Vol. 161, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1985, p. 1-60021-9193/85/010001-06$02.00/0Copyright © 1985, American Society for Microbiology

Isolation of a Versatile Serratia marcescens Mutant as a Host andMolecular Cloning of the Aspartase Gene

TSUTOMU TAKAGI* AND MASAHIKO KISUMIResearch Laboratory ofApplied Biochemistry, Tanabe Seiyaku Co., Ltd., Yodogawa-ku, Osaka, 532 Japan

Received 17 April 1984/Accepted 4 October 1984

An extracellular nuclease-deficient, antibiotic-sensitive, and restrictionless mutant was isolated from thewild-type strain of Serratia marcescens Sr41 by four rounds of mutagenesis. The mutant was transformedefficiently with plasmid DNAs prepared from Escherichia coli and S. marcescens, and was used as a host for thecloning of the aspartase gene (aspA+) of S. marcescens. Cells carrying the cloned aspA+ gene on a multicopyplasmid produced ca. 39-fold more aspartase than did control cells, and the level of enzyme overproduction wasin proportion to the copy number of the aspA+ recombinant plasmid. Aspartase was identified as a polypeptideof molecular weight 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Serratia marcescens strains have been used for manufac-turing various amino acids (7-10) and also for basic research(23). Although this microorganism has many properties incommon with other enteric bacteria, including Escherichiacoli, recombinant DNA techniques have not been applied,possibly because S. marcescens has obstacles such as anextracellular nuclease and a restriction endonuclease whichdegrades a transforming DNA. Recently, Reid et al. havereported a successful transformation in which they usedseveral strains of S. marcescens which produced the nu-clease (17). However, the transformation efficiency variedgreatly among the strains used, and even the best value wasfar lower than that in the E. coli transformation system.Thus, it is quite possible that the extracellular nucleaseinterferes with efficient transformation, as is observed inNeurospora crassa (21). It is also claimed that the extracel-lular nuclease causes a low recovery of plasmid DNA fromS. marcescens HY cells (22).

S. marcescens Sr41 used for the construction of aminoacid-producing mutants has the restriction endonucleasewhich is not related to the SmaI endonuclease (unpublishedobservation). The endonuclease is an obstacle to using S.marcescens as a host for recombinant DNA manipulations,since the enzyme will interfere with the introduction offoreign DNA into the microorganismn. In fact, Eichenlauband Steinbach described unsuccessful transformation of S.marcescens with a plasmid DNA prepared from E. coli (6).To develop the recombinant DNA techniques in S. marces-cens, a mutant deficient in extracellular nuclease and restric-tion endonuclease should be isolated as a favorable host forapplication of the techniques to this microorganism.

This paper describes the isolation of an extracellularnuclease-deficient, restrictionless, and antibiotic-sensitivemutant which can be used as an indispensable host in cloningexperiments with S. marcescens. The experiment, in whichan aspartase gene of S. marcescens was cloned on a multi-copy plasmid with the aid of the mutant, is also described todemonstrate the usefulness of the mutant.

MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and

plasmids used in this work are listed in Table 1. The glt-aspAmutant TK237 was isolated as described by Marcus and

* Corresponding author.

Halpern (13; Taniguchi et al., unpublished data). The doublemutant is necessary as the host for cloning the aspA+ gene.The glt mutation causes constitutive uptake of glutamate andmakes the cell grow on glutamate as the sole carbon andenergy source. Aspartase (EC 4.3.1.1) participates, as aconstituent of the "deamination cycle," in the metabolism ofglutamate taken up from the medium (13). The aspA muta-tion produces defective aspartase, thus blocking the utiliza-tion of glutamate. Hence, the aspA+ gene can be cloned inthe double mutant by selecting AspA+ transformants whichcan grow on glutamate as the sole carbon and energy source.

