JOURNAL OF BACTERIOLOGY, Aug. 1987, p.

3593-3600

Vol. 169, No. 8

0021-9193/87/083593-08$02.00/0 Copyright 1987, American Society for Microbiology

Clustering of Mutations Blocking Synthesis of Xanthan Gum by Xanthomonas campestris

LINDA THORNE, LISA TANSEY, AND THOMAS J. POLLOCK* Syntro Corporation, San Diego, California 92121
Received 24 February 1987/Accepted 27 April 1987

as

Mutations that block the synthesis of xanthan gum by Xanthomonas campestris li1459S-4L-II were isolated nonmucoid colonies after treatment with ethyl methanesulfonate. Complete libraries of DNA fragments from wild-type X. campestris were cloned into Escherichia coli by using a broad-host-range cosmid vector and then transferred into each mutant strain by conjugal mating. Cloned fragments that restored xanthan guim synthesis (Xgs+; mucoidy) were comnpared according to restriction pattern, DNA sequence homology, and complementation of a subset of Xgs mutations. Groups of clones that contained overlapping homologous DNA were found to complement specific Xgs- mutations. The results suggest clustering of the genetic loci involved in xanthan synthesis. The clustering occurred within three unlinked regions. Two forms of complementation were observed. In most instances, independently isolated cosmid clones that complemented a single mutation were found to be partially homologous. Less frequent was the second form of complementation, in which two cosmid clones that lacked any homologous sequences restored the mucoid phenotype to a single mutant. Finally, xanthan production was measured for wild-type X. campestris carrying multiple plasmid copies of the cioned xanthan genes.
strains during sequential passage or prolonged growth remains a problem (5). The synthesis of xanthan is probably very similar to exopolysaccharide synthesis by other gram-negative bacteria, such as species of Rhizobium, Pseudomonas, Klebsiella, and Escherichia (20). The synthetic pathway can be considered in three parts: (i) the uptake of simple sugars and their conversion to nucleotidyl derivatives, (ii) the assembly of pentasaccharide subunits attached to an isopentenyl pyrophosphate carrier, and (iii) the polymerization of pentasaccharide repeat units and their secretion. Compared with the more advanced molecular genetic understanding of colanic acid synthesis by E. coli (12, 17, 22) or alginate synthesis by Pseudomonas aeruginosa (7, 8), little is known about the genes, enzymes, or mechanisms that control the synthesis of xanthan by X. campestris. However, a genetic understanding would benefit not only the study, of phytopathogenesis but also could have commercial implications by improving the productivity of X. campestris B1459 for xanthan synthesis. In this report, we describe the cloning and expression of fragments of DNA from wild-type X. campestris B1459 that complement several mutations that block xanthan synthesis. The cloned DNA was physically mapped and arranged into functional complementation groups. One cluster of mutations that affected xanthan synthesis contained three complementation groups. A second unlinked locus had two complementation groups, and a third was represented by a single mutation. Furthermore, we show the effect of multiple copies of these genes on xanthan accumulation by wild-type X. campestris.
MATERIALS AND METHODS

Many microorganisms produce extracellular polysaccharides. The main reason is unknown, but two possibilities seem likely. Polysaccharides may serve as a protective barrier against adverse environmental conditions (25) or may facilitate colonization of or attachment to surfaces (6). The bacterium Xanthomonas campestris pv. campestris is the causal agent of black rot of crucifers (24). Other species of Xanthomonas, or close relatives, are also phytopathogens that cause severe agricultural damage if unchecked. One of the products of X. campestris that is thought to contribute to pathogenicity is its extracellular polysaccharide, xanthan (or xanthan gum). Plugging of xylem plant tissue appears to be caused by synthesis of xanthan by colonizing X. campestris since it is found at the site of the lesion (21). However, a direct link between the ability to synthesize xanthan and pathogenesis is not yet established. Related studies with Rhizobium species do establish a link between exopolysaccharide synthesis and nodulation of pea and bean plants (4, 15). Xanthan itself is useful as a specialty polymer for a growing list of commercial applications (14). The exploitation of xanthan resulted from a successful screening effort by the Northern Regional Research Center to find useful watersoluble polysaccharide products to replace existing gums from plant and algal sources. The Center discovered X. campestris NRRL B1459, a strain which produces a polymer that exhibits three desirable properties: (i) high viscosity at low concentration, (ii) pseudoplasticity, and (iii) insensitivity to a wide range of temperature, pH, and electrolyte concentrations (13). Because of its special rheological properties, xanthan is used in food, cosmnetics, pharmaceuticals, paper, paint, textiles, adhesives, and in tertiary oil recovery. In addition, the polymer is readily produced by fermentation from b-glucose (18). An increase in usage would result from improvements -in the overall productivity of the viscous fermentation process and from the development of new genetically stabilized strains. Degeneration of production
*

