECTOR 273

h

i 7 _FIG. 4. Pattern of fruiting body formation in TnV insertioh mutants. To induce fruiting body formation, we spotted 10 ,ul of cell suspension

(1,000 Klett units) on CF-agar as a starving condition and incubated it at 30°C. After 100 h, fruiting body formation was observed under adissecting microscope. Panel 1, wild-type DZF1; panels 2 through 7, mutants 221, 328, 338, 530, 773, and 786, respectively.

normnal fruiting bodies. Development-deficient mutants werefirst isolated on coli-agar plates. These mutants also showedunusual morphologies on CF-agar plates (Fig. 4). The wild-type strain (DZF1) aggregated and formed mounds within 24h after spotting on CF-agar plates, and fruiting bodiescontaining thousands of myxospores were formed within 48h. On the other hand, at 72 h after spotting, mutants 221, 328,and 530 had still not formed fruiting bodies, and the edgesand surfaces of the spots were just starting to become rough.At 100 h, a few fruiting bodies were formed, but only aroundthe spots (Fig. 4, panels 2, 3, and 5, respectively). Even after7 days, no additional significant morphological changes wereobserved. Mutants 338 and 773 aggregated poorly and wereunable to form normal fruiting bodies (Fig. 4, panels 4 and 6,respectively); they had very few myxospores, even after 7days. Mutant 786 moved actively and formed a tangled flarepattern (Fig. 4, panel 7). No myxospores were found in thismutant at 7 days after spotting.One-dsp cloning of developmentll genes. The results de-

scribed above suggest that in mutants 221, 328, and 530, thesame gene required for differentiation was mutagenized byTnV insertion. In mutants 221 and 530, TnV was inserted inthe same size Sall (Fig. 5, lanes 1 and 5, respectively),BamHI (lanes 2 and 6), EcoRI (lanes 3 and 7), and KpnI(lanes 4 and 8) fragments. An additional 4.3-kb KpnI frag-ment hybridized with pTF1 at very low intensity. We clonedTnV-inserted DNA fragments of these two mutants by theone-step cloning method depicted in Fig. 1. Total SalIdigests of chromosomal DNA from mutants 221 and 530

were ligated and used to transform E. coli HB101. Seven andsix Kmr transformants were obtained for mutants 221 and530, respectively. All Kmr transformants were found tocarry a plasmid containing the 15-kb Sall fragment expectedfrom Southern blot hybridization (Fig. 5, lanes 1 and 5).

1 2345678

23.1 -

9.4 -

66 -

4.4-

FIG. 5. Localization of TnV insertion on chromosomes of mu-tants 221 and 530. TnV insertions in mutants 221 (lanes 1 through 4)and 530 (lanes 5 through 8) were localized by Southern blothybridization with nick-translated pTF1 as a probe. Lanes: 1 and 5,SalI digests; 2 and 6, BamHI digests; 3 and 7, EcoRI digests; 4 and8, KpnI digests. Molecular standards of DNA fragments are shownin kilobases on the left. The arrow on the right represents a KpnIfragment hybridizing with the probe at low intensity.

VOL. 164, 1985

on April 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

274 FURUICHI ET AL.

12345678

23.1- A*m Rto

9.4- S.

6.6-

4.4-

n.N --

-

2.3-2.0-

FIG. 6. Identification of the target fragment of TnY insertion inmutants 221 and 530. Chromosomal DNA of DZF1 was digestedwith Sall (lanes 1 and 5), BamHI (lanes 2 and 6), EcoRI (lanes 3 and7), and KpnI (lanes 4 and 8) followed by Southern blot hybridizationwith the nick-translated Sall clone (see the text) of mutants 221(lanes 1 through 4) and 530 (lanes 5 through 8). The numbers on theleft show DNA sizes in kilobases.