Media. L broth (12) was routinely used as a rich medium.The glutamate medium contained 7 g of K2HPO4, 3 g ofKH2PO4, 1 g of (NH4)2SO4, 0.1 g of MgSO4 * 7H20, and 10g of sodium L-glutamate per liter, and was supplemented asrequired with 1 mM L-threonine, 1 mM L-leucine, and 0.3mM thiamine. Cells for the extracellular nuclease assay weregrown in the medium described previously (15). For theaspartase assay, cells were cultured in ASP medium (pH 7.0)containing 20 g of corn steep liquor, 20 g of Meast (EbiosuYakuhin Co. Ltd., Tokyo, Japan), 11.4 g of fumaric acid, 5g of ammonium fumarate, 2 g of K2HPO4, and 0.5 g ofMgSO4 7H20 per liter. To isolate transformants of E. coliK-12 strains, ampicillin and kanamycin were added to Lbroth at 50 and 20 ,ug/ml, respectively. Media were solidi-fied, when necessary, with 15 g of agar per liter.

Isolation of mutants. Exponentially growing cells weremutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (ata final concentration of 100 ,ug/ml in L broth, treated at 30°Cfor 10 min). An extracellular nuclease-deficient mutant wasselected on DNA-nutrient agar plates as described previ-ously (24). Mutants showing enhanced sensitivity to antibi-otics were isolated as follows. Mutagenized cells werespread on L agar plates containing 2.5 ,ug of ampicillin orkanamycin per ml. After 2 days of incubation at 30°C,smaller colonies among those that appeared were picked andtested for sensitivity to the corresponding antibiotic at 20jig/ml. Restrictionless mutants were isolated as follows.Mutagenized cells were transformed with pVT116 DNA (1,ug) which was prepared from E. coli C600r-m- by themethod described below. The resulting transformants, whichprobably include restrictionless mutants, were cured bybeing subcultured in the absence of ampicillin. The curedstrains were examined for a transformation efficiency with

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TABLE 1. Bacterial strains and plasmidsStrain Characteristicsa Reference or source

S. marcescensSr4l8000 Wild type Matsumoto et al. (14)IT101 Nuc- Mutagenesis of 8000TT102 Nuc- Aps Mutagenesis of TT101TT122 Nuc- Aps Km' Mutagenesis of TT102TT392 Nuc- Aps Kms r- Mutagenesis of TT122

E. coli K-12C600r-m- thr-l leuBI thi-J

supE44 lacYltonA21 hsdR hsdM

TK237 C600r-m- glt aspA This laboratoryPlasmidspBR322 Apr Tcr Bolivar et al. (1)pACYC177 Apr Kmr Chang and Cohen (2)pVT116 Apr 12.4-kb fragment of S.

marcescens chro-mosome insertedinto HindIlI site ofpACYC177; thislaboratory

pVT104 Apr aspA+ 11.6-kb fragment of S.marcescens chro-mosome insertedinto HindIII site ofpACYC177; thisstudy

pVT128 Apr aspA+ Deletion derivative ofpVT104; this study

pVT150 Apr aspA+ Subcloning of aspA+into pBR322 frompVT128; this study

a Symbols used for relevant genotypes and phenotypes were as follows:Nuc-, no production of extracellular nuclease; Aps, enhanced sensitivity toampicillin; Kms, enhanced sensitivity to kanamycin; r-, defect of a hostrestriction enzyme; glt, constitutive uptake. of glutamate; aspA, defect ofaspartase.

pVT116 DNAs prepared from C600r-m- and the transform-ants obtained previously.

Determination of antibiotic sensitivity. Approximately 5 x106 cells were inoculated into 3 ml of L broth containingantibiotic at various concentrations. After 20 h of incubationat 30°C with shaking, growth was turbidimetrically deter-mined with a Hitachi electric photometer.

Isolation of chromosomal DNA. Chromosomal DNA wasprepared by the phenol method as described by Saito andMiura (19) from cells in the early exponential phase.