Corresponding author.
3593

Bacterial strains and plasmids. X. campestris B1459S-4LII (our strain X55) obtained fromn the Northern Regional Research Center was the Xgs+ (positive for xanthan gum synthesis) parent of all X. campestris strains (5) used in this study. Strain X59 was a spontaneous rifampin-resistant derivative of X55 that was also fully Xgs+. Rif' derivatives of X55 arose at a frequency of about io0- and were selected on

3594

THORNE ET AL.
TABLE 1. Bacterial strains and plasmids

J. BACTERIOL.

Strain or plasmid

Genotype or phenotype"

Reference or source

X. campestris X55 X59

Xgs+, prototroph
Xgs+, prototroph, Rif Xgs-, prototroph, Rif' Xgs -, auxotroph, Rif' Xgs-, prototroph, Rif Xgs-, auxotroph, Rif' Xgs- prototroph, Rif' Xgs, prototroph, Rif' Xgs, auxotroph, RifP Xgs --, prototroph, Rif' Xgs-, prototroph, Rif' Xgs-, auxotroph, Rif' Xgs-, prototroph, RifP
F- hsd2O (rB mM) recAl3 ara-14 proA2 lac Yl gaIK2 rpsL20 (streptomycin resistance) xyl-5 mtl-l supE44 thi leu XrecAl endAI gyrA96 thi hsdRJ7 supE44, relAl A((Iac-proAB) [F'traD36, proAB lacIqZAM15] F- hsdR5J4 (rK MK-) suipE44 siipF58 X - galK2 galT22 metBI trpR55 lacYI Alac IZY-6 X b221 rex::Tn5 (Kanr) c1857 Oam29 Pam8O

X59ml X59m8 X59m9 X59mll X59m31 X59m45 X59m48 X59m65 X59m82 X59m96 X59m145
E. coli HB101

5 This This This This This This This This This This This This

work work work work work work work work work work work work

Bethesda Research Laboratories Bethesda Research Laboratories L. Enquist

19; R. Haselkorn 9 11 23 This work

JM109
LE392 Bacteriophage

Plasmids pRK311 pRK2013 pUC13 cl-c82

RK2 origin, Tra' Mob- Tetr X cos lacZ(o() ColEl origin, Imm+ Ampr Tra- Mob' Kanr Ampr, ColEl origin pRK311, complementing insert

a Abbreviations: Xgs, xanthan gum synthesis; Rif, rifampin resistance; Tetr, tetracycline resistance; Kanr, kanamycin resistance; Ampr, ampicillin resistance; Imm+, colicin El immunity; Tra and Mob, transfer and mobilization functions of RK2 plasmid, respectively.

agar plates containing Luria broth supplemented with rifampin at 60 jig/ml. The Rif' phenotype of X59 was useful as a marker to distinguish progeny from contaminants after mutagenesis and was necessary as a counterselection for E. coli Rif' donors in conjugal matings. X55 and X59 formed indistinguishable mucoid colonies on nutrient and minimal agar plates. Bacteriophage X b221 rex::TnS c1857 Oam29 Pam8O (19) was the source of Tn5 for mapping by insertional gene inactivation. Strain LE392 was the permissive host for propagating the phage. Bacterial strains and plasmids are listed in Table 1. Growth media. Xanthomonas species were cultured by shaking in liquid YT medium at 30C with rifampin at 50 ,Lg/ml, tetracycline at 7.5 pg/ml, or kanamycin at 50 pg/ml added for plasmid maintenance. YT medium contains tryptone (16 g/liter; Difco Laboratories), yeast extract (10 g/liter; Difco), and NaCl (5 g/liter). All nutrient agar plates contained tryptose blood agar base (TBAB; Difco) plus starch at

