Vol. 47, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1984, p. 253-2570099-2240/84/020253-05$02.00/0Copyright C) 1984, American Society for Microbiology

Genetic Construction of Lactose-Utilizing Xanthomonas campestrisPATRICIA M. WALSH,t* MICHAEL J. HAAS, AND GEORGE A. SOMKUTI

Agricultural Research Service, Eastern Regional Research Center, U.S. Department of Agriculture, Philadelphia,Pennsylvania 19118

Received 1 July 1983/Accepted 1 November 1983

Xanthomonas campestris, the producer of xanthan gum, possesses a 3-galactosidase of very low specificactivity. Plasmid pGC9114 (RP1: :Tn951), generated by the transposition of the lactose transposon Tn951 toRP1, was conjugally transferred into XN1, a nalidixic acid-resistant derivative of X. campestris NRRL B-1459S-4L. Transfer occurred on membrane filters and in broth. The ,B-galactosidase gene of Tn951 was

expressed in X. campestris. The specific activity of ,-galactosidase in transconjugants was over 200-foldhigher than that in XN1, and transconjugants grew as well in lactose-based media as in glucose-based media.The lactose-utilizing transconjugants could potentially be used to produce xanthan gum from cheese whey.

Xanthomonas campestris is a gram-negative bacterium ofindustrial importance due to its role in plant pathogenicityand its production of xanthan gum. Xanthan gum is a high-molecular-weight (12), extracellular polysaccharide com-posed of pentasaccharide repeating units containing D-glu-cose, D-mannose, D-glucuronic acid, acetal-linked pyruvicacid, and O-acetyl groups (13). Xanthan gum has manyapplications in agriculture; in the manufacture of abrasives,ceramics, food, paint, and textiles; and in petroleum produc-tion (14). Many researchers have studied the fermentationconditions required for optimal gum production (3, 15, 21,24). Souw and Demain (24) found that a 4% sucrose orglucose medium with a suitable nitrogen source gives thebest gum yields.

Currently there is a great deal of interest in using industrialwastes as nutrient sources in bioconversions. Whey is anutrient-rich by-product of the dairy industry, and its dispos-al is a major pollution problem. The composition of whey isvariable but generally includes 4 to 5% lactose, 0.8 to 1%protein, and smaller amounts of organic acids, minerals, andvitamins (4). No significant amount of xanthan gum isproduced when X. campestris is grown in lactose medium(25) probably because of the low substrate affinity of the ,B-galactosidase of X. campestris (9). Xanthan gum can besuccessfully produced from whey, if the lactose is firsthydrolyzed to glucose and galactose (4, 25). The directutilization of whey for xanthan gum production would bemore economical. An approach to this end is to engineer X.campestris to hydrolyze lactose more efficiently.

In this paper, we describe the conjugal transfer ofpGC9114, a plasmid containing the lac transposon Tn951 (7),into XN1, a nalidixic acid-resistant (NaP) derivative of X.campestris NRRL B-1459S-4L. The P-galactosidase gene ofTn951 was expressed in the transconjugants, which grew aswell in lactose broth as in glucose broth.

MATERIALS AND METHODSBacterial strains and plasmids. Strains used in this study

are listed in Table 1. Plasmid pGC9114 is a derivative of RP1,which confers resistance to ampicillin, kanamycin, andtetracycline, and was formed by the transposition of Tn951to RP1 (7). Cultures were maintained in Luria-Bertani (LB)

* Corresponding author.t Present address: Department of Food Science, Cornell Univer-

sity, Ithaca, NY 14853.

