Vol. 43, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1982, p. 1272-12770099-2240/82/061272-06$02.00/0

Plasmid DNA in Streptococcus cremoris Wg2: Influence of pHon Selection in Chemostats of a Variant Lacking a Protease

PlasmidROEL OTTO,'* WILLEM M. DE VOS,2 AND JONAH GAVRIELI3

Department of Microbiology1 and Department of Genetics,2 University of Groningen, 9751 NN Haren, TheNetherlands, and Eidgenossische Anstalt fur Wasserversorgung Abwasserreinigung und Gewasserschutz,

CH-8600 Dubendorf, Switzerland3

Received 7 December 1981/Accepted 2 March 1982

Cleared lysates of a proteolytic (Prt+) strain and a naturally occurring non-proteolytic (Prt-) variant of Streptococcus cremoris Wg2 contain equal amountsof covalently closed circular plasmid DNA. An analysis of this plasmid DNA byagarose gel electrophoresis revealed the presence of at least five different plasmidspecies in the Prt+ strain and only three plasmid species in the Prt- variant.Curing studies with acriflavine indicated that a 16-megadalton plasmid determinedproteolytic activity in the Prt+ strain. In energy-limited chemostats inoculatedwith both strains it was observed that the Prt+ strain was replaced by the Prt-variant. This effect was most apparent when the pH of the culture was fixed at avalue above 6.3. No selection for the Prt- variant was observed at pH 5.9. Sincethe two types of organisms contain equal amounts of plasmid DNA, it wasconcluded that the energy gain of the PrtV variants at pH values above 6.0probably has to be found in protein synthesis rather than in plasmid DNAsynthesis.

Preparation of starter cultures of lactic strep-tococci in the dairy industry is a large-scalebatch process. To produce high-quality starters,strain characteristics such as acid production,proteolytic activity, and phage resistance haveto be controlled carefully. Before the prepara-tion of the bulk starter, the mother culture issubjected to a series of transfers. Usually thenumber of transfers is kept to a minimum inorder to minimize contamination with lyticphages. Although in this respect the continuouscultivation of bulk starter cultures offers someclear advantages over the traditional batch pro-cedure (automated process control), the applica-tion of this technique has not found acceptancebecause during continuous cultivation the origi-nal population of starter streptococci is oftenreplaced by a mutant population with undesir-able characteristics. Using a continuous cultureof Streptococcus lactis C2 fed with glucosebroth, McKay et al. (25) showed that the originalpopulation was replaced by lactose-negative mu-tants. The irreversible loss of enzymes acting inlactose metabolism prompted these authors tosuggest that plasmids are involved in the lactosemetabolism of S. lactis C2. In a later series ofreports by the research group of McKay (22, 24),convincing evidence supporting this view wassupplied. It was also shown that the informationfor another important characteristic of starter

streptococci, proteolytic activity, is carried byplasmids (5, 16, 19, 22, 23). It has been knownfor a long time that lactic streptococci becomeless proteolytic during a series of transfers inmilk. This decrease in proteolytic activity hasbeen attributed to a shift from a proteolyticphenotype (Prt+) to a non-proteolytic phenotype(Prt-) (9, 10, 12). The selection of Prt- variantsin continuous cultures is less well documented.The irreversible loss of proteolytic activity uponprolonged cultivation in continuous cultures hasbeen reported by Oberman and Malanowska(28).

In this report we present information on theselection of Prt- variants of Streptococcus cre-moris Wg2 in lactose-limited continuous cul-tures and the effect of pH on the selection ofthese variants. In addition, we provide evidencethat a plasmid determining proteolytic activity ispresent in S. cremoris Wg2.

MATERIALS AND METHODSBacterial strains. Prt+ and Prt- variants of S. cre-

moris Wg2 were obtained from the Dutch Institute forDairy Research (Ede, The Netherlands). Both orga-nisms were routinely maintained in sterile 10% (wt/vol) reconstituted skim milk supplemented with 0.1%tryptone (Difco Laboratories, Detroit, Mich.) andstored at -20°C.

Culture conditions and media. Chemostat cultures

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were grown anaerobically under an N2 atmosphere inglass chemostats with working volumes of 200 ml at30°C and controlled pH, as described by Laanbroek etal. (17). The chemostats were fed with MRS broth (3)containing 0.25% (wt/vol) lactose or yeast extractbroth containing 0.25% (wt/vol) lactose, 0.2% (wt/vol)yeast extract, and 10 mM potassium phosphate bufferadjusted to the required pH (pH 5.9, 6.3, or 6.8).

