Abstract:
Increasing the molecular length of xanthan polymer makes a higher viscosity xanthan composition. Xanthan with higher specific viscosity characteristics provides more viscosity at equivalent concentration in food, industrial and oilfield applications. Methods for increasing the viscosity of xanthan include inducing particular key genes and increasing copy number of particular key genes.

Description:
[0001]    This application claims the benefit of provisional application U.S. Ser. No. 60/456,245 filed Mar. 21, 2003.  
         [0002]    A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. 
     
    
     
       FIELD OF THE INVENTION  
         [0003]    The invention relates to the field of microbial products. In particular it relates to microbial products having improved properties for various industrial purposes.  
         BACKGROUND OF THE INVENTION  
         [0004]    The chemical structure of xanthan is composed of a linear cellulosic (1→4)-β-D-glucose polymer with trisaccharide side chains composed of mannose, glucuronic acid and mannose, attached to alternate glucose residue in the backbone. (Milas and Rinaudo, Carbohydrate Research, 76, 189-196, 1979). Thus xanthan can be described as a branched chain polymer with a pentasaccharide repeat unit; normal xanthan typically has 2000-3000 pentasaccharide repeat units. The xanthan polymer is typically modified by acetylation and pyruvylation of the mannose residues.  
           [0005]    The fermentation of carbohydrates to produce the biosynthetic water-soluble polysaccharide xanthan gumBy the action of  Xanthomonas  bacteria is well known. The earliest work was conducted by the United States Department of Agriculture and is described in U.S. Pat. No. 3,000,790 . Xanthomonas  hydrophilic colloid (“xanthan”) is an exocellular heteropolysaccharide.  
           [0006]    Xanthan is produced by aerobic submerged fermentation of a bacterium of the genus  Xanthomonas . The fermentation medium typically contains carbohydrate (such as sugar), trace elements and other nutrients. Once fermentation is complete, the resulting fermentation broth (solution) is typically heat-treated. It is well established that heat treatment of xanthan fermentation broths and solutions leads to a conformational change of native xanthan at or above a transition temperature (TM) to produce a higher viscosity xanthan. Heat treatment also has the beneficial effect of destroying viable microorganisms and undesired enzyme activities in the xanthan. Following heat-treatment, the xanthan is recovered by alcohol precipitation. However, heat treatment of xanthan fermentation broths also has disadvantages, such as thermal degradation of the xanthan. Heating xanthan solutions or broths beyond TM or holding them at temperatures above TM for more than a few seconds leads to thermal degradation of the xanthan. Degradation of xanthan irreversibly reduces its viscosity. Accordingly, heat treatment is an important technique with which to control the quality and consistency of xanthan.  
           [0007]    Xanthan quality is primarily determined by two viscosity tests: the Low Shear Rate Viscosity (“LSRV”) in tap water solutions and the Sea Water Viscosity (“SWV”) in high salt solutions. Pasteurization of xanthan fermentation broths at temperatures at or above TM has been found to yield xanthan of a higher viscosity as indicated by higher LSRV and SWV values.  
           [0008]    Xanthan polymer is used in many contexts. Xanthan has a wide variety of industrial applications including use in oil well drilling muds, as a viscosity control additive in secondary recovery of petroleum by water flooding, as a thickener in foods, as a stabilizing agent, and as a emulsifying, suspending and sizing agent (Encyclopedia of Polymer Science and Engineering, 2nd Edition, Editors John Wiley &amp; Sons, 901-918, 1989). Xanthan can also be used in cosmetic preparations, pharmaceutical vehicles and similar compositions.  
           [0009]    There is a need in the art to produce a xanthan polymer with higher specific viscosity characteristics in the unpasteurized state. Such a higher specific viscosity xanthan polymer could provide more viscosity at equivalent xanthan concentrations, for example, for food, industrial, and oilfield applications.  
