APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1985, p. 441-4460099-2240/85/080441-06$02.00/0Copyright © 1985, American Society for Microbiology

Recombinant Plasmid Conferring Proline Overproduction andOsmotic Tolerance

MICHAEL W. JAKOWEC,'t LINDA TOMBRAS SMITH,'* AND ABHAYA M. DANDEKAR2

Plant Growth Laboratory1 and Department ofPomology,2 University of California, Davis, California 95616

Received 29 January 1985/Accepted 21 May 1985

A recombinant plasmid carrying the proBA (pro-74) mutant allele which governs osmotic tolerance andproline overproduction was constructed by using the broad-host-range plasmid vector pQSR49. The physio-logical, biochemical, and genetic properties of strains carrying the pQSR49 derivatives pMJ101 and pMJl,mutant and wild type, respectively, were investigated. pMJ101 conferred enhanced osmotolerance comparedwith strains carrying the wild type, pMJl. These results are in contrast to those obtained previously withstrains carrying recombinant plasmids based on pBR322 that failed to confer the osmotic tolerance phenotype.y-Glutamyl kinase (first step in proline biosynthesis) from strains carrying pMJ101 was 200-fold less sensitiveto feedback inhibition than was the wild-type enzyme. As expected, the intracellular proline levels of strainscarrying pMJl01 were more than an order of magnitude higher than those of the wild type. An analysis of copynumber revealed that the pQSR49 constructs were present in the cell at a level six- to eightfold lower than thoseof the pBR322 recombinants, which may account for the difference in phenotype. We found that the geneticstability of the pQSR49 derivative in a variety of gram-negative bacteria was dependent on the insertorientation and the presence of foreign DNA on the plasmid. These factors may be significant in future studiesaimed at expanding the osmotolerance phenotype to a broad range of gram-negative bacteria.

The ability of bacterial cells to adapt to changes in theosmotic strength of their environment is of crucial impor-tance to their survival. Bacteria have thus evolved a varietyof adaptive mechanisms, one of which is the intracellularaccumulation of osmoprotective compounds (17). In thismechanism it is thought that the cell accumulates low-molecular-weight nitrogenous compounds, such as proline,to balance the osmotic strength of the cytosol with that of theenvironment (4, 23). Indirect evidence for this adaptiveprocess was reported by Measures (23) and Tempest et al.(29), who surveyed a variety of bacteria grown under con-ditions of high osmolarity and found that they accumulatedhigh levels of proline or other amino acids. The osmoprotec-tive property of proline was first demonstrated by Christian(5, 6), who reported that addition of proline to media ofinhibitory osmotic strength increased the growth rate ofSalmonella oranienburg cells. These observations formedthe basis for the isolation of a rare, proline-overproducingmutation that also confers osmotolerance (7).

This mutation, designated pro-74, was located on theEscherichia coli episome, F'128, harbored by Salmonellatyphimurium; thus, the conjugal transfer of the mutant F'plasmid to different strains of enteric bacteria such asSalmonella spp. (7) and Klebsiella pneumoniae (18) resultedin proline overproduction and osmotolerance. In addition,this mutation led to the enhancement of nitrogenase activityof K. pneumoniae grown in media of high osmolarity (inhib-itory to growth) (18).A 10.4-kilobase (kb) fragment of F'128 containing the

pro-74 mutation was cloned into the plasmid pBR322 andfound to contain the proBA genes (20). For comparativepurposes, the 10.4-kb DNA fragment containing wild-typeproBA genes from F'128 was also cloned into pBR322.proB codes for the first enzyme in the proline biosynthetic

* Corresponding author.t Present address: Department of Molecular Biology, University

of Southerm California, Los Angeles, CA 90089.