Differential count of the Prt+ strain and the Pr-variant of S. cremoris Wg2. Samples from culturescontaining the Prt+ strain and the Prt- variant of S.cremoris Wg2 were diluted with sterile 0.1% tryptone(14). These diluted cell suspensions were suspended intransparent citrate-containing milk agar (1). In thismedium the Prt+ strain and the Prt- variant can bedistinguished easily, since Prt+ strains form normal-sized colonies (diameter, 2 to 3 mm) after 48 h ofincubation at 30°C, whereas the Prt- variant formssmaller colonies (diameter, 0.5 mm).Treatment with acriflavine. Two-drop portions of an

actively growing culture of Prt' S. cremoris Wg2 inMRS broth were inoculated into 10-ml portions ofMRS broth containing different acriflavine concentra-tions. After incubation for 18 h at 30°C, each culturewas diluted and suspended in citrated milk agar. Afterincubation for 48 h at 30°C, the plates were scored forthe presence of Prt- variants.

Preparation of cell suspensions for the determinationof proteolytic activity. S. cremoris was cultured inpasteurized (10 min, 80°C) 10o (wt/vol) reconstitutedskim milk. After 12 h of incubation at 30°C, the cellswere harvested, washed, and concentrated as de-scribed by Exterkate (7).The method of Exterkate (7) was used to determine

the proteolytic activity of intact S. cremoris Wg2 cells.Isolation of plasmid DNA. S. cremoris was grown at

30°C to late log phase in modified Elliker broth (6, 15).Cleared lysates were prepared by the method ofKlaenhammer et al. (15). Covalently closed circular(CCC) plasmid DNA was isolated from cleared lysatesby cesium chloride-ethidium bromide (EtBr) gradientcentrifugation (29). CsCI-EtBr gradients were pre-pared with 8 ml of cleared lysate and 1 ml of EtBr (5mg/ml), and the solution was adjusted to a refractiveindex of 1.395 (20°C) with solid CsCl. The gradientswere centrifuged for 65 h at 35,000 rpm in polyalomertubes in a Beckman type 5OTi fixed-angle rotor at150C.The CCC plasmid DNA band was collected, the

EtBr was extracted with CsCI-saturated isopropanol,and the CsCl was removed by dialysis against 10 mMTris-hydrochloride buffer (pH 7.4) containing 1 mMEDTA. In some instances the plasmid DNA wasconcentrated by ethanol precipitation (20).

Radioisotope labeling of plasmid DNA and prepara-tion of total lysates of S. cremoris. For radioisotopelabeling, cells were grown for 6 h in 100 ml of modifiedElliker broth containing 1 mCi of [3H]thymidine (6.7Ci/mmol) and 25 mg of deoxyadenosine. The cellswere harvested, washed with TES buffer (50 mMNaCl, 5 mM EDTA, 30 mM Tris, pH 8.0), and thensuspended in 5 ml of TES buffer containing 25%sucrose, lysozyme (1 ml of a 30-mg/ml solution in TESbuffer), 0.4 ml of 1 M EDTA (potassium salt), and 0.1ml of an RNase preparation (10 mg/ml of TES buffer,previously heated for 3 min at 1000C), and the suspen-

sion was incubated for 20 min at 30°C. Lysis waseffected with 1% NaCl.

Protein was digested with 0.5 ml of a pronase Ppreparation (grade B; 6 mg/ml in TES buffer; previous-ly autodigested for 1 h at 37°C; Calbiochem, La Jolla,Calif.). The total lysates were prepared for CsCl-EtBrgradient centrifugation as described above. After cen-trifugation the tubes were fractionated from the top,and the fractions were assayed for acid-precipitableradioactivity (4). The fraction of CCC plasmid DNAwas estimated from the amounts of radioactivity pres-ent at the positions of the chromosomal and CCCplasmid DNAs.