         BRIEF SUMMARY OF THE INVENTION  
         [0010]    In a first embodiment an unpasteurized xanthan composition is provided. The composition can be provided by a cell which over-expresses gumB and gumC. It has an intrinsic viscosity which is at least 20% greater than xanthan from a corresponding strain which does not over-express gumB and gumC.  
           [0011]    In a second embodiment a xanthan composition is provided. It comprises a population of xanthan molecules having a range of molecular lengths. At least 1% of the population has a length greater than 3 um as measured by atomic force microscopy.  
           [0012]    In a third embodiment of the invention a method is provided for producing a xanthan polymer preparation having increased viscosity relative to that produced by a wild-type strain. The amount of gene product of gumB and gumC is selectively increased in a  Xanthomonas campestris  culture. The amount of a gene product of orfX is not selectively increased. Nor is the amount of a product of a gene selected from the group consisting of gumD-gumG selectively increased. A higher viscosity xanthan polymer preparation is thereby produced by the culture.  
           [0013]    In a fourth embodiment of the invention a method is provided for producing a xanthan polymer preparation having increased viscosity relative to that produced by a wild-type strain. A  Xanthomonas campestris  strain is cultured in a culture medium under conditions in which it produces a xanthan polymer. The strain selectively produces relative to a wild-type strain more gene product of gumB and gumC but not of orfX nor of a gene selected from the group consisting of gumD-gumG.  
           [0014]    In a fifth embodiment of the invention an unpasteurized xanthan composition is provided. The composition is made by a cell which over-expresses gumB and gumC. The composition has a seawater viscosity which is at least 10% greater than xanthan from a corresponding strain which does not over-express gumB and gumC.  
           [0015]    The present invention thus provides the art with xanthan compositions which have increased viscosity relative to those similarly produced by corresponding wild-type strains. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0016]    [0016]FIG. 1 shows genetic constructs relative to a genetic map of the gumB-M operon, also known as the xpsB-M (xanthan polysaccharide synthesis) operon.  
         [0017]    [0017]FIGS. 2A and 2B show Western blot analyses of gumB and gumC protein product expression, respectively.  
         [0018]    [0018]FIG. 3 shows an intrinsic viscosity plot for xanthan gum samples, one of which over-expresses gumB and gumC gene products due to the presence of a plasmid carrying extra copies of the genes. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0019]    It is a discovery of the present inventors that overexpression of gumB and gumC gene products relative to other genes in their operon, yields xanthan products with higher viscosity on a per weight basis. While applicants do not wish to be bound by any particular theory of operation, it appears that a shift in the ratio of certain gene products leads to a shift in the size distribution of xanthan polymer molecules. A significant number of molecules are of higher molecular length than when xanthan is made by a wild-type cell. These longer molecules lead to a higher viscosity of the population or preparation.  
         [0020]    It is known in the art that increases in viscosity can be obtained by pasteurizing xanthan preparations. See Talashek et al., U.S. Pat. No. 6,391,596. However, the increased viscosity found as the result of overexpression of gumB and gumC is observed even in the absence of pasteurization. Nonetheless subsequent pasteurization of the products of the present invention will yield an even more viscous preparation.  
         [0021]    Overexpression of both gumB and gumC appear to be required to achieve the increased viscosity. When either gene was tested alone, the increase was not observed. The overexpression of gumB and gumC can be assessed relative to other genes of the gumB-M operon. While overexpression relative to any of those genes may be sufficient to achieve the effect, overexpression with respect to orfX and gumD may be particularly significant. OrfX is a small open reading frame that was previously published as a segment of the genome designated as gumA, immediately upstream of gumB. Recently two open reading frames have been discerned in the former gumA region, ihf and orfX Overexpression relative to all of the genes gumD-gumM may be desirable.  
         [0022]    Overexpression of the desired gene products may be achieved by any means known in the art, including, but not limited to, introducing additional copies of the genes encoding the desired gene products to a  Xanthomonas campestris  cell or other bacterium that makes xanthan, and induction of the desired gene products using for example an inducible promoter. Other bacteria that make xanthan include those that have been genetically engineered to contain the xanthan biosynthetic genes. The gumB and gumC genes can be introduced on one or more vectors, i.e., in combination or individually.  