pathway, -y-glutamyl kinase, which catalyzes the productionof y-glutamyl phosphate and ADP from glutamate and ATP(1, 12). The second enzyme, the proA gene product, isglutamate-semialdehyde dehydrogenase, which catalyzesthe NADPH-dependent reduction of -y-glutamyl phosphateto glutamate semialdehyde (2, 13). The DNA sequences ofboth proA and proB (9; A. H. Deutch, K. E. Rushlow, andC. J. Smith, manuscript in preparation) are known. Nor-mally, proline biosynthesis is controlled at the enzyme levelby feedback inhibition of -y-glutamyl kinase by proline (1).pro-74 codes for a mutation resulting in a mutated derivativeof -y-glutamyl kinase that is about 2 orders of magnitude lesssensitive to feedback inhibition than is the wild-type enzyme(17).

Unexpectedly, insertion of the pro-74 mutation into theplasmid vector pBR322 results in proline overproduction,but not osmotic tolerance (20). This loss of osmotoleranceled us to construct other recombinant plasmids for thefollowing reasons: (i) to determine the factors that allowexpression of the osmotic tolerance phenotype, and (ii) toexpand the expression of this phenotype to a broad spectrumof gram-negative bacteria. For the studies reported here, thebroad-host-range plasmid vector pQSR49 (24), a derivativeof the Pseudomonas plasmid R1162 (IncP-4), was selectedfor the insertion of the segment of DNA governing prolineoverproduction and osmotic tolerance. A recombinant plas-mid containing the wild-type gene was also constructed andcharacterized. These plasmids have been designated pMJ101and pMJ1 for the mutant and wild type, respectively. Theproperties of these broad-host recombinant plasmids andtheir ability to confer osmotic tolerance on their bacterialhosts are described. Preliminary results of this research havebeen presented previously (M. W. Jakowec, DNA 3:97,1984).

(This work was completed by M. W. J. in partial fulfill-ment of the requirements for the degree of Master of Scienceat the University of California, Davis.)

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442 JAKOWEC, SMITH, AND DANDEKAR

TABLE 1. Bacterial strains and plasmidsBacterial strain . Source orRelevant characteristics rerncor plasmid reference

E. coliHB101 galK2 rpsL20(Strr) proA2 ATCC collection

lacYl hsdS20 (rBmB ) recAJ3 ara-14xyl-5 mtl-l supE44

CSH26 i(/ac-proBA) thi ara Lab collectionCHS26(rif) CHS26 Rifr This work

K. pneumoniaeM5A1 Wild type Lab collectionM5A1 (pro-) Pro- Lab collection

S. typhimuriumLT2(rif) Rifr K. SandersonSA2513(rif) Pro- Rifr K. Sanderson

pLA1 pBR322 containing 20proBA

pLA101 pBR322 containing pro- 2074 mutation in proBA

pQSR49 Broad host range cloning 23vector derived fromRSF1010, Strr Cbr

pRK2013 ColEl replicon 11containing transferfunction of RK2

pMJ1 pQSR49 containing This workproBA

pMJ2 Same as pMJ1 This workwith insert in oppositeorientation

pMJ101 pQSR49 containing pro- This work74 mutation in proBA

pMJ102 Same as pMJ101 with This workinsert in oppositeorientation

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Bacterialstrains and plasmids used in this study are described in Table1. The complex medium used was L broth, and the minimalmedium was M63 (25). The Klebsiella minimal medium usedin this study was described previously (16). Solid media alsocontained 15 g of agar per liter. Supplements, when used,were 2 mM proline, 0.6 mM thiamine, 1 mM L-azetidine-2-carboxylate, 50 mg of streptomycin sulfate per liter, 50 mg ofsodium ampicillin per liter, 25 mg of tetracycline per liter, and100 mg of rifampin per liter.For monitoring plasmid stability, bacterial strains harbor-

ing plasmids were grown to stationary phase in L broth orM63 medium with appropriate antibiotics. Cells werewashed, suspended in sterile saline, and diluted to about 200cells per ml with L broth. They were grown at 37°C in ashaker bath, and aliquots were withdrawn at various times,diluted with saline, and plated onto L broth plates. Cellnumbers determined from colony counts were used to cal-culate generation number. Each colony was screened for theinsert by growth on minimal plates and for the vector bygrowth on L broth plates with antibiotics.