glucose (LBG) broth which contains 1% tryptone (DifcoLaboratories), 0.5% yeast extract (Difco), 0.5% NaCl, and0.2% glucose. Antibiotics (Sigma Chemical Co.) were addedto growth media at the following concentrations (microgramsper milliliter): nalidixic acid (Nal), 50; erythromycin (Ery),200; kanamycin sulfate (Kan), 100; and dihydrostreptomycinsulfate (Str), 500.Mating procedure. The filter mating procedure used was

that described by Lai et al. (16) with some modifications.From an overnight culture of Escherichia coliJC3272(pGC9114), a 2% transfer was made into 10 ml of LBbroth which contains 1% tryptone (Difco), 0.5% yeast ex-tract (Difco), and 0.5% NaCl in a 125-ml flask. This culturewas incubated at 37°C with shaking, until it reached anoptical density (600 nm) of ca. 0.68. Donor and recipientcultures were combined in a ratio of 1:10 (vol/vol), and 3 mlof this mixture was collected on a sterile membrane filter(type HA; 0.45 ,um; Millipore Corp.). Filters were placed onLB agar, incubated at 28°C for 30 min, and then washed in 2ml of sterile distilled water. Serial dilutions of the wash wereplated onto LBG agar containing Kan and Str, Kan and Nal,and Nal. After 72 h at 28°C, recipients and kanamycin-resistant (Kmr) Nalr transconjugants were counted. E. colidonors were counted after 24 h at 37°C. Controls were donorand recipient strains filtered and incubated separately. TheLac phenotype of the parent strains and of the transconju-gants was tested on Xgal-glucose agar (20) containing thelactose operon inducer isopropyl-p-D-thiogalactoside (IPTG)at a concentration of 1 mM and on peptone-phenol red-lactose agar (2). After 24 to 48 h at 28°C, the plates wereexamined for color changes.For the CL355 x XE1 matings, donors and recipients

were mixed in a ratio of 1:2 (vol/vol). Filter matings weredone as described above. Serial dilutions of the filter washwere plated on LBG agar containing Kan and Nal, Kan andEry, and Ery. Colonies were counted after 72 h at 28°C.For broth matings, donor and recipient strains were grown

as described for filter matings. A mating mixture was pre-pared by combining 0.3 ml of donor cells with 2.7 ml ofrecipient cells in a 125-ml Erlenmeyer flask. The mixturewas incubated at room temperature on a rotary shaker at 30rpm. After 4 h, the flask was vigorously agitated with aVortex mixer, and samples were removed, diluted, plated,and incubated as described for filter matings. Controlsconsisted of donor and recipient cultures incubated separate-ly.

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254 WALSH, HAAS, AND SOMKUTI

TABLE 1. Bacterial strains used in this studyStrain designation Chromosomal markers Plasmid composition Source/comment

E. coliJC3272(pGC9114) his lys trp lac AX74 Stri pGC9114 G. CornelisJC3272 his Iys trp lac AX74 StrF NDa Spontaneous Lac- derivative of

JC3272(pGC9114)

X. campestrisNRRL B-1459S-4L ND Northern Regional Research Center, U.S.

Department of AgricultureXN1 Nalr ND Spontaneous mutant of NRRL B-1459S-4LXE1 Eryr ND Spontaneous mutant of NRRL B-1459S-4LCL355 Nalr pGC9114 JC3272(pGC9114) x XN1 transconjugantCL381 Nalr pGC9114 JC3272(pGC9114) x XN1 transconjuganta ND, No plasmids were detected.

Plasmid stability. Transconjugant CL355 was plated onLBG agar containing Kan. A Kmr colony was picked intoLBG broth containing Kan. After incubation at 28°C withshaking for ca. 16 h, the culture was transferred into LBGbroth without Kan. The culture was successively subcul-tured in the absence of antibiotic when it reached an opticaldensity (600 nm) of 0.6 to 0.8. Before each transfer, theculture was diluted and plated on LBG agar. After incuba-tion for 48 h at 28°C, 100 colonies were picked onto LBGagar containing Kan. After the colonies were incubated for48 h at 28°C, the numbers of kanamycin-sensitive (Kms) andKmr colonies were scored.Enzyme assays. Fresh overnight cultures of X. campestris