Isolation of plasmid DNA. Cells were grown in L broth at30°C for 18 h. The cells were lysed by the addition of 1%sodium dodecyl sulfate to the cell suspension, which hadbeen treated with 2 to 4 nmg of lysozyme per ml for 30 min atroom temperature in 50 mM Tris-hydrochloride-50 mMEDTA-10% sucrose (pH 8.0). Potassium acetate was addedto the lysate to 0.5 M. The mixture was incubated at 0°C for2 h or more and then was centrifuged at 95,800 x g for 30min at 0°C. The supernatant was treated with phenol, andthe plasmid DNA was purified by ethidium bromide-cesiumchloride centrifugation.Enzymes and chemicals. Restriction endonucleases, T4

DNA ligase, and bacterial alkaline phosphatase were pur-chased from Takara Shuzo Co., Ltd., Kyoto. Other chemi-cals were also commercially obtained and not further puri-fied.

Transfoirmation. S. marcescens and E. ccli cells weregrown in 20 ml of L broth at 30'C with aeration until the cell

density reached 5 x 108 to 10 x 108 cells per ml. The culturewas centrifuged for 10 min at 0°C. The pellet was suspendedin 20 ml of ice-cold 0.1 M MgCl2, centrifuged for 10 min at0°C, and then suspended and incubated at 0°C in 10 ml of 0.1M CaCl2-0.25 M sucrose for 1 h. The cells were collected bycentrifugation at 0°C, resuspended in 2 ml of ice-cold 0.1 MCaClI-0.25 M sucrose, and incubated at 0 to 5°C for 24 to 72h. Plasmid DNA was added to 0.2 ml of CaCl2-treated cells,and the mixture was incubated at 0°C for 1 h, followed by a2-min heat pulse at 42°C. The cells were plated onto selec-tive plates after 90 min of incubation in L broth. The plateswere incubated for 1 day at 30°C.

Construction of recombinant plasmid colony collection. ThepACYC177 vector was first passed through TT101 for themodification. The chromosomal DNA prepared from TT101was partially digested with the endonuclease HindIII, fol-lowed by ligation with the HindIII-digested phosphatase-treated pACYC177 DNA. The DNA thus ligated was trans-formed into TT122, and then Apr Kms transformants wereselected. Thus, 3,000 transformant colonies were collectedand found to contain recombinant plasmids 4 to 25 kilobasepairs (kb) in total size. Digestion, ligation, and the phospha-tase treatment of DNA, agarose gel electrophoresis, andminiscreen preparation of plasmid DNA were as describedpreviously (5).

Cloning and subcloning of the aspA+ gene. TK237 wastransformed with each of the 10 mixtures of recombinantplasmid DNAs which were prepared from the mixed culturesof 300 colonies out of the colony collection constructed asdescribed above. The resulting transformants were selectedon the glutamate medium containing 25 p.g of ampicillin perml to clone the aspA+ gene. Subclones derived from pVT104(see Fig. 1) were isolated by screening recombinant plasmidDNAs in TK237.

Determination of copy number. Cells were grown withshaking in L broth or in ASP medium to a density of ca. 109cells per ml, washed twice with 10 mM Tris-hydrochloride-1mM EDTA (pH 7.5), and lysed with lysozyme-sodiumdodecyl sulfate. The lysate was treated with 50 ,ug of RNaseA (Sigma Chemical Co.) per ml at 37°C for 1 h and then with200 ,g of proteinase K per ml at 37°C for 1 h. Copy numberwas determined by the method of Projan et al. (16), using thesample prepared as described above.Enzyme assays. Extracellular nuclease activity was as-

sayed by the method of Nestle and Roberts (15) and ex-pressed as units of optical density at 260 nm of acid-solublematerials enzymatically released per 20 min per ml ofsupernatant fluid. Aspartase activity was measured by themethod of Sato et al. (20) and expressed as micromoles ofL-aspartic acid formed per minute per milligram of protein.Other methods. Sodium dodecyl sulfate-polyacrylamide

gel electrophoresis was performed as described by Laemmli(11). Protein concentrations were determined with Bio-Radprotein reagent.