Some plasmids had additional Sall fragments of varioussizes in addition to the 15-kb fragment (data not shown). Thepresence of TnV in the 15-kb Sall fragment was confirmedby BglII, HindlIl, and XhoI digestions, which generated theexpected 3-, 3.6-, and 5.0-kb fragments, respectively, corre-

sponding to the internal fragments of TnV (Fig. 2; data notshown). Since TnV was 6 kb in size, the size of the originalchromosomal Sall fragment was concluded to be 9 kb. Toconfirm this, we carried out Southern blot hybridization as

shown in Fig. 6. The Sall digest ofDZF1 chromosomal DNAwas hybridized with nick-translated plasmid DNA frommutant 221 or 530. A positive band was detected at 9 kb (Fig.6, lanes 1 and 5), indicating that TnV was in fact inserted intothe 9-kb Sall fragment of the M. xanthus chromiosome. Fromthe size of the BamHI (7.9 kb), EcoRI (20 kb), and KpnI (30kb) positive bands in Fig. 5, it was also concluded that TnVwas insetted into 1.9-kb BamHI, 14-kb EcoRI, and 24-kbKpnI fragments of the chromosome in both mutants. Thiswas also confirmed by Southern blot hybridization (Fig. 6).These results clearly demonstrate that the two independentmutants, 221 and 530, had TnV inserted in the same gene.Fine analysis of these plasmids, however, revealed that theTnV insertion sites were not identical (data not shown).

DISCUSSION

TnV, a transposon carrying the replication origin, was ableto transpose into many different sites in the M. xanthuschromosome. The TnV transposition frequency was compa-rable to that of P1::Tn5. Once transposed, TnV was stablymaintained in M. xanthus. The TnV-inserted DNA frag-ments were then cloned into an E. coli host directly from themutant chromosomal DNA as Kmr transformants. Since theDNA of TnV does not contain pBR322 or X phage se-

quences, the entire plasmid DNA of the TnV-inserted clones

can be used as a probe to screen a genomic library to isolateintact target genes. To efficiently clone TnV-inserted frag-ments, the digestion of mutant chromosomal DNAs has to bewith restriction enzymes which do not cleave TnV itself. Sofar, we were able to clone TnV-inserted DNA fragmentsranging from 0.5 to 35 kb in size (data not shown).

Since P1 and Tn5 have a wide host range (3), this strategymay be applied to many other bacterial species as a powerfulmethod for mutagenesis and one-step cloning of a gene ofinterest. For this purpose, conjugative plasmids such asRP4, which are known to have a wide host range, can beused as a vehicle instead of the phage P1 used in this study(3). The replication region of pSC101 used in this study lacksthe par locus that is required for stable maintenance ofplasmids during cell growth (18). However, by applyingselection pressure (in this study, kanamycin resistance) westably maintained TnV-derivative clones in E. coli.Of 855 independent Kmr mutants, 6 were identified as

developmental mutants on the basis of morphology duringdifferentiation. Three of them had a TnV insertion within a0.25-kb region of the same restriction fragment. Genetic andbiochemical analyses of the developmental genes cloned inthis study are now in progress.

ACKNOWLEDGMENTSWe thank Rolf Sternglanz, Dale Kaiser, and Douglas Berg for

providing D110 (polA), P1::TnS, and pBR322::TnS, respectively, andMartin Teintze and Anil Dhundale for critical reading of themanuscript.

This work was supported by Public Health Service grantGM-26843 from the National Institutes of Health.

LITERATURE CITED1. Avery, L., and D. Kaiser. 1983. In situ transposon replacement

and isolation of a spontaneous tandem genetic duplication. Mol.Gen. Genet. 191:99-109.

2. Bachmann, B. J. 1972. Pedigrees of some mutant strains ofEscherichia coli K-12. Bacteriol. Rev. 36:525-557.

3. Berg, D. E., and C. M. Berg. 1983. The prokaryotic transposableelement Tn5. Biotechnology 1:417-435.

4. Birnboim, H. C., and J. foly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

5. Boyer, H., and D. Roulland-Dussoix. 1969. A complementationanalysis of the restriction and modification of DNA in Esche-richia coli. J. Mol. Biol. 41:459-472.