         [0023]    Inducible promoters which can be used according to the invention include any that are known in the art, including the lac promoter, the ara promoter, the tet promoter, and the tac promoter. Natural and artificial inducing agents for these promoters are known in the art, and any can be used as is convenient. Additional copies of genes can be introduced on plasmids or viral vectors, for example. Additional copies of the desired genes can be maintained extrachromosomally or can be integrated into the genome.  
         [0024]    Recovery of xanthan from a culture broth typically involves one or more processing steps. The xanthan may be heat-treated. The xanthan may be precipitated with an alchohol, such as isopropyl alcohol, ethyl alcohol, or propyl alcohol. Typically the cells are not specifically removed from the culture broth.  
         [0025]    Xanthan molecules produced biosynthetically typically have a distribution of sizes. The increased viscosity of the present invention may be achieved by increasing the number of molecules having a much longer than average length, or by increasing to a greater degree the number of molecules having a somewhat longer than average length. The number of molecules which have increased length need not be huge. At least 1, 3, 5, 7, 9, or 11% of the molecules with an increased length may be sufficient. The molecules of increased length may be greater than 3, 4, 5, 6, 7, 8, or 9 um, as measured by atomic force microscopy. The percentage of the mass of the total xanthan population contributed by the molecules which are longer than 3, 4, 5, 6, 7, 8, or 9 um will be greater than their number proportion in the population. Thus at least 1, 3, 5, 10, 15, 20, or 25% of the total mass of the xanthan molecules may be contributed by molecules having a greater than 3 um length.  
         [0026]    Intrinsic viscosity measurements are yet another way to characterize the preparations of the present invention. Increases seen using this type of measurement may be as great as 5, 10, 15, 20, 25, 30, or 35% over that produced by wild-type strains. Proper controls for comparison purposes are those corresponding strains which are most closely related to the strains being tested. Thus if testing strains that have additional copies of gumB and gumC, the best control will have the same genetic complement but for the presence of the additional copies of gumB and gumC. If testing cultures that have been induced by an inducer to produce more gumB and gumC gene product, then the best control will be cultures of the same strain that have not been induced. Sea water viscosity can also be used to characterize preparations of the present invention. Increases seen using this type of measurement may be as great as 5, 10, 15, 20, 25, 30, or 35% over that produced by wild-type strains.  
         [0027]    Xanthan is used as a component in a number of products to improve properties. The properties may include viscosity, suspension of particulates, mouth feel, bulk, to name just a few. Other properties include water-binding, thickener, emulsion stabilizing, foam enhancing, and sheer-thinning. Such products include foods, such as salad dressings, syrups, juice drinks, and frozen desserts. Such products also include printing dyes, oil drilling fluids, ceramic glazes, and pharmaceutical compositions. In the latter case, xanthan can be used as a carrier or as a controlled release matrix. Other products where xanthan can be used include cleaning liquids, paint and ink, wallpaper adhesives, pesticides, toothpastes, and enzyme and cell immobilizers.  
         [0028]    While the invention has been described with respect to specific examples including presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described systems and techniques that fall within the spirit and scope of the invention as set forth in the appended claims.  
       EXAMPLES  
     Example 1  
     Strain Construction  
       [0029]    To isolate a fragment carrying the complete gum gene region of  X. campestris , a genomic library of the wild type  X. campestris  strain, NRRL B-1459 (1), was constructed with the broad-host-range cosmid vector pRK311 (2) by cloning of total DNA partially digested with Sau3AI. This library was mated en masse from  E. coli  S17-1 (3) to the Gum −    X. campestris  mutant 2895 (4). One of the cosmids isolated from several mucoid exconjugants termed pIZD15-261 (5) contains a 16-kb fragment encompassing the complete gum region. See FIG. 1 for a graphic representation and Table 1 for a listing of the genes of the operon.  