RESULTSIsolation of the extracellular nuclease-deficient mutant. We

first examined whether S. marcescens Sr41 produces theextracellular nuclease, because most strains of S. marces-cens produce the nuclease (18). The wild-type strain of S.marcescens Sr41 produced a considerable amount of theextracellular nuclease (Table 2). This nuclease might frus-trate our attempts to transform S. marcescens Sr41 strainswith plasmid DNA and to prepare the DNA from the cells.Thus, we attempted to isolate an extracellular nuclease-de-ficient mutant. Only one colony of such a mutant was

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TABLE 2. Effect of extracellular nuclease on efficiency oftransformation with pACYC177

No. ofStrain Nuclease transformants peractivity' ml of transformation

culture'8000 27.1 1.3 x 103TT1O1 <0.5 1.4 x 104

a Activity is expressed as units of optical density at 260 nm per 20 minutesper milliliter of supernatant fluid.bTo 0.2 ml of competent cells, 0.5 ,ug of pACYC177 DNA prepared from

TT101 was added. Transformants in 0.1 ml of transformation culture werescored on L broth agar containing 50 Fg of kanamycin per ml.

obtained among 3,068 colonies screened on the DNA-nutri-ent agar plates. Transformation of the mutant TT101, whichlacks the ability to produce the nuclease, was 10 times moreefficient than that of the wild-type strain (Table 2). Thismutant can serve as a useful host for transformation exper-iments, although even the increased frequency is two tothree orders of magnitude lower than that in E. coli K-12.

Isolation of the antibiotic-sensitive mutants. S. marcescensstrains, even when no extrachromosomal resistance factor isobserved, are generally more resistant to various antibioticsthan is E. coli K-12, and then the higher concentration isrequired for selection of transformants, as shown by Reid etal. (17). In the experiments shown in Table 2, the transform-ants of TT101 were scored on L agar plates containing 50 ,ugof kanamycin per ml. When ampicillin resistance was usedas a selective marker, 500 ,ug of the antibiotic per ml wasrequired to select the transformants. The concentrationswere higher than those used for E. coli K-12 strains.For ease of manipulation, we attempted to isolate the

mutants showing enhanced sensitivity to the antibiotics. Wefirst isolated the mutant showing the enhanced sensitivity toampicillin. The mutant TT102 was unable to grow in thepresence of 12.5 ,ug of ampicillin per ml, whereas the parentstrain TT101 grew even with 100 ,ug of the antibiotic per ml(Table 3). Next, the mutants showing enhanced sensitivity tokanamycin were isolated from TT102; one of them, TT122,showed a higher sensitivity to kanamycin than did TT102(Table 3). The mutants were readily isolated by the methoddescribed above. Using the mutant TT122 as the host, wecould isolate the transformants with vector pACYC177 byselecting for resistance to ampicillin at 25 ,ug/ml or tokanamycin at 12.5 ,ug/ml. The concentrations of the antibi-otics employed for TT122 were significantly reduced ascompared with those used for TT101, and similar to thoseused for E. coli K-12. On the other hand, we could notisolate mutants showing enhanced sensitivity to tetracycline.S. marcescens Sr4l was resistant to 100 ,ug of the antibioticper ml.

Isolation of the restrictionless mutant. When pBR322 orpACYC177 was used for transformation of TT122, thefrequency of transformants was two to three orders ofmagnitude lower with the plasmid DNAs prepared from E.coli than with those from S. marcescens Sr41. In addition,when the larger plasmid pVT116 DNA prepared fromC600r-m- cells was used, we obtained no transformants atall. These findings suggest that S. marcescens Sr4l containsa restriction endonuclease. Thus, we attempted to isolaterestrictionless mutants. Such mutants are expected to betransformed with plasmid DNAs from E. coli and S. marce-scens at a similar frequency. By using the method describedabove, we isolated eight mutants showing the expectedproperties from among 51 transformants obtained from the

TABLE 3. Enhanced sensitivity to antibiotics in mutants ofS. marcescens Sr4l

Growtha in L broth containingAntibiotic Strain (parent) antibiotic (jig/ml)