1% (wt/vol). Selection plates for conjugal matings contained tetracycline at 7.5 pg/ml, kanamycin at 50 ,ug/ml, and rifampin at 50 ,ug/ml. Minimal agar plates contained M9 inorganic salts (1) plus glucose, mannose, and fructose at 1% (wt/vol) as the carbon source. Liquid medium for shake flask experiments to measure xanthan accumulation was referred to as XG004 and consisted of 1x basic salts, 0.5% (wt/vol) tryptone, 0.25% (wt/vol) yeast extract, 1x trace minerals, 0.01% (wt/vol) CaCl2, and 2% (wt/vol) glucose. Basic salts (10x) consisted of 6.8 g of KH2PO4, 0.2 g of MgSO4 7H20, 2.2 g of L-glutamic acid, 2 g of citric acid in 100 ml of deionized H20 with pH adjusted to 7 with NaOH at 30C. Trace minerals (1,OOOx) was 2.25 g of FeCl3 6H2O, 1.41 g of MnSO4- H20, 2.2 g of ZnSO4- 7H2O, 0.25 g of CuSO4 5H2O, 0.4 g of CoCl2 6H.O, 0.26 g of

Na2MoO4 2H2O, 0.4 g of H3BO3, and 0.06 g of KI per liter of deionized H2O (with HCI added to solubilize the salts). Escherichia coli was grown in Luria broth at 37C with tetracycline at 10 ,ug/ml and kanamycin at 50 jig/ml as appropriate or on agar plates containing Luria broth or TBAB. Mutagenesis of X. campestris. About 2 x 109 freshly grown cells (A600 of 2) were suspended in 2 ml of minimal salts medium and shaken at 30C with 0 to 40 [lI of ethyl methanesulfonate for 1, 2, or 3 h. Samples of 0.5 ml were taken from each treatment, washed two times with YT medium, suspended in 2 ml of YT medium, and shaken overnight at 30C. Dilutions were spread on TBAB plus 1% (wt/vol) starch plates. After 3 days, non-wild-type colonies (about 1% of the total) were saved. Most of the mutants were nonmucoid. Only one mutant was selected from each treatment with ethyl methanesulfonate, unless colony morphology was clearly distinctive. The mutants, designated X59ml to X59m150, were tested for retention of the Rif' marker of the parent X59, for the presence of cleared zones around colonies on plates containing starch, and for the ability to utilize different carbon sources. Mutagenesis with bacteriophage X carrying TnS was as previously described (19). DNA isolation and recombinant DNA techniques. Total DNA from strain X59 (Xgs+) was prepared by the boiling method (16) or the Birnboim and Doly procedure (3). Frequently used plasmids were further purified by equilibrium sedimentation in density gradients of CsCl containing ethidium bromide (16). Total DNA was partially digested with Sau3A restriction endonuclease, and large fragments of 20 to 30 kilobase pairs (kbp) were purified by velocity sedimentation in neutral sucrose gradients. This ensured that only contiguous chromosomal DNA fragments were inserted