Restriction endonuclease digestion and gel electropho-resis of plasmids from S. cremoris. Digestion of plasmidDNA with restriction endonuclease EcoRI was per-formed by the method recommended by the manufac-turer (Boehringer, Mannheim, Germany).Gel electrophoresis was performed on horizontal

0.8% (wt/vol) agarose gels (11 by 11 cm; Bio-RadLaboratories, Richmond, Calif.). Tris-acetate buffer(40 mM Tris-acetate, 20 mM sodium acetate, 2 mMEDTA, pH 7.8) was used throughout. Gels were runwith 0.2% bromphenol blue as the tracking dye at 100mA for 3 to 4 h at room temperature. Subsequently,the gels were stained with EtBr (final concentration, 1,ug/mi), and individual plasmid bands were visualizedwith a long-wave UV lamp. Plasmid DNA was extract-ed from gels by the method of Thuring et al. (31).For molecular weight determinations of restriction

fragments, an EcoRI digest of bacteriophage SPP1DNA was added to provide markers (30). The molecu-lar weights of the intact plasmids were determined bymigration relative to reference plasmids. Staphylococ-cus aureus plasmids pC194 (1.8 megadaltons [13]),pUB110 (2.8 megadaltons [18]), pSA2100 (4.7 mega-daltons [13]), and pRN4008 (25.4 megadaltons [27])and Escherichia coli plasmid pJN67 (8.7 megadaltons[32]) were used for this purpose.

Materials. [3H]thymidine was obtained from NewEngland Nuclear Corp., Boston, Mass. All other mate-rials were reagent grade and were purchased fromcommercial sources.

RESULTSProteolytic activity of Prt+ S. cremoris Wg2 and

its Prt- variant. In an extensive study of theproteolytic system of Prt+ S. cremoris HP,Exterkate (7) demonstrated the presence inwhole cells of two cell wall-bound proteolyticactivities, an acid protease with a pH maximumof 5.2 which could be detected at 40°C and aneutral protease for which the optimum condi-tions were pH 7.5 and 30°C.

In a later report Exterkate (8) extended theseobservations to other Prt+ strains of S. cre-moris. We tested Prt+ and Prt- S. cremoris Wg2variants previously grown in milk for proteolyticactivity against 1"CH3-labeled casein. Our re-sults (Fig. 1) revealed clear differences betweenPrt+ and Prt- S. cremoris Wg2. Proteolyticactivity could not be detected in the Prt- variantunder any conditions, whereas considerable pro-

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1 2

oc pWV04\ccc pWVO5QocpWV03 ,

cccpWVO4ccc pWV03\ocpWV02

cccpWVO21

fcocpWVO/1cccpWVO-.'

FIG. 1. Proteolytic activity of Prt+ S. cremorisWg2 (solid lines) and its Prt- variant (dashed lines)determined at 30°C (0) and 40°C (0) as a function ofpH. Proteolytic activity is expressed as acid-solubleradioactivity.

teolysis was observed in the Prt+ parentalstrain.

Plasmid quantity of Prt+ and PrtN S. cremorisWg2. To determine whether the absence ofproteolytic activity in Prt- S. cremoris Wg2 wasdue to a loss of plasmid DNA, cleared lysates ofboth Prt+ and Prt- variants were prepared.These preparations were analyzed for the pres-ence of plasmid DNA by CsCl-EtBr centrifuga-tion. Both strains contained CCC plasmid DNA,as shown by the existence of a dense peakseparate from the chromosomal DNA peak. Toestimate the amount of plasmid DNA in each ofthe strains, the DNA was labeled with [3H]thy-midine, and the plasmid DNA was isolated fromtotal lysates. The amount of plasmid DNA wasestimated from the radioactivity recovered fromthe plasmid peak. This analysis showed that 3.3and 3.5% of the total DNA radioactivity were

present in the plasmid peaks of the Prt+ strainand the Prt- variant, respectively, indicatingthat there was no significant difference in plas-mid quantity between the two strains.

Characterization of plasmid DNAs from Prt+and Prt- S. cremoris Wg2. To determine whetherthe CCC plasmid DNAs present in Prt+ and Prt-S. cremoris Wg2 were composed of differentplasmid species, the plasmid DNAs were puri-fied from cleared lysates of the two strains andanalyzed by agarose gel electrophoresis (Fig. 2).To identify the CCC and open circular (OC)forms of the plasmid species, individual plasmid

FIG. 2. Agarose gel electrophoresis of plasmidDNA from Prt+ (lane 1) and Prt- (lane 2) S. cremorisWg2. Plasmid DNA was purified from cleared lysatesby CsCI-EtBr gradient centrifugation and electropho-resed on a 0.8% agarose gel. The positions of the CCCand OC forms of the individual plasmids of the Prt+and Prt- variants are indicated. OC pWV03 and CCCpWVW4 migrated to the same position.