Determination of intracellular proline. To measure intra-cellular proline, soluble molecules were extracted from thecells with 70% ethanol (7), and the amount of soluble prolinewas determined by a bioassay. E. coli CSH26, which carries

a deletion of proBA, was used as an indicator strain. Thecellular extract containing proline was added to about 104cells per ml, and the optical density at 550 nm was deter-mined after 12 h of incubation at 37°C with shaking.DNA extraction and restriction analysis. Restriction

endonucleases EcoRI and HindIII were purchased fromBethesda Research Laboratories, Inc., and BglII was pur-chased from New England BioLabs. T4 DNA ligase was agift from G. Heidecker. Conditions for restriction endonu-clease digestions and ligations of DNA were as specified bythe manufacturers.The rapid DNA isolation procedure developed by

Birnboim and Doly (3) was used to determine plasmid size.Electrophoresis of DNA fragments was carried out with0.8% agarose-89 mM Tris-89 mM boric acid-2.5 mM EDTA(pH 8.3). Samples were run at approximately 5 V/cm,stained with ethidium bromide, and photographed. X phageDNA digested with HindIII was used as a standard formolecular weight determinations.

Triparental mating. Recombinant plasmids were trans-ferred by using the triparental mating procedure of Ditta etal. (10), in which E. coli HB101 (pRK2013) was used as thesource of the mobilizing plasmid pRK2013 (11). Transconju-gants were selected on solid media for resistance to thefollowing antibiotics: for E. coli CSH26(rif) rif str, L broth;for K. pneumoniae M5A1 (pro-) str, Klebsiella minimalmedium; for S. typhimurium LT2(rif) and SA2513(rif) rif stramp, L broth medium. The transfer frequency was deter-mined by comparing the number of recipients receiving theplasmid with the total number of recipients.

Construction of pMJl and pMJ101. Plasmid DNA wasisolated from E. coli CSH26(pLA1) and CSH26(pLA101),which harbor wild-type and mutant proBA, respectively.pLA1 and pLA101 are recombinant plasmids that wereconstructed by inserting the DNA into the EcoRI site ofpBR322 (20). The plasmid DNA from the above strains wasdigested with the restriction endonuclease EcoRI. Althoughthe fragment containing proBA was reported to be 10.4 kb(20), it appeared slightly larger to us (10.6 kb). The 10.6-kbDNA fragment containing proBA was separated from vectorDNA by agarose gel electrophoresis (0.8% agarose run in 40mM Tris-acetate-2 mM EDTA). The DNA was removedfrom the gel by electroelution and purified by passagethrough a NACS column (Bethesda Research Laboratories).This fragment was ligated to pQSR49 which had beendigested with EcoRI. The ligation mixture was transformedinto E. coli CSH26(AproBA) as recipient by using the CaC12method described by Mandel and Higa (21). Recombinantscontaining wild-type proBA were selected on solid minimalmedium containing streptomycin and ampicillin. For mutantrecombinants the solid medium also contained L-azetidine-2-carboxylic acid, to select for proline overproduction (7).Transformants were subsequently screened for tetracyclinesensitivity to demonstrate loss of the pBR322 vector. Re-striction analyses of the transformant colonies were carriedout with EcoRI to characterize the recombinant plasmids.Enzyme and protein assays. The enzyme activity of y-

glutamyl kinase, the proB gene product, was measured bythe method of Hayzer and Leisinger (12), in which y-glutamyl phosphate is converted to its hydroxamate. Toderepress nitrogenase activity, K. pneumoniae cultures weregrown as described by Le Rudulier and Bouillard (16), andthe nitrogenase activity was determined by the reduction ofacetylene to ethylene (26). Protein concentrations of bacte-rial extracts and whole cells were determined by the methodof Lowry et al. (19).