VOL. 169, 1987

XANTHAN SYNTHESIS GENES OF X. CAMPESTRIS

3595

in the cloning vector upon ligation. The cloning vector was the broad-host-range cosmid pRK311 constructed by Ditta et al. (9). DNA fragments to be cloned were inserted into the BamHI sequence of the multiple cloning site within the lacZ portion of the vector. Using the in vitro packaging kit of Stratagene, we selected for insertions of DNA of about 20 to 25 kbp into the cosmid vectors. The pRK311 vector also carries a selectable tetracycline resistance gene. After in vitro ligation and packaging, E. coli JM109 was transfected with phage particles and tetracycline-resistant colonies were individually saved. Each tetracycline-resistant colony contained the plasmid vector plus a 20- to 25-kbp insertion of X. campestris DNA. A library of fragments of DNA inserted in pRK311 resulted from pooling the clones and was named L[X59]. Since the number of clones in the library exceeded 1,000, we were at least 99.9% certain of having all fragments of the X. campestris chromosome represented at least once. Three different libraries were used in this work. Restriction enzymes (from Boehringer GmbH) were used according to the instructions of the manufacturer. DNA sequence homology was demonstrated by the blotting method of Southern (16) by utilizing Zeta-Probe (Bio-Rad Laboratories) for DNA immobilization. DNA for use as a hybridization probe was labeled with [32P]dCTP by using a nick translation reagent kit from Bethesda Research Laboratories, Inc.). Fragments of DNA were separated by electrophoresis through agarose gels (0.6 to 0.7% [wt/vol]) in Tris acetate buffer (16). Conjugation and complementation of Xgs- mutants. The complete library (or a specific element of the library) was transferred from E. coli to X. campestris by a triparental mating scheme (10). From fresh overnight cultures, 109 recipient cells (X. campestris Xgs- mutants), 5 x 108 donor cells (JM109 containing L[X59], the library), and 5 x 108 helper cells (E. coli HB101 containing plasmid pRK2013) were mixed and passed through a type HA 0.45-p.m-poresize Millipore filter. The filters were incubated on TBAB plates overnight at 30C, and the cells were then washed into 2 ml of selecting medium (TBAB plus tetracycline at 7.5 ,ug/ml and rifampin at 50 ,ug/ml). The cells were diluted by 104- to 105-fold and spread on selection plates containing antibiotics. Complementation (restoration of the Xgs+ phenotype in an Xgs- mutant) occurred at a frequency of 0.1 to 0.5%. The Xgs+ exconjugants were purified, and the recombinant plasmid was isolated and transferred back to E. coli JM109 for storage and further purification. Subsequent matings with a purified member of the library raised the Xgs+ frequency in the exconjugants to 100%. Measurement of xanthan accumulation. Strains to be tested for xanthan production were grown in liquid YT medium overnight and then counted and diluted to 107/ml. Flasks (125-ml capacity) containing 10 ml of medium XG004 were inoculated with equal numbers of cells (108) and shaken at 28C at 250 rpm. At the time of sampling, 20 ml of isopropyl alcohol was added to each flask to precipitate the exopolysaccharides. The precipitate was collected on a Whatman 934-AH 2.4-cm-diameter filter, which was then dried in a vacuum oven and weighed. RESULTS Isolation of mutants deficient in xanthan gum synthesis (Xgs-). A collection of Xgs- mutants was generated by exposing strain X59 (and less frequently X55) to ethyl methanesulfonate. After growth at 30C for 3 days, nonmucoid colonies were selected and purified for further

use. In most cases, the nonmucoid colonies were distinctively different in appearance from the wild type, although some independently isolated mutants displayed similar nonmucoid appearance. The latter could be distinguished by plating on different carbohydrate sources and as a function of time of growth. In contrast to parent strain X59, four mutants (m8, mll, m48, and m96) did not grow on minimal agar plates supplemented with either glucose or mannose. We included mannose in this survey because xanthan contains two mannose residues per pentasaccharide repeat. Isolate m65 had a greatly reduced growth rate, and isolate m45 failed to grow at all on minimal mannose medium. As an alternative to chemical mutagenesis, we exposed the library of X. campestris DNA to transposon Tn5 in E. coli. After mating the mutated library back into X. campestris, we selected for the pRK311 vector (Tet) and Tn5 (Kanr). When the tetracycline selection was removed, the plasmid was lost within a few generations. Among the Tets Kanr segregants, we found Xgs- mutants. In work to be reported elsewhere (L. Tansey, L. Thorne, and T. Pollock, unpublished data), we demonstrated that the appearance of the Xgs- phenotype was associated with homologous recombination between the mutated plasmid and the chromosome of the exconjugant. By cloning fragments of the chromosomal DNA that carried the Kanr marker (Tn5), we obtained the flanking X. campestris DNA. ComplementAtion of Xgs- defects by cloned normal DNA. As yet there is no DNA transformation method that works as efficiently for X. campestris as does intergenic conjugal mating. The RK2-derived pRK311 cosmid is a broad-hostrange vector that is not self-transmissible. For pRK311carrying inserts of X. campestris DNA to be transferred by conjugation between E. coli and X. campestris, a second helper plasmid must be present (pRK2013) which carries necessary transfer factors but which has a limited host range that does not include X. campestris. Transfer of recombinant plasmids was as described in Materials and Methods. About 15 independently isolated Xgs- mutants were complemented and restored to mucoidy (Xgs+) by conjugal mating with the complete library of X. campestris genes. Generation of the gene library is described in Materials and Methods. The frequency of complementation was about 0.1% for most matings, as would be expected if there was only one copy of each gene per chromosome. A photograph of a set of related colonies is shown in Fig. 1 (top to bottom): X59-pRK311 (a wild-type strain carrying a cosmid vector), X59m45-pRK311 (a mutant carrying a cosmid vector), and X59m45-c45 (a mutant carrying a cosmid vector plus complementing cloned DNA). The presence of the c45 cosmid restored the mucoid appearance, as seen for the wild-type X59. The mucoid phenotype for X59m45-c45 depended on the continued presence of the recombinant plasmid, as judged by the maintenance of the Tetr gene of the plasmid. A similar overall pattern was seen for all complemented Xgsmutants. Several mucoid exconjugants were picked and purified by replating. Plasmid DNA was prepared for each and transformed into E. coli and then mated back into the original Xgs- mutant strain to prove that the Xgs+ phenotype was caused by the plasmid. In each case, this resulted in 100% complementation, and all of the tetracyclineresistant exconjugants carried the same recombinant cosmid. The transformants of E. coli also served as a source of DNA for restriction mapping and DNA hybridization tests by Southern blotting. A summary of the complementation data is included in Table 2. During the course of this work, we found a useful shortcut