100 50 25 12.5 6.25 3.13 0

Ampicillin 8000 (wild type) + + + + + + + + + + + + +TT1O1 (8000) + ++ ++ ++ ++ ++ ++TT102 (TT101) - - - - + ++ ++TT122 (TT102) - - - - + ++ ++

Kanamycin 8000 (wild type) - - + + + + + + + +TT102 (TT101) - - + + + + + + + +TT122 (TT102) - - + ++

a Growth was turbidimetrically determined. Symbols: -, no growth; +,optical density at 660 nm <0.5; ++, optical density at 660 nm > 0.5.

three separate transformation cultures. The representativemutant TT392 was efficiently transformed with both types ofpVT116 DNA, whereas the parent strain TT122 was nottransformed with the DNA prepared from E. coli (Table 4).In addition, pVT116 DNA prepared from TT392 was foundto transform the restriction-positive strain TT122. Theseresults demonstrate that TT392 is a restrictionless, modifi-cation-positive mutant. The others were mutants like TT392(data not shown). The mutant strain TT392 was used in thefollowing cloning experiments.

Primary cloning of the aspA+ gene of S. marcescens. Arecombinant aspA+ plasmid would be isolated by shotguncloning experiments with TK237. In strain TT122, however,we had constructed the recombinant plasmid colony collec-tion which would serve for cloning of genes specific to S.marcescens. Thus, 10 mixtures of recombinant plasmidDNAs prepared from the colony collection were screened bytransformation for a possible complementation of the aspAmutation in TK237. AspA+ Apr transformants of TK237were obtained from 4 of the 10 transformation cultures. Allof 12 transformants tested contained the recombinantplasmid, which was composed of an 11.6-kb HindIII frag-ment of the chromosome and the 3.7-kb HindIII-digestedpACYC177. These individual recombinant plasmids con-ferred the AspA+ phenotype on TK237 upon a secondtransformation. Thus, the 11.6-kb fragments were consid-ered to be an identical DNA segment carrying the aspA+gene of S. marcescens. One of the recombinant plasmidswas designated pVT104 and used for further study.

Expression of the S. marcescens aspA+ gene in E. coli K-12.As described above, pVT104 complemented the aspA muta-tion of TK237. This indicated that the aspA+ gene of S.marcescens Sr41 was cloned in pVT104 and that the gene

TABLE 4. Transformation of the restrictionless mutant TT392with pVT116a DNAs

No. of transformantsb (per ml oftransformation culture) with pVT116 DNA

Strain prepared from:

C600r-m- TT392

TT122 0 400TT392 700 490

a A 12.4-kb Hindlll fragment of chromosome DNA of TT101 was clonedinto the HindIlI site of pACYC177 (unpublished data).

b Transformation was carried out as described in the footnotes to Table 2and the test. Transformants were scored on L broth agar containing 50 ,ug ofampicillin per ml.

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TABLE 5. Expression of the aspartase gene of S. marcescens inE. coli

taof ~~Stability ofStrain Sp actao plasmidbaspartase (%)

TK237 <0.2 NDCTK237(pACYC177) <0.2 91TK237(pVT104) 43.1 53C600r-m- 13.4 NDC600r-m-(pACYC177) 13.2 99C600r-m-(pVT104) 124.2 62

a Specific activity is expressed as micromoles of L-aspartic acid formed perminute per milligram of protein.

b Stability of the plasmid is expressed as the percentage of ampicillin-resistant cells in the culture used for aspartase assay.cND, Not determined.