APPL. ENVIRON. MICROBIOL.

RECOMBINANT PLASMID CONFERRING OSMOTIC TOLERANCE

Copy number estimation. L broth (100 ml) supplementedwith 50 ,ug of ampicillin per ml was inoculated to an opticaldensity of 0.04 at 550 nm with a fresh culture of E. coliCSH26 harboring pLA1, pLA101, pMJ1, or pMJ101. Thecells were grown at 37°C for 3 to 4 h to a cell density of 1 x108 to 2 x 108 cells per ml. The number of viable cells weredetermined, after appropriate dilutions, on L agar platessupplemented with 50 jig of ampicillin per ml. The remainderof each culture was used for plasmid isolation by the alkalinelysis procedure (22). Samples of the purified plasmids weresuspended in 1 mM Tris-hydrochloride-0.1 mM EDTA (pH8.0) and brought to a final volume proportional to the numberof cells from which each plasmid was isolated (100 ,lI per1010 cells). Thus, the concentration of plasmid from differentcultures could be directly compared. A 16-,u portion of eachDNA sample was digested with the restriction endonucleaseEcoRI, and the fragments were resolved by agarose gelelectrophoresis with 0.8% agarose. The DNA was stainedwith 0.5 jxg of ethidium bromide per ml for 30 min, rinsed for30 min with distilled water, and photographed with type 55Polaroid film. The negative was scanned densitometricallywith a Biomed model SL-TRFF soft laser scanningdensitometer. Areas under the curves representing the10.6-kb insert were calculated and converted to micrograms.A scan from an EcoRI digest of 1 ,ug of a highly purifiedpLA101 DNA sample was used as a standard. The concen-tration of the DNA standard was determined by assumingthat the absorbance at 260 nm is 1 optical density unit per 50,ug of double-stranded DNA (22). The copy number wasestimated by assuming that the molecular weight of eachbase is 300.

RESULTSConstruction and stability of recombinant plasmids. The

strategy for the construction of the recombinant plasmids is

E E

Eco R I ,Digest ,

E E

a~~~~~~~E

T4 DNALigase

B

E pMJBg

E

B

H

B B

E p E

B~~~~BH E

Bg H

B

E pM 102)

H B

Bg

FIG. 1. Strategy for insertion of proline proBA genes intopQSR49. The proBA operon is indicated by the heavy black line.pLAi and pLA101 were digested with EcoRI, and the 10.6-kbfragment of DNA containing proBA was purified. These fragmentswere ligated to pQSR49 previously digested with EcoRI. Plasmidswith inserts in both orientations were isolated.

FIG. 2. Restriction endonuclease analysis of recombinant plas-mids. Lanes 1 and 2, EcoRI digests of pLA1 and pLA101, respec-tively; lane 3, EcoRI digest of pQSR49; lane 4, HindIll total digestof bacteriophage X DNA. Lanes 5 through 14, digests of pMJ1,pMJ2, pMJ101, and pMJ102 as follows: 5, EcoRI digest of pMJ1; 6,BamHI digest of pMJ1; 7, HindIll digest of pMJ1; 8, EcoRI digest ofpMJ2; 9, BamHI digest of pMJ2; 10, EcoRI digest of pMJ101; 11,BamHI digest of pMJ101; 12, HindIIl digest of pMJ101; 13, EcoRIdigest of pMJ102; 14, BamHI digest of pMJ102.