                                 TABLE 1                           List of genes designations in the chromosomal region       encoding xanthan polysaccharide synthesis              X. campestris                      ATCC13951     X. campestris         (NRRL B-   pv.  campestris     Chromosomal       1459)   ATCC33913   Location*   Function               inf   himA (XCC2457)   2918744-2918448   integration host factor,                   alpha chain       orfX   (XCC2456)   2918464-2918111   transcriptional regulator       xpsB   gumB (XCC2454)   2917444-2916806   xanthan export       xpsC   gumC (XCC2453)   2916731-2915385   xanthan export       xpsD   gumD   2915139-2913688   glucosyl transferase           (XCC2452)       xpsE   gumE (XCC2451)   2913602-2912307   xanthan polymerization       xpsF   gumF (XCC2450)   2912307-2911216   acetyl transferase       xpsG   gumG (XCC2449)   2911216-2910149   acetyl transferase       xpsH   gumH   2910078-2908939   mannosyl transferase           (XCC2448)       xpsI   gumI (XCC2447)   2908939-2907893   mannosyl transferase       xpsJ   gumJ (XCC2446)   2907893-2906397   xanthan export       xpsK   gumK (XCC2445)   2906014-2905130   glucuronic transferase       xpsL   gumL (XCC2444)   2905086-2904295   pyruvyl transferase       xpsM   gumM   2904284-2903496   glucosyl transferase           (XCC2443)       orf165   (XCC2442)   2903458-2902964   unknown conserved                   hypothetical                          
 
         [0030]    For the construction of the pBBR5-BC plasmid, a 4026 bp fragment from pIZD15-261 digested with SpeI-BglII was cloned between the XbaI and BamHI sites of pKmob19 (8), giving rise to pGum02-19S (5). A 2855 bp fragment was released from plasmid pGum02-19S by digestion with SphI. This fragment was cloned into pUC 18 (9), which was previously digested with SphI, forming pUC18-BCAS.  
         [0031]    The final plasmid (pBBR5-BC) was constructed by cloning the HindIII-XbaI fragment, containing the gum promoter and gumB and gumC genes, into HindIII-XbaI digested pBBR1-MCS5 (10) (GenBank accession no. U25061).  
         [0032]    The nucleotide sequence of the resulting pBBR5-BC plasmid is shown in SEQ ID NO: 1. (The predicted amino acid sequences of gumB and gumC are shown in SEQ ID NOs: 2 and 3, respectively. This broad-host-range, medium-copy-number plasmid is 7.6 kb in length and is compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons. The presence of an origin of transfer (mobRK2) enables its transference by conjugation into a wide range of bacteria when the RK2 transfer functions are provided in trans. It also carries the gentamicin resistance gene and it contains the pBluescript II KS multiple cloning site located within the gene encoding the LacZ a peptide (pBluescript II KS from Stratagene, La Jolla, Calif., USA).  
         [0033]    To verify the expression of GumB and GumC proteins from pBBR5-BC, the plasmid was introduced into  X. campestris  mutant 1231, in which the entire gum (xps) gene cluster was deleted. Both proteins were detected by Western blot in the mutant strain.  