Plosmid VectorH

pVTI 04pVTI 25pVTI 28pVTI 30pVTI 33pVTI50

pACYCI77pACYCI77pACYCI77pACYCI77pBR322pBR322

0

Insert DNASo S SaE

- "/E

- -

2 4 6 8 10

aspAH

--

12 kbFIG. 1. Physical map of the insert DNA in plasmid pVT104 and

the recombinant plasmids constructed from it by deletion andsubcloning. The vector into which each of the inserts was clonedand the corresponding phenotype are shown alongside. Insertspresent in plasmids are indicated by heavy lines. The abbreviationsused for the restriction sites are: H, HindIll; Sa, Sall; S, SmaI; E,EcoRI.

was expressed in E. coli K-12. Further evidence for thesepoints was provided by assaying the aspartase activity(Table 5). No detectable activity was observed in TK237 andTK237(pACYC177). In contrast, TK237 carrying pVT104showed high aspartase activity. Furthermore, the aspartaseactivity was 9 times higher in C600r-m-(pVT104) than incontrol strains. The amplified level was lower than thatexpected upon the plasmid copy number (22 copies perchromosome [2]). To make this point clear, we examined theplasmid stability. After ca. 10 generations of growth, 40 to50% of cells lost the recombinant plasmid (Table 5). Theresult evidently indicates that the instability of pVT104 isresponsible for the lower amplified level.

Subcloning of the aspA+ gene from pVT104. A restrictionmap of the insert DNA in pVT104 is shown in Fig. 1. Todetermine the location of the aspA+ gene on the insert, weconstructed several recombinant plasmids carrying portionsof the aspA+ region (Fig. 1). Two deletion derivatives,pVT125 and pVT128, were obtained from pVT104 by usingendonucleases SmaI and HindlIl. Plasmid pVT130 was

constructed by deleting a 1.4-kb EcoRI segment frompVT128. The EcoRI segment was subcloned into pBR322 togive pVT133. A 2.8-kb SalI-EcoRI-EcoRI fragment was

subcloned in the SalI-EcoRI interval of pBR322 to generatepVT150. Among the plasmids obtained as described above,plasmids pVT128 and pVT150 were found to carry theaspA+ gene upon both a possible complementation of theaspA mutation and an overproduction of aspartase. Fromthese results, we concluded that the structural gene foraspartase was located on the 2.8-kb fragment of the insertand that the coding region was cut with the endonucleaseEcoRI.

Overproduction of aspartase in S. marcescens. The wild-type strain was used instead of TT392 as the host to excludeany unexpected effect of the mutagenesis on overproductionof the aspartase. The recombinant aspA+ plasmids were

isolated in the E. coli mutant. Therefore, we were unable totransform directly the larger plasmids pVT104 and pVT128into the wild-type strain of S. marcescens Sr4l whichcontains a restriction activity. Accordingly, the recombinantplasmids were first transformed into the restrictionless mu-

tant TT392, followed by transformation of the wild-typestrain with the plasmid DNAs prepared from TT392. Theresulting transformants, 8000(pVT104), 8000(pVT128), and8000(pVT150), were examined for overproduction of theaspartase.When cells were grown at 30°C, the aspartase activity was

7, 9, and 19 times higher in the strains carrying pVT104,

pVT128, and pVT150, respectively, than in the control strain(Table 6). The stability of the plasmids was also examined.After ca. 10 generations of growth, ca. 50, 30, and 1% of thecells lost the recombinant plasmids pVT104, pVT128, andpVT150, respectively. Thus, the smallest plasmid pVT150was the most stable of the three plasmids and conferred thehighest aspartase activity on the host. The variety of theoverproduction level was due to the variety of stability of theplasmids. When the strain harboring pVT150 was incubatedat 40°C, it produced aspartase at up to 39 times the controllevel. The overproduction was twofold over that in the cellsgrown at 30°C.

Total cellular proteins in cells carrying the aspA+ recom-binant plasmids were analyzed on a 12.5% sodium dodecylsulfate-polyacrylamide gel. Overproduction of the aspartaseresulted in a substantial accumulation of polypeptide ofmolecular weight 52,000, and its amount augmented relativeto the activity (Fig. 2).Copy number of plasmids. We measured copy numbers of

plasmids in S. marcescens Sr4l to determine whether thelevel of aspartase overproduction was in proportion to thecopy number and whether the copy number of pACYC177and pBR322 vectors in S. marcescens Sr4l was similar tothat in E. coli K-12 (Table 7). In strain 8000 carryingpVT150, the copy number was ca. twofold higher in the cellsgrown at 40°C than in those grown at 30°C. The twofoldamplification is in agreement with the findings of aspartaseoverproduction described above. This indicates that in thestrain carrying pVT150, the temperature-dependent overpro-duction of the aspartase is related to the copy number rather