3596

THORNE ET AL.

J. BACTERIOL.

exconjugants over the Xgs- background. This as yet unexplained phenomenon seemed to correlate with the relative viability of the particular Xgs- recipient. For example, the frequency of Xgs+ exconjugants was elevated by more than 20-fold for a poorly growing mutant, m8, when the library
was first exposed to Tn5 not show this effect.

in E. coli. More viable mutants did

carrying

FIG. 1. Colony morphologies of X. campestris B1459 derivatives recombinant plasmids. Panels: top, X59-pkK311 (Xgs+); middle, X59m45-pRK311 (Xgs-); bottom, X59m45-c45 (Xgs+). The strains were plated on TBAB plus starch-, tetracycline, and rifampin and incubated at 30C for 5 days.

Alignment of cloned inserts and Xgs- mutations by restriction mapping, DNA hybridization, and genetic complementation. When more than one recombinant plasmid from the complete library complemented the same mutation, we usually found that they were either indistinguishable sibling clones or shared considerable DNA hotnology. DNA hybridization analyses that demonstrate this point are shown in Fig. 2. Several recombinant plasmids were digested with a mixture of EcoRI and Hindlll enzymes. HindIII cleaves in the multiple-cloning site to one side of the cloned insert, and EcoRI cleaves to the other side and also once within the vector. This produces two fragments of vector DNA of about 8 and 13 kbp (Fig. 2, marked v). The digestion products were separated by electrophoresis through agarose gels, with panels A, B, and C containing the same digestion products. A photograph of one of the gels after staining with ethidium bromide is shown in panel C. Most of the bands are from the cloned X59 DNA. For panel A, the hybridization probe was the radiolabeled c45 plasmid, and for panel B, the probe was c31. The hybridization pattern indicated that cosmids c8, c31, c45, and c65 carried overlapping segments of chromosomal DNA. The region in common between these four clones included restriction fragments of 0.6 and 1.2 kbp and part of the 4.3-kbp fragnment. There was no homology between the c45 probe and the cloned inserts in c9 (lane 2), c39 (lane 7), or c89 (lane 0, ethidium stain only). The cosmids c39 and c89 each complemented a respective mutant (m39 and m89) but have not been studied in detail. The hybridization results shown in Fig. 2 and similar blotting analyses for other mutants and cloned inserts were used to compile the three genetic maps shown in Fig. 3. The approximate map position for each was determined from the physical boundaries of the overlapping cloned fragmentg of DNA and the pattern of complementation by that DNA. Overlapping cloned fragments were aligned according to restriction pattern and DNA homology. Superimposed on this alignment are the results of comnplementation experiments, with + signifying restoration of the Xgs+ phenotype to an Xgs- mutant. In one case, we subcloned the compleTABLE 2. Complementation of Xgs- mutations by wild-type cloned X. campestris DNAa Cloned fragment
cl c8

that allowed cloning and mapping of xanthan genes in one step. We mutagenized the entire library of X. campestris genes in E. coli by infecting with bacteriophage X: .TnS (19). The mutagenized library was then mated with the X. campestris Xgs- recipients to yield exconjugants that grew on mediumi containing rifampin (the recipient selection), tetracycline (the plasmid marker), and kanamycin (the Tn5 marker). Restriction analysis of several Xgs+ Tetr Kanr isolates allowed us to determine the position of TnS on each plasmid and to map the xanthan genes. About one-half of the TnS insertions were found in the vector portion of the recombinant plasmid, and the other half were in the cloned X. campestris DNA. Since- we selected for mucoid exconjugants, the TnS insertions were outside the complementing xanthan gene. The shortened process described here also resulted in an unexpectedly higher frequency of Xgs+