6. Campos, J. M., J. Geisselsoder, and D. R. Zusman. 1978.Isolation of bacteriophage Mx4, a generalized transducingphage for Myxococcus xanthus. J. Mol. Biol. 119:167-178.

7. Cohen, S. N., and A. C. Y. Chang. 1973. Recircularization andautonomous replication of a sheared R-factor DNA segment inEscherichia coli transformants. PrQc. Natl. Acad. Sci. U.S.A.70:1293-1297.

8. Cohen, S. N., and A. C. Y. Chang. 1977. Revised interpretationof the origin of the pSC101 plasmid. J. Bacteriol. 132:734-737.

9. Hagen, D. C., A. P. Bretscher, and D. Kaiser. 1978. Synergismbetween morphogenetic mutants of Myxococcus xanthus. Dev.Biol. 64:284-296.

10. Hirschel, B. J., and D. E. Berg. 1982. A derivative of TnS withdirect terminal repeats can transpose. J. Mol. Biol. 15:105-120.

11. Inouye, M., S. Inouye, and D. R. Zusman. 1979. Gene expres-sion during development of Myxococcus xanthus: pattern ofprotein synthesis. Dev. Biol. 68:579-591.

12. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. Arestriction enzyme cleavage map of Tn5 and location of a regionencoding neomycin resistance. Mol. Gen. Genet. 177:65-72.

13. Kaiser, D., C. Manoil, and M. Dworkin. 1979. Myxobacteria:cell interactions, genetics, and development. Annu. Rev. Mi-crobiol. 33:595-639.

14. Kleckner, N., J. Roth, and D. Botstein. 1977. Genetic engineer-

J. BACTERIOL.

on April 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ONE-STEP CLONING VECTOR

ing in vivo using translocatable drug-resistance elements: new

methods in bacterial genetics. J. Mol. Biol. 116:125-159.15. Kuner, J., and D. Kaiser. 1981. Introduction of transposon TnS

into Myxococcus for analysis of developmental and other non-

selectable mutants. Proc. Natl. Acad. Sci. U.S.A. 78:425-429.16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular

cloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

17. Marinus, M. G., and N. R. Morris. 1975. Pleiotropic effects of aDNA adenine methylation mutation (dam-3) in Escherichia coliK-12. Mutat. Res. 28:15-26.

18. Meacock, P. A., and S. N. Cohen. 1980. Partitioning of bacterialplasmids during cell division: a cis-acting locus that accom-

plishes stable plasmid inheritance. Cell 20:529-542.19. Miller, J. H. 1972. Experiments in molecular genetics, p.

201-205. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

20. Nakamura, K., and M. Inouye. 1981. Inactivation of the Serratiamarcescens gene for the lipoprotein in Escherichia coli byinsertion sequences, IS1 and ISS; sequence analysis ofjunction

points. Mol. Gen. Genet. 183:107-114.21. Nakamura, K., Y. Masui, and M. Inouye. 1982. Use of a lac

promoter-operator fragment as a transcriptional control switchfor expression of the constitutive ipp gene in Escherichia coli. J.Mol. Appl. Genet. 1:289-299.

22. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977.Labeling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.133:237-251.

23. Rosner, J. L. 1972. Formation, induction and curing ofbacteriophage P1 lysogens. Virology 49:679-689.

24. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

25. Vieira, J., and J. Messing. 1982. The pUC plasmids, and M13mp7-derived system for insertion mutagenesis and sequencingwith synthetic universal primers. Gene 19:259-268.

26. Yee, T., and M. Inouye. 1981. Reexamination of the genome sizeof myxobacteria, including the use of a new method for genomesize analysis. J. Bacteriol. 145:1257-1265.

VOL. 164, 1985 275

on April 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from