TABLE 6. Overproduction of aspartase by the wild-type strain ofS. marcescens carrying the aspA+ recombinant plasmid

Growth Sp acta of Stability ofStrain temp C~C) aspartase plamid

8000 30 12.7 NDC8000 40 13.0 ND8000(pACYC177) 30 11.1 998000(pBR322) 30 12.3 1008000(pVT104) 30 93.5 548000(pVT128) 30 114.9 748000(pVT150) 30 241.5 998000(pVT150) 40 492.7 100

a Specific activity is expressed as micromoles of L-aspartic acid formed perminute per milligram of protein.

b Stability of the plasmid is expressed as described in the footnotes to Table5.

c ND, Not determined.

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MUTANT AS HOST FOR CLONING IN S. MARCESCENS 5

I Z34 5 6 7w .

14.4 K

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel analysis oftotal cellular proteins. Cells were grown overnight in ASP medium,harvested by centrifugation, and washed twice with O.9o NaClbefore being suspended in 50 mM potassium phosphate-100 mMKCl-5 mM P-mercaptoethanol (pH 7.5). Extracts were prepared bysonicating the cells. Electrophoresis was carried out according toLaemmli (11), with a 12.5% sodium dodecyl sulfate-polyacrylamidegel. Protein bands were visualized with Coomassie blue stain. Thepositions of marker protein bands run on the same gel are shownwith their corresponding molecular masses in kilodaltons (K). Lane1, 8000 grown at 30°C; lane 2, 8000 grown at 40°C; lane 3,8000(pBR322) grown at 30°C; lane 4, 8000(pACYC177) grown at30°C; lane 5, 8000(pVT104) grown at 30°C; lane 6, 8000(pVT128)grown at 30°C; lane 7, 8000(pVT150) grown at 30°C; lane 8,8000(pVT150) grown at 40°C.

than a transcription and translation efficiency. A similartemperature-dependent amplification of copy number wasobserved also in pBR322 carried by E. coli C600r-m- and inplasmids pACYC177, pBR322, and pVT150 carried byTT392. In addition, the extracellular nuclease produced bystrain 8000, in the method employed here to determine thecopy number, was found not to interfere with a reproduciblerecovery of the plasmid DNA.

DISCUSSION

The unreproducible and low transformation efficiencyobserved in S. marcescens has been considered to be due, atleast in part, to the presence of extracellular nucleaseproduced by most strains of this species, although there wasno direct evidence for this relationship. Isolation of thenuclease-deficient mutant which showed 10-fold higher trans-

TABLE 7. Copy numbers of plasmids

Growth Copy numberHost strain Plasmid Medium temp ( of plasmid piertm(C)chromosome'TT392 pACYC177 L broth 30 13.1 ± 5.3 (4)TT392 pBR322 L broth 30 14.4 ± 2.2 (4)TT392 pVT150 L broth 30 10.6 ± 1.8 (4)8000 pVT150 ASP medium 30 12.2 ± 1.4 (4)C600r-m- pBR322 L broth 30 14.7 ± 4.2 (5)TT392 pACYC177 L broth 40 20.1 ± 4.5 (4)TT392 pBR322 L broth 40 19.7 ± 4.5 (4)TT392 pVT150 L broth 40 20.1 ± 7.6 (4)8000 pVT150 ASP medium 40 25.0 ± 8.4 (5)C600r-m- pBR322 L broth 40 31.4 ± 7.6 (5)

a Copy number per chromosome was calculated by taking the molecularweight of the S. marcescens chromosome to be 2.5 x 109, pACYC177DNA as2.46 x 106, pBR322DNA as 2.88 x 106, and pVT15ODNA as 4.22 x 106.Values are the mean ± standard deviation of independent trials; the number oftrials is shown in parentheses.