outlined in Fig. 1. The wild-type and mutant segments ofDNA were each ligated to an EcoRI digest of pQSR49.Because the DNA fragments can be inserted in either of twoorientations, four recombinant plasmids resulted. Digestionwith the restriction endonuclease BamHI was used to deter-mine the orientation of the insert (Fig. 2). As the wild-typeand mutant inserts yield identical restriction patterns withseveral restriction endonucleases (20; A. M. Dandekar, M.W. Jakowec, and L.-S. Gong, Fed. Proc. 42:2162, 1983), theorientation of both the wild-type and the mutant plasmidscan be determined with the same restriction enzymes. In oneorientation, BamHI digestion of pMJ1 (wild type) andpMJ101 (mutant) resulted in two fragments of DNA, oneapproximately 20 kb in length and the other 5 kb, whereasBamHI digestion of the recombinants in the opposite orien-tation (pMJ2 and pMJ102 for wild type and mutant, respec-tively) resulted in an 11- and a 14-kb fragment of DNA.Although the pMJ1 orientation occurred more frequentlythan that of pMJ2 (18 of 22 confirmed insertions), thisorientation is even more favored with the proline-overproducing derivatives, for which 40 of 41 insertionswere of the pMJ101 type.An experiment was carried out to determine whether the

genetic instability of one of the orientations might accountfor this biased ratio. Stability was simply defined as theability of the bacterial cell to maintain the vector with theinsert under nonselective conditions (i.e., in complex mediacontaining no antibiotics). Under these conditions, it wasfound that over a period of 23 generations, pMJ1 and pMJ101were maintained intact (Fig. 3); however, cells carryingpMJ2 or pMJ102 showed an 80% loss of the insert after 23generations. The vector alone, however, was stably main-tained, a point that is discussed below.

Analysis for production of an altered form of -y-glutamylkinase. It was previously reported that -y-glutamyl kinasefrom the proline-overproducing mutant containing pro-74 isabout 2 orders of magnitude less sensitive to feedbackinhibition than is the wild-type enzyme (17). To demonstratethat no changes in the sensitivity to feedback inhibitionoccurred during the construction and handling of pMJ1 and

443VOL. 50, 1985

444 JAKOWEC, SMITH, AND DANDEKAR

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Generation

FIG. 3. Analysis of the stability of plasmWiin E. coli CSH26 grown under nonselecti,CSH26 containing either pMJ101 (@) or pMJlbroth, and the percentage of the cells carryidetermined as a function of the number ofresults were obtained with pMJ1 and pMJ2 (

pMJ1O1, -y-glutamyl kinase activity wfunction of proline concentration in-y-Glutamyl kinase activity from E. colcreased to half maximum at about 0.05 n-y-glutamyl kinase from strain CSH2(about a 200-fold increase in proline conca similar level of inhibition (Fig. 4). Tithat this construct harbors the original Imutation.Phenotype of strains carrying pMJl

experiments in this section are concern

15

of recombinant plasmids to different species of entericbacteria and the analysis of the resulting phenotype. Theresults show that pMJ1O1 confers proline overproductionand, unlike the pro-74 clone in pBR322 (20), confers osmotictolerance.The recombinant plasmids were transferred to E. coli, S.

typhimurium, and K. pneumoniae strains at frequencies ashigh as 30% (percent transconjugants per recipient) (Table2). Both pMJ1 and pMJ1O1 were transferred at similarfrequencies, indicating that the mutation itself did not alterthe transfer rate from species to species. Transfer to twononenteric, gram-negative bacteria, Pseudomonas putidaand Alcaligenes eutrophus, was attempted, but neither wild-type nor mutant recombinant plasmid could be detected inthese hosts. This result is discussed below.