in LBG broth were transferred (1%) twice in minimal salts(10) supplemented with 0.4% Casamino Acids (Difco), 0.4%glucose, and 80 jxg of tryptophan per ml. Cultures wereincubated overnight at 28°C with shaking. The second sub-culture was incubated until it reached an optical density (600nm) of 0.5 to 0.8. E. coli strains were subcultured twice at37°C with shaking in minimal A medium (20) supplementedwith 20 ,ug of vitamin B1 per ml, 0.4% glucose, 1 mMMgSO4, 0.4% Casamino Acids, and 80 jig each of trypto-phan, lysine, and histidine per ml as described by Miller (20).Where indicated in Table 2, strains were grown in thepresence of 1 mM IPTG. The cells were harvested bycentrifugation, washed in 5 ml of physiologically bufferedsaline containing 0.68% KH2PO4 and 0.876% NaCl (pH 7.2),and suspended in 4 ml of physiologically buffered saline. To1 ml of cell suspension, 2 drops of chloroform and 1 drop of0.1% sodium dodecyl sulfate solution were added, and thecells were agitated with a Vortex mixer for 10 s (20). Fordetermination of the amount of protein in the sample, 100 RIof the chloroform-sodium dodecyl sulfate-treated cells wasanalyzed by using a protein assay (Bio-Rad Laboratories)and following the directions of the manufacturer for themicroassay. Bovine serum albumin was used as a proteinstandard. Samples of the treated cells were assayed for 1B-galactosidase by the method of Miller (20). A unit of 1B-galactosidase was defined as the amount of enzyme whichproduced 1 nmol of o-nitrophenol per min at 28°C and pH7.0.Growth curves. Overnight cultures of CL355 and XN1 in

LBG broth were subcultured twice in minimal salts (10)supplemented with 0.4% Casamino Acids, 80 jig of trypto-phan per ml, and either 0.4% glucose or 0.4% lactose. Thecultures were incubated at 28°C with shaking. At the start ofthe second transfer and thereafter, growth of the culture wasmonitored by measuring the optical density (600 nm).

Plasmid analysis. X. campestris strains were grown over-

night at 28°C with shaking in 100 ml of succinate broth (16) ina 1-liter flask. Cells were harvested by centrifugation. Plas-mid DNA was isolated by the method of Meyers et al. (19)with the exceptions that RNase digestion of the samples wasomitted and chloroform-isoamyl alcohol (24:1) was substitut-ed for phenol in the deproteinization step. Ethanol-precip-itated DNA was suspended in TES buffer (50 mM NaCl, 5mM disodium EDTA, 30 mM Tris, pH 8.0) and furtherpurified by CsCI-ethidium bromide buoyant density gradientcentrifugation (8).Plasmid DNA was isolated from JC3272(pGC9114) grown

in antibiotic medium D (BBL Microbiology Systems) by theprocedure of Clewell and Helinski (5) with the modificationsof Macrina et al. (17). Plasmids were isolated from clearedlysates by ultracentrifugation in CsCl-ethidium bromidebuoyant density gradients (8). Ethidium bromide was re-moved from the purified plasmid samples (8), and afterethanol precipitation, the DNA was dissolved in 10 mMTris-0.1 mM disodium EDTA, pH 8.0.

Restriction enzyme digestions were performed accordingto the directions of the supplier (Bethesda Research Labora-tories). Restriction enzyme-generated fragments and plas-mid DNA were analyzed in a 16-cm vertical gel containing0.8% agarose (SeaKem) in Tris-borate buffer (89 mM Tris,2.5 mM disodium EDTA, 89 mM boric acid, pH 8.0).Bacteriophage lambda DNA digested with HindIll was usedto estimate the molecular weights of the restriction frag-ments. Gels were electrophoresed at 40 mA, constant cur-rent, for 3 h and then stained in a solution of 0.5 ,g ofethidium bromide per ml. The gels were destained in distilledwater and photographed under UV light.