Mutant

c9

cll

c31

c45

c65

c82

ml m8 m9 mll m31 m45 m48 m65 m82 m96 m145

a

+
-

+I+

+I+

+I+

+I+I
+ +
+

+ + -

+ + -

+
+

+ -

_ +
-

+ +/+
-

+ -

+ NT

+/I
+ -

+/+ -

+
+

Xgs- mutants that received a recombinant cosmid by mating were scored for mucoid (+) or nonmucoid (-) appearance by visual inspection of colonies. +/-, Partial mucoidy; NT, not tested.

VOL. 169, 1987

A 1

XANTHAN SYNTHESIS GENES OF X. CAMPESTRIS

B

3597

234 567

2 3 4 56 7

mO 1 2 3 4
1297w73..

6 7

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1.79 S ~*-1.2'.

FIG. 2. Analysis of cloned X. campestris DNA insertions for plasmids that complement Xgs- mutations. Plasmid DNA was prepared from E. coli JM109 and digested with a mixture of EcoRI and HindlIl restriction endonucleases. The DNA fragments were separated by agarose gel electrophoresis in two gels. After the gels were stained with ethidium bromide and photographed, the fragments were transferred to nitrocellulose and hybridized to a nick-translated radiolabeled DNA probe. (A) Autoradiograph resulting from hybridization with the radiolabeled plasmid c45. (B) Autoradiograph resulting from hybridization with plasmid c31. (C) Photograph of the ethidium-stained gel also shown in panel B. Both gels were nearly identical in appearance. Lanes: 0, c89; 1, c45; 2, c9; 3, c65; 4, c8a::TnS; 5, c8b::TnS; 6, c31; 7, c39; m, molecular markers with sizes given in kilobase pairs. v, Fragment from the vector plasmid pRK311. For c8a::Tn5, the insertion was within the 10.5-kbp fragment, and for c8b: :TnS, the insertion was in the 0.9-kbp fragment, with neither insertion affecting complementation of mutant m8.

menting region on a small fragment (Fig. 3, see c9e) to refine the map. The approximately 120 kbp of unique sequences cloned and shown in Fig. 3 represent about 5% of the chromosome of X. campestris. In addition, two other unlinked mutant loci have been identified (m39 and m89) but not characterized. Xanthan synthesis by exconjugants of X59 with multiple copies of complementing cloned genes. Assuming that there is a rate-limiting enzymatic step in the xanthan biosynthetic pathway, we reasoned that by introducing multiple copies (the pRK311 copy number is about 5) of a gene that codes for a limiting enzyme, we could raise the extent or rate of xanthan synthesis. We transferred each complementing clone by mating into X59, already Xgs+, and measured xanthan synthesis. For a control, we used X59 bearing the vector alone, pRK311. The cells were grown in shake flasks at 30C, starting from inocula of 107 cells per ml. The amount of xanthan was determined by standard methods, i.e., precipitation of the exopolysaccharides by 2 volumes of isopropanol, followed by drying and weighing. For most of the complementing clones, the extra gene copies either had no detectable effect or caused a decrease in xanthan yield. For those that repeatedly accumulated amounts of xanthan significantly higher than the control, representative xanthan and cell growth data are given in Fig. 4. In no case was the increase in accumulation greater than 20%'o. However, the

rate of xanthan accumulation between 24 and 36 h for X59-c45 was twice that for the control X59-pRK311. When X59 without pRK311 was included in the time course experiments, we found that the large vector plasmid itself had a negative effect on xanthan accumulation (data not shown). The final extent of xanthan accumulation in these shake flask tests was limited by the amount of the carbon source in the medium (2% [wt/vol] glucose, 0.25% [wt/vol] tryptone, and 0.25% [wt/vol] yeast extract). Based on the yield of 2.0 g of xanthan in a 10-ml culture (X59-c45), the carbon conversion was about 80% or near the theoretical maximum.