Properties of E. coli and K. pneumoniae strains containingpMJ1 or pMJ1O1 are given in Table 3. Intracellular prolineconcentration, growth rates, and nitrogenase activity were

'2 determined in the absence and presence of osmotic stress.16 20 24 The entries in the first two columns of Table 3 show the

intracellular proline concentrations in E. coli and K. pneu-is pMJ1O1 and pMJ102 moniae harboring the recombinant plasmids. These datave conditions. E. coli illustrate two points. First, the mutation confers proline102 (0) was grown in L overproduction and second, the intracellular proline concen-ing intact plasmid was tration is further increased by increases in the osmoticgenerations. Identical strength of the growth medium. For E. coli strains grown in[data not shown). the absence of added salt, strain CSH26(pMJ1) (wild type)

contained 8.8 nmol of proline per mg of protein, whereasstrain CSH26(pMJ1O1) (mutant) contained about 10 times

ias monitored as a that level of proline. At 0.55 M NaCl, E. colithe assay mixture. CSH26(pMJ101) cells contained about 40-fold more proline{i CSH26(pMJ1) de- than did the wild-type strain CSH26(pMJ1). Hence, thenM proline, whereas mutant maintains a higher level of proline than does the wild6(pMJ1O1) required type in minimal medium, but this difference increases:entration to achieve severalfold when cells are grown in a medium of highhese results confirm osmotic strength.feedback-insensitive A modulation of the intracellular proline concentration

caused by the osmotic strength of the growth medium wasand pMJ1Ol. The also observed with K. pneumoniae. In the absence of added

led with the transfer NaCl, strain M5A1(pMJ1O1) (mutant) contained 25 times theamount of proline as did strain M5Al(pMJ1) (wild type).This value increased about 80-fold when cells were grown in0.65 M NaCl. Interestingly, the wild type, K. pneumoniaeM5Al, without plasmid, contained about half the amount ofproline as did K. pneumoniae cells with pMJ1.To correlate intracellular proline levels with protection

against osmotic stress, growth rates of strains containing therecombinant plasmids were measured at low and high os-motic strength (Table 3). E. coli CSH26(pMJ1) had a growth

Proline Concentration (mM)FIG. 4. Inhibition of -y-glutamyl kinase by proline. The specific

activity of -y-glutamyl kinase from E. coli CSH26 containing eitherpMJ1 (0) or pMJ101 (0) was determined as a function of prolineconcentration in the assay mixture, as described in the text.

TABLE 2. Transfer of broad-host-range plasmids containingproline genes into enteric bacterial recipientsa

Donor Recipient Transfer frequencyb (%)

pMJ1 E. coli CSH26(rif) 39pMJ1 K. pneumoniae M5A1 (pro-) 12pMJ1 S. typhimurium LT2(rif) 3.7pMJ1 S. typhimurium SA2513(rif) 4.3pMJ101 E. coli CSH26(rif) 31pMJ101 K. pneumoniae M5A1 (pro-) 14pMJ101 S. typhimurium LT2(rif) 5.9pMJ101 S. typhimurium SA2513(rif) 6.1

a The triparental mating procedure of Ditta et al. (10) was used with E. coliCHS26 harboring either pMJ1 or pMJ101 as donor.

b Transfer frequtency is expressed as the percentage of transconjugants perrecipient.

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APPL. ENVIRON. MICROBIOL.

RECOMBINANT PLASMID CONFERRING OSMOTIC TOLERANCE

TABLE 3. Intracellular proline concentration, growth rates, and nitrogenase activity of E. coli and K. pneumoniae harboring therecombinant plasmids pMJ1 and pMJlOla

Intracellular proline (nmol/mg Growth rate (generations/h) Nitrogenase activity (nmoL/hof protein) with: with: per mg of protein) with:

Strain_____________ __

No added NaCl added No added NaCl added No added 0.4 M NaCINaCi NaCi NaCl added

E. coli CSH26(pMJ1) 8.8 20b 0.83 0.17bE. coli CSH26(pMJ101) 90 730b 0.56 0.42b

K. pneumoniae M5A1 4.5 4.3c 0.12c 2,100 185K. pneumoniae M5A1(pMJ1) 8.3 8.9c 0.12c 1,640 110K. pneumoniae M5Al(pMJ101) 210 700C 0.25c 1,440 357

a Experiments with E. coli and K. pneumoniae were carried out in M63 medium (25) and Klebsiella medium (16), respectively. The amount of salt added to themedium in each experiment was chosen to yield the maximum difference in the parameter measured between strains with pMJ1 and pMJ101. To determine growthrates, 0.2% inocula (grown in minimal media) were used. The growth rates of K. pneumoniae grown without salt were similar to those found previously when usingF'128 and F'128 containing pro-74 (18).b0.55 M NaCl added.c 0.65 M NaCl added.