                             TABLE 2                           Bacterial strains and plasmids used or constructed in this work.                    Source       Bacterial strain or plasmid   Relevant characteristics   (reference)                 E. coli.                 DH5α   F - endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 ΔlacU169   New England           (φ80dlacZΔM15)   Biolabs       S17-1   E. coli 294 RP4-2-Tc::Mu-Km::Tn7   (3)       JM109   F′ traD36 proA + B +  lacl q  Δ(lacZ)M15/Δ(lac-proAB) glnV44 e14   New England           gyrA96 ompT hsdS B (r B   −  m B   − ) gal [dcm] [lon]   Biolabs       BL2I(DE3)   F - ompT hsdS B (r B   −  m B   − ) gal [dcm] [lon] (DE3)   Novagen         X. campestris         NRRL B-1459   Wild type.   (1)       2895   Rif r  xpsI-261   (11)       1231   Tc::Tn 10 ΔxpsI   C.P. Kelco       XWCM1   Mutant of NRRL B-1459   C.P. Kelco       PRM-1   Mutant of NRRL B-1459   C.P. Kelco       Plasmids       pRK311   oriV(RK2) Tc r  oriT(mob + ) tra −  λcos lacZ(α)   (2)       pIZD15-261   Cosmid based on pRK311 carrying the  X. campestris  gum   (5)           region.       pK19mob   Km r , pK19 derivative, mob-site   (8)       pgum02-19AS   pK19mob vector carrying the gum fragment 770-4795 a     (5)       pUC18   Ap r , Co1E1, lacZα +     (9)       pUC18-BCAS   pUC18 vector carrying the gum fragment 770-3610 a     This work       pBBR1-MCS5   Gm r , pBBR1CM derivative, mob-site, lacZα +     (10)       pBBR5-BC   PBBR1-MCS5 carrying the gum fragment 770-3610 a     This work       pQE-Xps#6   pQE30 vector carrying the gum fragment 1336-1971 a     C.P. Kelco       pQE30   Ap r     Qiagen       pREP4   Km r     Qiagen       pET-C   pET22b(+) vector carrying the gum fragment 2135-3319 a     This work       pET22b+   Ap r     Novagen       pH336   pRK290 carrying gum BamHI fragments1-15052 a     Synergen       pCOS6   pRK293 carrying Sa1I fragments 1-14585a and upstream xps I DNA   CP Kelco       pFD5   pRK404 carrying partial BamHI gum fragment 318-3464 a     Ielpi       pCHC22   pRK293 carrying Sa1I fragments 1-9223a and upstream xps I DNA   (4)       pBBR-prom   pBBR1-MCS5 carrying gum fragment 1000-1276 a     This work       pBBR5-B   pBBR1-MCS5 carrying gum fragment 770-1979 a     This work       pBBR-promC   pBBR1-MCS5 carrying gum fragment 1979-3459 a     This work                          
 
         [0034]    Bacterial strains, plasmids, and growth conditions. The strains and plasmids used in this study are listed in Table 2 . E. coli  strains were grown in Luria-Bertani medium at 37° C.  X. campestris  strains were grown in TY (5 g of tryptone, 3 g of yeast extract, and 0.7 g of CaCl 2  per liter of H 2 O) or in YM medium (12) at 28° C. Antibiotics from Sigma (St. Louis, Mo.) were supplemented as required at the following concentrations (in micrograms per milliliter): for  X. campestris , gentamicin, 30; and tetracycline, 10; for  E. coli , gentamicin, 10; kanamycin, 30; ampicillin, 100; and tetracycline, 10.  
         [0035]    DNA biochemistry. Plasmid DNA from  E. coli  and  X. campestris  was prepared by using the QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany). DNA restriction, agarose gel electrophoresis and cloning procedures were carried out in accordance with established protocols (13). All constructs were verified by DNA sequencing. Plasmid DNA was introduced into  E. coli  and  X. campestris  cells by electroporation as instructed by Bio-Rad (Richmond, Calif.) (used parameters:  E. coli:  200 Ω, 25 μF, 2500V and  X. campestris:  1000 Ω, 25μF, 2500V).  
         [0036]    Analysis of nucleotide and protein sequences. The nucleotide and amino acid sequences were analyzed by using the MacVector Sequence Analysis Software (Oxford Molecular Limited, Cambridge, UK).  
       Example 2  
     Western Analysis of gumB and gumC Expression  
       [0037]    Western Analysis confirmed that gumB and gumC gene products are being over-expressed in the  X. campestris  strain with extra copies of gumB and gumC. See FIG. 2.  