bands were extracted (31) from an agarose gel,and the individual bands were electrophoresed.Since only CCC forms can give rise to twoseparable bands (one containing intact CCCDNA and one containing OC DNA as a conse-quence of limited nicking during the extraction

TABLE 1. Sizes of plasmids from Prt+ and Prt-variants of S. cremoris Wg2aPresence in: Mol wt Mol wt determined

Plasmid Prt+of intact from the EcoRIPlasmi

+ Prt plasmid restrictionvariant variant (x 106) fragments (xl10)

pWV05 + 16.0 15.6 (4)bpWV04 + + 11.5 11.2 (3)pWV03 + _ 6.1 NDcpWV02 + + 2.9 NDpWVO1 + + 1.5 ND

a The molecular weights of the intact plasmids andthe restriction fragments were estimated by migrationrelative to reference plasmids having known molecularweights (see text) or by calculating the sum of theindividual EcoRI fragments of each plasmid, whichwere calibrated with the EcoRI fragments of SPP1DNA (30).

b The numbers in parentheses are the numbers ofrestriction fragments.

c ND, Not determined.

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procedure), these forms can be distinguishedeasily from the OC forms. In this way weidentified five different plasmids in the Prt+strain and three different plasmids in the Prt-variant (Table 1).The plasmid DNA was further characterized

by determining the molecular weights of theCCC forms and their sensitivities to restrictionendonuclease EcoRI. Isolation of individualplasmids from an agarose gel (31) and subse-quent EcoRI digestion revealed that onlypWV04 and pWV05 contained recognition sitesfor EcoRI (three and four sites, respectively)which permitted determinations of the molecularweights of these plasmids by summation of themolecular weights of the individual fragments.Our results (Table 1) could lead to speculationthat plasmids pWV02, pWV03, and pWV04 arestable multimeric forms of plasmid pWV01.However, further characterization of these plas-mids with different restriction enzymes (data notshown) indicated that the plasmids are not relat-ed and represent unique species. In addition, insome plasmid preparations of both strains smallamounts of plasmid species with high molecularweights were observed (Fig. 3). These plasmidswere not analyzed further.

Conversion of Prt+ S. cremoris Wg2 to Prt-mutants by treatment with acriflavine. Our re-sults suggested that one or perhaps two plasmids(pWV05 and pWV03, both absent in the Prt-variant) are involved in the proteolytic activityof the Prt+ strain. This assumption was support-ed by curing studies. The curing agent acrifla-

1 2 3 4 5 6 7 8

pWV055_a

FIG. 3. Agarose gel electrophoretic patterns ofCsCI-EtBr-purified plasmid DNAs from acriflavinetreated Prt+ S. cremoris Wg2 cells. Lanes 3, 4, 5, 6, 7,and 8 contained plasmid DNAs from mutants S2, S3,S8, S9, S23, and S32, respectively. Also shown are theresults of an analysis of the Prt+ variant (lane 2) andthe naturally occurring Prt+ variant of S. cremoris(lane 1). In this gel small amounts of slowly migratingplasmid species are visible; these species were notanalyzed further (arrows on the right side). The posi-tion of CCC pWV05 is indicated.

vine had a clear effect on the ability of the Prt+parent strain to produce large colonies on ci-trate-containing milk agar after 48 h of incuba-tion at 30°C; 2 to 2.5 ,ug of acriflavine per ml wasoptimal for the production of small colonies oncitrate-containing milk agar (data not shown).Evidence that these small colonies were Prt-came from growth studies in milk. Isolated smallcolonies failed to produce sufficient acid tocoagulate milk within 24 h. On the other hand,these variants coagulated milk fortified with 1%casein hydrolysate.

Finally, six small colonies were picked ran-domly, and their plasmid profiles and casein-hydrolyzing activities were determined. None ofthe isolates showed any proteolytic activity at40°C and pH 5.5 or at 30°C and pH 7.5 (data notshown), indicating that both proteolytic activi-ties were lost. Figure 3 shows the plasmid pro-files of the mutants (S2, S3, S8, S9, S23, and S32),together with the profiles of the Prt+ parentalstrain and the naturally occurring Prt- variant.This figure clearly shows that the loss of proteo-lytic activity was accompanied by the loss ofpWV05; pWV03 was cured only in Sq. Fromthese results we concluded that pWV05 codesfor cell wall-bound proteolytic activity in S.cremoris Wg2. The molecular weight of thisplasmid is approximately 16.0 x 106.