DISCUSSION Little is known about the genes, enzymes, or mechanisms that control the synthesis of xanthan gum by X. campestris. However, a considerable body of information exists with respect to polysaccharide synthesis among several gramnegative bacterial species (20). The synthesis of colanic acid by E. coli K-12 is probably closely related to that of xanthan. Both require the conversion of glucose to UDPglucose, UDPglucuronic acid, and GDPmannose. In addition, for colanic acid synthesis, GDPmannose must be converted to GDPfucose in two enzymatic steps and UDPglucose must be converted to UDPgalactose in one step. Colanic acid is made from GDPfucose, UDPgalactose, UDPglucose, and

3598

THORNE ET AL.
II

J. BACTERIOL.

5 kb

I
-W

m45 m48
I

R+H

III~m3
WlffU UM

1$ m31M96 m65
I
-

HRR

AI I
1 1

c65, c31
c8
+
+

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I :1
+

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0)

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c9H7 c82
c9

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Jim@{ m145 ii
R B
+
.
.

.+

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C9e

+4

FIG. 3. Compilation of three physical maps for X. campestris DNA insertions in vector pRK311 and complementation groups. The line marked R + H shows the order and position of restriction cleavage sites for EcoRI (R) and HindlIl (H) enzymes deduced from the overlapping maps given below for individual cloned inserts. Parentheses at the ends of the clone maps signify that we could not distinguish between an end generated by cleavage within the cloned insert from one generated by restriction in the adjoining multiple cloning site. The tentative map positions for Xgs- mutations are indicated above the physical maps. Unordered loci are enclosed within braces ({}). A summary of the complementation results from Table 2 is superimposed on the clone maps as + (positive complementation) or - (lack of complementation; Xgs-). Cosmids ci to c82 are independent isolates from the library of cloned DNA fragments. Cosmid c9e is a subclone generated by in vitro restriction and ligation. Cosmids c9H7 and c1H5 were obtained by screening the library for DNA hybridization to c9 and ci, respectively.

UDPglucuronic acid; xanthan is made from the latter two nucleotidyl sugars plus GDPmannose. The pentasaccharide repeat of xanthan and the hexasaccharide repeat of colanic acid are both assembled first as covalent attachments to an isopentenyl pyrophosphate carrier. The enzymatic polymerization of repeat units and eventual secretion of the polysaccharide are also likely to follow similar mechanisms. However, in contrast to the synthesis of colanic acid by E. coli, which can vary conditionally over a 100-fold range, the synthesis of xanthan appears to be more constitutive, varying at most by a few fold. The genes involved in a biosynthetic pathway are often contiguous in enteric bacteria. Classic examples are the E. coli trp and his operons. Similarly, several genes essential for capsular polysaccharide synthesis (colanic acid) and responsive to regulation by lon have been discovered at 45 min on the E. coli map and were given the name cps (22). Likewise, mutations that affect the synthesis of alginic acid by P. aeruginosa are also clustered (8). Therefore, we expected clustering of mutations affecting xanthan synthesis, and we found such a clustering for X. campestris. However, not all of the genes essential to xanthan accumulation were found to be clustered. The physical mapping and genetic complementation re-

sults described in this work define at least three unlinked loci carrying xanthan genes. We were able to subdivide one locus into three distinct complementing regions. Likewise, a second locus was found to contain two complementation groups. We used the physical boundaries of complementing cloned DNA to map the Xgs- mutations into complementation groups. The genes carried by these complementing cloned fragments can code for enzymes in the xanthan synthetic pathway (20), auxiliary structural proteins, or positive regulatory factors (such as the rcs loci that control colanic acid synthesis in E. coli [12]). The complementing genes that we isolated by this method were dispensable for growth of X. campestris. Our strategy for gene isolation would not yield coding sequences for enzymes that were essential to the synthesis of the cell wall polysaccharides peptidoglycan and lipopolysaccharide. Two different examples of likely common steps are the conversion of glucose-1P to UDPglucose and recycling of the oligosaccharide carrier molecule isopentenyl pyrophosphate. Barrere et al. (2) recently described the cloning of a fragment of DNA from X. campestris that restores mucoidy to two independently isolated mutants. The EcoRI restriction fragments from their cloned insert appear very similar to those of our c8 clone. Consistent with our results, they found