       Example 3  
     Intrinsic Viscosity determination  
       [0038]    Xanthan samples prepared from  X. campestris  strains with (XWCM1/pBBR5BC) and without (XWCM1) multiple, plasmid encoded copies of the gumB and gumC genes were compared. Shake flask fermentations, using glucose as a carbon source, were carried out to obtain xanthan from these strains.  
         [0039]    Intrinsic viscosity was determined by measuring viscosity on both purified and unpurified xanthan samples. An increase in the intrinsic viscosity for xanthan from  X. campestris  strain with multiple copies of gumB and gumC was observed. Intrinsic viscosity is proportional to the molecular weight for a given polymer type when measured under identical solvent and temperature conditions. Therefore, xanthan from  X. campestris  strain with multiple copies of gumB and gumC is of higher molecular weight compare to xanthan from control strain.  
         [0040]    Methods: Five shake flasks each of the two broths were tested. The broths of each type were combined and the total volume measured. The broth was then precipitated in isopropyl alcohol. (Note: It was estimated that the broth contained approximately 3% gum. Measuring the total broth volume and multiplying by 3% gave the approximate dry gum weight. This approximation was used to calculate the amount of water required to produce approximately a 0.5% gum solution). The wet fibers of the precipitate were then immediately rehydrated with mixing in 0.01M NaCl to produce approximately a 0.5% gum solution. The fibers were mixed for three hours with good shear using a 3-blade 2 inch diameter propeller stirrer, then allowed to stand overnight. The following procedure was used to prepare the samples for intrinsic viscosity measurements.  
         [0041]    Filter the ˜0.5% gum solution, prepared above, using a Gelman Science 293 mm pressure filtration unit. The solution is first filtered through a 20 μ Magna nylon filter (N22SP29325) . The filter is pressurized to ˜60 psi, and the solution collected into clean beakers. (Note: the filters are changed when the flow rate is reduced to ˜5 drips per minute.  
         [0042]    Following the first filtration step, the samples are filtered two more times using the above filtration unit. First, through a Millipore 8.0 μ filter (SCWP 293 25), then through a Gelman Versapor® 293 mm 1.2 μ filter (66397). The filtered sample is recovered in clean beakers following each filtration step.  
         [0043]    After filtration, ˜600 ml of the gum solution is placed into Spectra/Por® dialysis tubing 28.6 mm diameter Spectrum #S732706 (MWCO 12,000 to 14,000). The tubing is cut into lengths of ˜18-20 inches, and a knot tied in one end. The solution is added to the tubing, filling it to within ˜2 inches from the end. Tie a second knot in the tubing such that as little air as possible is trapped in the tubing. Continue until all the gum solution is in dialysis tubing.  
         [0044]    Rinse the outside of the tubing containing the gum solution for ˜1 minute with de-ionized water, then place the tubing into a container of 0.1M NaCl. The salt solution should completely cover the dialysis tubing.  
         [0045]    Allow the tubing to sit in the 0.01M NaCl solution for 4 days, changing the NaCl solution daily. After the 4 days, cut open one end of the tubing and carefully transfer the gum solution to a clean beaker.  
         [0046]    Solids are run on the filtered dialyzed solution using the following procedure:  
         [0047]    Using an analytical balance capable of weighing to ±0.0002 g, weigh and record the weight of a clean aluminum weighing dish VWR Cat #25433-008. (A)  
         [0048]    Using a clean pipet add approximately 10 ml of the gum solution to the aluminum pan and record the exact weight of the combined pan and gum solution. (B)  
         [0049]    Place the pan with the solution into a 105° C. drying oven and allow to stand for 24 hours.  
         [0050]    Remove the pan from the oven after 24 hours, cool and reweigh. Record the weight of the pan and remaining dried gum. (C)  
         [0051]    Subtract the weight of the aluminum pan (A) from the weight of the pan plus the gum solution (B). Subtract the weight of the aluminum pan (A) from the weight of the dried gum plus the pan (C). Divide the first value (B-A) into the second (C-A). Multiply this value by 100 to obtain the % solids.  