RESULTSStrain construction. E. coli JC3272(pGC9114) was mated

with XN1, a Nalr mutant of X. campestris NRRL B-1459S-4L. After a 30-min filter mating, Kmr Nalr transconjugantsappeared at a frequency of 4.5 x 10-1 per donor. Four-hourbroth matings resulted in 7.8 x 10-6 Kmr Nalr transconju-gants per donor. Filter matings between X. campestristransconjugant CL355 (Kmr NaI) and XE1, an erythromy-cin-resistant mutant (Ery9 of X. campestris NRRL B-1459S-4L, resulted in 4.2 x 10-1 Kmr Eryr transconjugants perdonor. All reported conjugation frequencies represent anaverage of two separate mating experiments.The Lac phenotype of the transconjugants was tested on

Xgal-glucose agar containing 1 mM IPTG and on peptone-phenol red-lactose agar. After 24 h on Xgal-glucose agar,Kmr Nalr transconjugants appeared deep blue as did

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TABLE 2. Expression of Tn951 in E. coli and X. campestris

Strain ,B-Galactosidase sp acta+IPTG -IPTG

E. coliJC3272(pGC9114) 2,060 52.2JC3272 1.32 0.691

X. campestrisXN1 18.8 16.0CL355 4,260 257.0CL381 4,810 NDba Specific activities are expressed as nanomoles of o-nitrophenol

minute-' milligram of protein-'. The values reported represent theaverage of at least two separate determinations.

b ND, Not determined.

JC3272(pGC9114), but XN1 was light aqua. After 24 h onpeptone-phenol red-lactose agar, Kmr Nalr transconjugantsand JC3272(pGC9114) had acidified the medium, causing acolor change to yellow, whereas it took XN1 several days tocause this change.

In the absence of Kan selection, pGC9114 was not stablymaintained in CL355. After ca. 20 generations, over 25% ofthe 100 colonies tested were Kms. All 25 of the Kms coloniesexamined were lactose negative (Lac-) on Xgal medium,whereas all 22 of the Kmr colonies examined were lactosepositive (Lac').

Expression of Tn951 in X. campestris. The specific activi-ties of P-galactosidase in induced (+IPTG) and uninduced(-IPTG) strains of E. coli and X. campestris were deter-mined (Table 2). The specific activity of P-galactosidase ineight independently isolated transconjugants tested wasgreater than 4,000 U/mg of protein. This was 200-fold higherthan the specific activity of the X. campestris parent strainXN1 and about twice as high as the specific activity of E. coliJC3272(pGC9114).Growth curves showed that CL355 grew much better in

lactose broth than did the parent strain XN1 (Fig. 1). Also,CL355 grew as well in lactose broth as it did in glucose broth(Fig. 1).

Plasmid analysis. Despite repeated attempts, no plasmidswere detected in recipient strain XN1 (data not shown). Thedonor strain JC3272(pGC9114) contained a single plasmid.The transconjugant CL355 contained a single plasmid ofapparently the same molecular weight as pGC9114 (Fig. 2).The BamHI-generated restriction pattern of the plasmidfrom CL355 was indistinguishable from that of pGC9114(Fig. 2).Two of the Kms Lac- derivatives of CL355 isolated during

the plasmid stability study were examined for plasmids. Noplasmids were detected (data not shown).

DISCUSSIONAlthough X. campestris synthesizes 3-galactosidase (11),

it is unable to produce significant amounts of xanthan gumfrom cheese whey (25), perhaps because of the low substrateaffinity of the enzyme (9). Using transposon Tn951, weconstructed a strain of X. campestris that can better hydro-lyze lactose and potentially could be used to producexanthan gum from cheese whey. Transposon Tn951 is a 16.6-kilobase transposon first isolated from Yersinia enteroco-litica (7). It contains DNA homologous to the E. coli lacoperon genes I, Z, and Y (7) and to the lac genes on plasmidsisolated from Salmonella spp., Klebsiella spp., and Proteus

spp. (6). Plasmid pGC9114 (RP1::Tn951) (7) was conjugallytransferred into X. campestris XN1. Transfer occurred at ahigh frequency on membrane filters, and broth matings werealso successful. Kmr Nal' X. campestris transconjugantswere able to transfer pGC9114 to another strain of X.campestris, demonstrating that the transfer of pGC9114 intomore industrially useful strains should be straightforward. Inthe absence of selection, the plasmid was not stably main-tained; Lac- Kms derivatives appeared at a high frequency.Maintaining the culture either in lactose broth or in thepresence of antibiotic should alleviate this problem.The 3-galactosidase gene of Tn951 was expressed in X.