rate of 0.17 generation per h in 0.55 M NaCl; introduction ofthe plasmid pMJ101 into CSH26 resulted in about a 2.5-foldenhancement of the growth rate (0.42 generation per h). Asimilar observation was made with K. pneumoniae cells,with wild-type strains showing a growth rate of 0.12 gener-ation per h in 0.65 M NaCl, whereas the strain harboringpMJ101 showed a growth rate of 0.25 generation per h. Thisincrease in growth rate is similar to that reported previously(18) for the pro-74 mutant allele transferred on F'128 to K.pneumoniae cells.

It has been reported that in K. pneumoniae cells, whole-cell nitrogenase activity is sensitive to osmotic stress andthat proline supplied in the medium or proline overproduc-tion conferred by the F' carrying pro-74 protects nitrogenaseactivity (18). These observations were further investigatedwith our recombinant plasmids. The time course of theinduction of nitrogenase activity from K. pneumoniae cellsgrown in 0.4 M NaCl was followed. When strains weregrown in minimal medium plus 0.4 M NaCl, the nitrogenaseactivity of the strain harboring pMJ101 was threefold greaterthan that of the strain containing pMJ1 and about twofoldgreater than that of strain M5A1 with no plasmid (Table 3).Hence, the pro-74 mutation on a recombinant plasmid(pMJ101) confers the osmotolerance phenotype on nitroge-nase activity.The results in Table 3 show that pMJ101 confers osmotic

tolerance in E. coli and K. pneumoniae cultures. Previously,Mahan and Csonka reported that pBR322 carrying pro-74(pLA101) does not confer osmotic tolerance in S.typhimurium or E. coli strains (20). They suggested that theloss of the phenotype may be due to the high copy number of

TABLE 4. Plasmid copy numiber of pQSR49 and pBR322derivatives in E. coli CSH26

Amt of DNA/ No. ofPlasmida 1.6 x 109 cells' copies/cell(jLg)pMJ1 0.16 9.5pMJ101 0.13 7.5pLA1 1.0 61pLA101 1.0 60

a Plasmids used were pQSR49 derivatives (pMJ1 and pMJ1l1) and pBR322derivatives (pLA1 and pLA101)." The amount of the 10.6-kb DNA fragment containing proBA was quanti-tated from every strain and used to determine the copy number.

the vector pBR322 (20; also see below). We determined thecopy numbers of these plasmids by quantitating from aknown number of cells the number of moles of the 10.6-kbinsert in a DNA restriction analysis (see above). The copynumber of pMJ1 or pMJ101 in E. coli CSH26 was 8 to 10 percell, whereas the copy number of pLA1 or pLA101 was 60(Table 4). Hence, the plasmid copy number with the pQSR49derivative is 6 to 8 times lower than that with the pBR322derivatives. These results are consistent with the hypothesisthat the osmotolerance phenotype is dependent on the copynumber of the plasmid that carries pro-74.

DISCUSSIONThere is increasing interest in the mechanism of osmotic

stress tolerance as well as potential applications of this field.The construction of a recombinant plasmid conferring os-motic tolerance represents a significant step toward en-hancement of stress tolerance in microorganisms. In thisreport we have shown that a recombinant plasmid carrying a10.6-kb DNA segment which contains the pro-74 mutationconfers both proline overproduction and osmotolerance toE. coli and K. pneumoniae. This is the first report of arecombinant plasmid conferring this phenotype.