         [0052]    Note: Solids were run in triplicate for each filtered dialyzed solution using the above procedure. The calculated % solids were than averaged for each sample and the averaged value was used.  
         [0053]    Based on the solids determination for each solution, the samples are diluted to 0.25% total gum concentration using 0.01M NaCl.  
         [0054]    Intrinsic viscosity measurements were made using the Vilastic Viscoelasticity Analyzer (Vilastic Scientific, Inc., Austin, Tex., fitted with the 0.0537 cm radius X 6.137 cm length tube. The instrument was calibrated with water prior to making measurements and verified after the measurements were completed. Measurements were conducted using the instruments TIMET software protocol, set to a frequency of 2.0 Hz, a constant strain of 1.0, and an integration time of 10 seconds. The temperature was maintained at 23.5° C. The samples were prepared by dilution of the 0.25% gum solution. Each dilution was mixed for 20 minutes, and allowed to stand refrigerated overnight before being measured. Six measurements were made for each dilution and averaged. Table 3 below shows the dilutions and the resultant averaged viscosities for each prepared sample.  
                                                                       TABLE 3                                           Viscosity           Dilutions   Measurements                0.25% X.G.   0.01 M NaCl   XWCM1    XWCM1/       Concentrations   (ml)   (ml)   Control   pBBR5-BC                    Solute 0.01 M   0   100   .921   .921       NaCl       0.0025%   1   99   1.114   1.165       0.0050%   2   98   1.326   1.486       0.0075%   3   97   1.537   1.829       0.0100%   4   96   1.762   2.181       0.0150%   6   94   2.302   2.963       0.0200%   8   92   2.920   3.901                  
 
         [0055]    Intrinsic viscosities were determined by plotting the reduced specific viscosity (η sp /c) against the gum concentration η sp /c=((η c −η o )/η o ) where η c =viscosit of the gum. The intercept yields the intrinsic viscosit{tilde over (y)}. See FIG. 3.  
         [0056]    The increase in intrinsic viscosity for the XWCM1/pBBR5-BC variant is believed due to an increase in molecular weight. Intrinsic viscosity is proportional to the molecular weight for a given polymer type when measured under identical solvent and temperature conditions as done in this experiment. The relationship between [η] and molecular weight is given by the Mark-Houwink equation [η] =kM a , where k and a are constants for a specified polymer type in a specified solvent at a specified temperature. Because the constant “a” is positive number, an increase in [η] can only be obtained by an increase in the molecular weight (M) unless the samples have a different molecular conformation in which case the Mark-Houwink equation is not obeyed.  
       Example 4  
     Procedure—Low Shear Rate Viscosity Measurement  
       [0057]    Low shear rate viscosity measurements were performed on purified xanthan samples. The procedure used to measure LSRV is detailed below. Increased viscosity for xanthan from a strain with multiple copies of gumB and gumC compared to xanthan from a control strain was observed. The data suggest that over-expression of both gumB and gumC is required for increased chain length; over-expression of either gumB or gumC individually is not sufficient to increase chain length.  
         [0058]    Material and Equipment:  
         [0059]    1. Standard (synthetic) Tap Water (water containing 1000 ppm NaCl and 40 ppm Ca ++  or 147 ppm CaCI 2 0.2H2O): Prepare by dissolving in 20 Liters of distilled water contained in a suitable container, 20 gm of reagent grade NaCl and 2.94 gm of reagent grade CaCl 2 0.2H 2 O.  
         [0060]    2. Balance capable of accurately measuring to 0.01 gm.  
         [0061]    3. Brookfield LV Viscometer, Spindle #1, and spindle Guard.  
         [0062]    4. Standard laboratory glassware.  
         [0063]    5. Standard laboratory stirring bench. An RAE stirring motor (C25U) and stirring shaft ({fraction (5/16)}″) with 3-bladed propeller may be substituted.  
         [0064]    Procedure:  
         [0065]    1. To 299 ml of synthetic tap water weighed in a 600 ml Berzelius (tall form) beaker, slowly add 0.75 gm (weighed to the nearest 0.01 gm) of product,  
         [0066]    while stirring at 800 rpm.  