campestris. The induced levels of ,3-galactosidase in X.campestris transconjugants were ca. 200-fold higher than inthe X. campestris parent strain, XN1. CL355, a Lac' X.campestris transconjugant, grew equally well in lactose andglucose broths. The expression of Tn9SJ in E. coli, Proteusmirabilis, and Pseudomonas spp. (2) and in Rhodopseudo-monas sphaeroides (23) has been studied. The lac genes areexpressed in all of these bacteria, and the specific activity ofP-galactosidase is much higher in E. coli than in the otherspecies. We found that the P-galactosidase specific activityin E. coli JC3272(pGC9114) is comparable to findings inpublished reports (2, 23); however, the high level of ac-tivity in Lac' X. campestris, twice that in E. coliJC3272(pGC9114), was unexpected based on the resultsobtained by others with Pseudomonas spp. (2). The unin-duced level of P-galactosidase in X. campestris CL355 wasapproximately five times greater than that in E. coli

1.0

E0

(0

-

cLAC-)

a-C0

00 2 4 6 8 10

TIME (hours)12 14

FIG. 1. Growth curves of the X. campestris parent strain XN1 inlactose broth (0) and of transconjugant CL355 in lactose broth (A)and in glucose broth (O).

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256 WALSH, HAAS, AND SOMKUTI

A .B C D E

FIG. 2. Agarose gel electrophoresis of plasmids isolated from E.coli JC3272(pGC9114) and X. campestris and their BamHI-generat-ed restriction fragments. (A) Lambda DNA digested with HindIII;(B) pGC9114, isolated from JC3272; (C) pGC9114 digested withBamHI; (D) plasmid profile of CL355; (E) BamHI-generated restric-tion fragmehts of plasmid isolated from CL355.

JC3272(pGC9114). The biochemical basis for these differ-ences was not studied.

In a separate series of experiments, we attempted toincrease the level of 3-galactosidase in X. campestris XN1by constructing and introducing recombinant plasmids con-sisting of a BamHI restriction ftagment containing the lacgenes of Tn951 (6) and of pKT230, a derivative of RSF1010(1) (data not shown). These recombinant Lac plasmids werestable in E. coli. In X. campestris XN1, deletion eventsoccurred which resulted in lower-molecular-weight plasmidsand greatly reduced ,B-galactosidase specific activity. Otherresearchers have reported similar results with similar cloningvectors. RSF1010-trp hybrid plasmids form deleted plasmidsin Pseudomonas aeruginosa although they are stable in E.coli (22); and insertions into R1162, which is identical toRSF1010, are not stable in Pseudomonas putida (18). Theexplanation for these events is not yet known but may berelated to plasmid organization (18). These instability prob-lems were not detected in X. campestris transconjugantscontaining pGC9114.

This study demonstrated that the 3-galactosidase gene ofTn951 was expressed in X. campestris. Transconjugants hadhigh levels of 3-galactosidase activity and grew well inlactose-based medium. Although we were not successful in

our attempts to inicrease P-galactosidase activity by cloningthe lac genes of Tn951, this may have been due to our choiceof cloning vector. Successful cloning of the genes mayfurther increase the level of 3-galactosidase in X. campes-tris. The ability of the Lac' transconjugants to producexanthan gum from lactose was not tested; however, ourstudy clearly showed that the direct utilization of whey forxanthan gum production is a real possibility.

ACKNOWLEIDGMENTSWe thank Marianne Bencivengo for excellent technical assistance

and G. Cornelis, M. Bagdasarian, and G. Jacoby for kindly provid-ing strains.P.M.W. was partially supported by a Dairy Research Foundation

Postdoctoral Fellowship.

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