VOL. 169, 1987

XANTHAN SYNTHESIS GENES OF X. CAMPESTRIS

3599

B
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I
68

Time (h) Time (h) FIG. 4. Time course of accumulation of xanthan by wild-type strain X59 carrying multiple copies of xanthan genes. Recombinant plasmids containing inserts of cloned X. campestris DNA that restored xanthan synthesis to Xgs- mutants were individually transferred to strain X59 and maintained by selection with tetracycline. Each inoculum was 107 bacteria, and at indicated times, samples were withdrawn to measure cell growth (for panel A, optical density at 600 nm) and xanthan accumulation in 10 ml of culture (panel B, isopropyl alcohol-precipitable exopolysaccharide). Symbols: 0, X59; A, X59-pRK311; A, X59-c45; O, X59-c9; 0, X59-cl; *, X59-c31. The upper curve in panel A represents four cultures. In panel B, the solid line is for X59-c45, the dashed line is for X59-pRK311, and the dotted line is for X59-c31.

that Tn5 insertions into several different locations of their clone cause a nonmucoid phenotype when the mutated region is recombined back into the wild-type chromosome. Thus, they also show evidence for clustered xanthan genes. We thought that even for a prolific producer of exopolysaccharide, such as X. campestris B1459S-4L-II, we might be able to identify a rate-limiting enzymatic step in the biosynthetic pathway by introducing into wild-type cells cloned normal genes on multiple-copy plasmids. The simple notion was that the extra gene copies carried by plasmid pRK311 might overcome a limiting function. When we quantitated xanthan production, reproducible changes of up to 20% in the extent of xanthan accumulation were observed and in one case the rate of accumulation was raised twofold. We also found that the large plasmid vector itself depressed cell growth and xanthan synthesis. However, we recognize that these results alone are not sufficient to refer to the number of copies of a particular gene as rate limiting, since several genes may be included on our large cosmid clones. Although suggestive, the phenotypic change between mutant (Xgs-) and complemented exconjugant (Xgs+) does not prove that a cloned fragment necessarily codes for a normal copy of a mutant product. We must first understand why two nonhomologous cloned fragments of normal X. campestris DNA appear to complement a single mutation. Mutant ml was complemented by clones that were not homologous at the DNA sequence level (cl and c8, c31, or c65) (Table 2). Likewise, mutants m9, mll, and m82 were also complemented by nonhomologous clones. Two reasons seem plausible. First, there may be gene products coded by certain cloned fragments that regulate the expression of multiple target genes. Second, it is possible that the elevated number of copies for one cloned gene might compensate for a mutant defect in a different gene, for example by altering the flow of substrates through a pathway. To discriminate between these possibilities, further stud-

ies are genes.

sIs.

needed to refine the mapping of the clustered xanthan The most important objective now is to identify the exact roles of these cloned gene products in xanthan syntheACKNOWLEDGMENTS

We are grateful to Ric Armentrout, Ron Brown, Joe Cappello, Franco Ferrari, Charlie Johnson, and Chuck Richardson for repeated helpful discussions during this work and to Marcia O'Neill for preparation of the manuscript. Some of the initial Xgs- mutants were isolated by W. Charles Johnson of Syntro. LITERATURE CITED 1. Anderson, E. H. 1946. Growth requirements of virus-resistant mutants of Escherichia coli strain "B". Proc. Natl. Acad. Sci. USA 32:120-128. 2. Barrere, G. C., C. E. Barber, and M. J. Daniels. 1986. Molecular cloning of genes involved in the production of the extracellular polysaccharide xanthan by Xanthomonas campestris pv. campestris. Int. J. Biol. Macromol. 8:372-374. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 4. Borthakur, D., C. E. Barber, J. W. Lamb, M. J. Daniels, J. A. Downie, and A. W. B. Johnston. 1986. A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not beans by R. phaseoli and is corrected by cloned DNA from Rhizobium or the phytopathogen Xanthomonas. Mol. Gen. Genet. 203:320-323. 5. Cadmus, M. C., S. P. Rogovin, K. A. Burton, J. E. Pittsley, C. A Knutson, and A. Jeanes. 1976. Colonial variation in Xanthomonas campestris NRRL B-1459 and characterization of the polysaccharide from a variant. Can. J. Microbiol. 22:942-948. 6. Costerton, J. W., R. T. Irvin, and K.-J. Cheng. 1981. The role of bacterial surface structures in pathogenesis. Crit. Rev. Microbiol. 8:303-338. 7. Darzins, A., and A. M. Chakrabarty. 1984. Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis

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