In a previous report, Mahan and Csonka (20) observedthat a pBR322-based plasmid vector carrying the pro-74mutation failed to give the osmotic tolerance phenotype.They hypothesized that this might be due to the overproduc-tion of the phoE gene product synthesized at inhibitorylevels as a result of the relatively high copy number of thisplasmid. We have found that the copy number of pMJ101conferring osmotic tolerance is almost an order of magnitudelower than that of the pBR322 recombinant clone whichlacks this phenotype. Thus our results are consistent withthe hypothesis that plasmid copy number is an importantparameter for osmotic tolerance involving the pro-74 muta-tion. However, the role of the phoE gene product remains tobe determined.

In addition to plasmid copy number, a number of otherfactors appear to play a significant role in osmotic tolerance;for example, the intracellular concentration of proline, incontrast to total proline production, is an important param-eter (Table 3, columns 1 and 2) (7, 8). The capacity tooverproduce proline, although essential, may not be effec-tive if the proline is excreted or degraded by the cell.Another factor that may be involved in this osmotic modu-lation of intracellular proline concentration is the biosynthe-

445VOL. 50, 1985

446 JAKOWEC, SMITH, AND DANDEKAR

sis of glutamate, the substrate of y-glutamyl kinase. Forexample, it has been shown that in Rhizobium spp. (14, 30)and other species (29), glutamate biosynthesis is regulatedby osmotic stress. By analogy, in E. coli the intracellularconcentration of the substrate glutamate might be limitingfor maximum -y-glutamyl kinase activity when the glutamatepool size is low in the absence of stress. To maintain themassive increase in the proline level required forosmoprotection, one or more of these systems may becomerate limiting and must be taken into account in studies aimedat genetic manipulation of this system.By constructing and testing both possible orientations of

the proBA genes, we have found that the orientation ofDNAinserts strongly influences the stability of replication. Thisproperty did not appear to be associated with the proline-overproducing mutation itself, because both wild-type andmutant plasmids showed identical orientation effects. Themechanism remains unclear.The availability of osmotic tolerance genes carried by

broad-host-range plasmids is the first step in the transfer ofthese genes to other species of bacteria. Our recombinantplasmids were stably transferred to enteric bacteria butcould not be transferred to and maintained in P. putida or A.eutrophus. However, both of these strains behave as recip-ients for the plasmid vector pQSR49 itself (24). During thecompletion of this work, Kim and Meyer (15) reported thatpQSR49 becomes unstable in P. putida only when theplasmid contains foreign (i.e., E. coli) DNA. They proposedthat the foreign DNA induces a specific mechanism foreliminating these plasmids. This mechanism may well be thecause for the lack of transfer of pMJ1 and pMJ101. We arepresently trimming down the size of the pro-74 insert andtesting recombinants of other broad-host-range vectors suchas pRK290 (10) and pSalS1 (27, 28) to increase the range ofbacteria into which these genes can be introduced.

ACKNOWLEDGMENTSWe acknowledge R. C. Valentine for many helpful discussions and

R. Meyer for supplying pQSR49.This work was supported by grants PCM8314246 (to L.T.S.) and

PFR7707301 (to R. C. Valentine) from the National Science Foun-dation.

LITERATURE CITED1. Baich, A. 1969. Proline synthesis in Escherichia coli: a proline

inhibitable glutamic acid kinase. Biochim. Biophys. Acta192:462-467.

2. Baich, A. 1971. The biosynthesis of proline in Escherichia coli,phosphate-dependent glutamate -y-semialdehyde dehydrogenase(NADP), the second enzyme in the pathway. Biochim. Biophys.Acta 244:129-134.

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

4. Brown, C. M., and S. 0. Stanley. 1972. Environment-mediatedchanges in the cellular content of the "pool" constituents andtheir associated changes in cell physiology. J. Appl. Chem.Biotechnol. 72:363-389.

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