formation efficiency than the wild-type strain confirmed theinhibitory role of the nuclease on transformation. When thismutant strain is used as the host, it is no longer necessary toheat cells at 65°C, as Reid et al. reported (17), to get a hightransformation efficiency. We also found that in S. marces-cens Sr4l, transformation efficiency in cells incubated for 24to 72 h in cold 0.1 M CaC12-0.25 M sucrose increased ca.10-fold over cells used immediately after treatment withcalcium chloride. Similar results were reported with E. coliK-12 cells (4). Thus, it is now possible to trarnsform S.marcescens cells at an efficiency roughly 100 times higherthan before. The extracellular nuclease also interferes with areproducible recovery of plasmid DNA from S. marcescenscells, as shown with strain HY (22). This problem could bepartly eliminated by washing cells before extracting DNA,as was done routinely in this experiment. By use of thenuclease-deficient mutant, however, such a problem couldbe totally solved, as described for strain HY (22).When the larger plasmids such as pVT104 (15.5 kb) and

pVT128 (9.0 kb) prepared from E. coli K-12 were used, wecould not transform the wild-type strain of S. marcescensSr41. This and other evidence leads us to the assumptionthat S. marcescens has a restriction enzyme which degradesincoming foreign DNAs. That this is the case was proved byisolating mutants which could be transformed, at a similarfrequency, by pVT116 DNAs prepared from E. coli and S.marcescens.The restrictionless, modification-positive mutant TT392 is

considered to be of great importance for the application ofrecombinant DNA techniques to S. marcescens Sr41, be-cause it can serve as an intermediate host for the transfer ofcloned genes from an E. coli strain to S. marcescens mutantswhich are useful for production of amino acids and othermetabolites. Thus, we now have favorable means to clone S.marcescens genes and to make them express in S. marces-cens mutant cells. The overall procedures may be summa-rized as follows: cloning of a S. marcescens gene in anappropriate E. coli mutant as host, transformation of TT392with the cloned DNA for its Serratia modification, and thesecond transformation of desired S. marcescens mutantswith the cloned DNA prepared from TT392. The successfulcloning of the aspA+ gene of S. marcescens and its expres-sion in the wild-type strain of S. marcescens proved thevalidity of these procedures. Thus, TT392 can provide theadvantage of using a wide variety of both mutants andcloning systems in E. coli for recombinant DNA manipula-tions in S. marcescens Sr4l.The reason pVT104 and pVT128 were unstable is not

clear. Vectors pACYC177 and pBR322 themselves werestable in S. marcescens Sr4l. The overproduction of aspar-tase and the larger overall size of the recombinant plasmidsare not responsible for their instability, because pVT150(overproduction of aspartase) and pVT116 (a larger overallsize; unpublished data) are stable. One possible explanationis that a product(s) of gene(s) carried by pVT104 and pVT128would interfere, at the amplified level, with stable replicationof the plasmids or with normal growth of host cells.

Strain C600r-m-(pVT104) was similar to 8000(pVT104)with regard to the overproduction of aspartase and theinstability of the plasmid. The copy number of pACYC177 inS. marcescens was also similar to that in E. coli describedpreviously (2). These results indicate that the aspartase geneof S. marcescens carried by pVT104 is expressed in E. colias well as in S. marcescens. In addition, we found that thelevel of the aspartase overproduction was in proportion tothe copy number of pVT150 per cell, calculated to be 19 and

VOL. 161, 1985

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6 TAKAGI AND KISUMI

40 in cells grown at 30 and 40°C, respectively, based on ourresults and those described by Cooper and Helmstetter (3).

In summary, the mutant TT392 has made it possible for S.marcescens Sr4l strains to serve as a good host for recom-binant DNA manipulations.

ACKNOWLEDGMENTSWe are grateful to Ichiro Chibata, senior manager of the Research

and Development Division of our company, for encouragement.

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