Continuous culture studies with Prt+ and Prt-S. cremoris Wg2. The spontaneous occurrence ofcells which cannot ferment lactose to lactate(Lac- variants) in glucose-limited continuouscultures of S. lactis is well known (21, 22).However, we never observed selection of suchLac- variants of S. cremoris in lactose-limitedcultures.When a lactose-limited chemostat fed with

yeast extract broth at a fixed pH of 6.3 wasinoculated with a mixture of approximately 95%Prt+ S. cremoris Wg2 and 5% Prt- S. cremorisWg2, we always observed rapid selection infavor of the Prt- variant (Fig. 4). Surprisingly,no selection for the Prt- variant was observedwhen the culture was kept at pH 5.9. In this casethe Prt- variant was actually washed out. At pH6.8 a slow increase in the Prt- variant wasobserved (Fig. 4). The experiments describedabove were carried out with chemostat culturesfed with yeast extract broth containing limitingamounts of lactose (0.25%, wt/vol). Similar re-sults were obtained with chemostat cultures fedwith MRS broth (lactose limiting), which rulesout a possible effect of medium composition onthe outcome of the selection experiments (datanot shown).

DISCUSSIONBy means of a gel electrophoretic analysis of

the plasmids present in Prt+ S. cremoris Wg2 at

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FIG. 4. Chemostat growth of S. cremoris Wg2 inyeast extract broth at 30°C showing the effect ofpH onthe selection of Prt- variants. At zero time the che-mostat was inoculated with a mixture of approximate-ly 95% Prt+ and 5% Prt-. The dilution rate was 0.11h-1. Symbols: A, pH 5.9; 0, pH 6.3; O, pH 6.8.

least five different plasmid species were detect-able. Two of these plasmids (pWV05 andpWV03) were missing in a naturally occurringPrt- variant (Fig. 2). When the Prt+ strain wascured with acriflavine, other Prt- variants wereobtained. It appeared that the loss of the proteo-lytic capacity of the Prt+ strain was in all casesaccompanied by the loss of pWV05 (Fig. 2 and3). This strongly suggests that this plasmid (mo-lecular weight, 16.0 x 106) specifies the proteo-lytic activity in S. cremoris Wg2. The molecularweight of pWV05 is approximately twice thatreported for the protease plasmid in S. cremorisHP (19). Since no restriction analysis of theprotease plasmid from the latter strain has beenperformed, the relationship between the prote-ase plasmids from the two strains remains to beestablished.The role of pWV03 (molecular weight, 6.1 x

106), which was also absent in some Prt- vari-ants, is unknown. Since the natural Prt- variantshowed increased sensitivity to copper ions (un-published data), pWV03 may determine heavymetal resistance in Prt+ strains of S. cremorisWg2.

It has been shown repeatedly that bacteriaeasily lose their plasmids when they are grownin culture media in which the information on theplasmids is not required (2, 11, 26, 33). There-fore, it was not surprising that Prt+ S. cremorisWg2 was rapidly replaced by the Prt- variant

when it was grown in a lactose-limited chemo-stat provided with sufficient amino acids (yeastextract). Although two plasmid species wereabsent in the natural Prt- variant used in thisexperiment, the total amounts of plasmid DNAin the two types of cells were similar, mostprobably as a consequence of a higher copynumber of plasmid pWV02 in the Prt- variant(Fig. 1). Therefore, the energy gain of the Prt-variant has to be found in protein synthesisrather than in plasmid DNA synthesis. Howev-er, the selective advantage of the Prt- variantunder energy-limiting conditions was expressedonly at pH values higher than 6.0. This indicatesthat at lower pH values another, still unknownfactor became more important and gave the Prt+strain a selective advantage. In this respect it isimportant to note that the maximum specificgrowth rate of the Prt- variant in energy-suffi-cient batch cultures was lower than that of thePrt+ strain at pH values below 6.0, whereas thereverse was true at higher pH values (Otto,Ph.D. thesis, University of Groningen, Haren,The Netherlands, 1981). These results demon-strate that it is possible to grow both Prt+ strainsand Prt- strains under controlled conditions.This information could be very useful to thedairy industry because it could facilitate theproduction of starter cultures of the desiredcomposition.

ACKNOWLEDGMENTThis study was supported financially by the Dutch Institute

for Dairy Research.

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