         [0067]    2. After stirring four hours at 800 rpm, remove the solution from the stirring bench, and allow to stand for 30 minutes.  
         [0068]    3. Adjust the temperature to room temperature and measure the viscosity using a Brookfield LV Viscometer with the No. 1 spindle at 3 rpm. Record the viscosity after allowing the spindle to rotate for 3 minutes.  
       Example 5  
     Quantification of Protein Expression  
       [0069]    Cell lysates were subjected to Western blot and immunodetection analysis to establish the level of plasmid encoded GumB and GumC. Four independent blots were analyzed. Although absolute values for the same sample were not reproducible in each quantification, the relative quantities between samples remained the same in all the measurements.  
         [0070]    Preparation of antibodies raised against GumB and GumC. An 1184 bp DNA fragment encoding amino acid residues 53-447 of the GumC protein was produced by PCR amplification. The following primers were used: F2135: 5′GGAATTCCATATGTTGATGCCCGAGAAGTAC-3′ (SEQ ID NO: 4) and B3319: 5′CGGGATCCTCAAAAGATCAGGCCCAACGCGAGG-3 (SEQ ID NO: 5)′. The PCR product was digested with NdeI and BamHL subcloned into pET22b(+) and the resulting plasmid (pET-C) introduced into the  E. coli  strain BL21 (DE3).  
         [0071]    [0071] E. coli  BL21(pET-C) grown in L-broth containing 50 μg carbenicillin ml −1  to OD 600  0.6 was induced with 1 mM IPTG for 3 h. Total cell lysates were prepared by treating with 1 mg lysozyme ml −1  in lysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA pH8, 100 mM NaCl, 1 mM PMSF, 0.1 mg DNase ml −1, 0.5 % Triton X-100) at 37° C. for 30 min, followed by sonication on ice. Cell debris was removed by low speed centrifugation (Eppendof, 4000×g, 5 min) and the supernatant was fractionated in a soluble and in a pellet (inclusion bodies) fraction by centrifugation at 14000×g for 10 min. Pellet fraction was washed twice with lysis buffer, in a volume identical to that of the original cell lysate, once with 2 mg DOC ml −1  in lysis buffer followed by three washes with water. After treatment, proteins were separated by SDS-PAGE and the major band containing the overproduced GumC protein was cut and eluted for immunizing rabbits.  
         [0072]    [0072] E. coli  JM109(pQE-Xps#6, pREP4) grown in L-broth containing 50 μg carbenicillin, 25 μg kanamycin ml −1  to OD 600  0.6 was induced with 1 mM IPTG for 3 h. Total cell lysates were prepared by treating with 1 mg lysozyme ml −1  in lysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA pH8, 100 mM NaCl, 1 mM PMSF, 0.1 mg DNase ml −1 , 0.5% Triton X-100) at 37° C. for 30 min, followed by sonication on ice. Cell debris was removed by low speed centrifugation (Eppendof, 4000×g, 5 min) and the supernatant was fractionated in a soluble and in an pellet (inclusion bodies) fraction by centrifugation at 14000×g for 10 min. Pellet fraction was washed twice with lysis buffer, resuspended in 6 M guanidine hydrochloride in 100 mM Phosphate buffer (pH7), 5 mM DTT, 5mM EDTA and inclusion bodies were chromatographed on an FPLC Superdex HR200 (Pharmacia Biotech) pre-equilibrated with buffer D (4 M GdnHCl, 50 mM Phosphate buffer (pH7), 150 mM NaCl). Fractions containing GumB were pooled and used to immunize mice.  
         [0073]    Construction of plasmids pFD5, pBBR-promC, and pBBR5-B. A 3141 bp fragment containing gumB and gumC genes was obtained by partial digestion of pIZD15-261 with BamHI (#318 and #3459) and cloned into BamHI-digested pRK404 to yield plasmid pFD5. A 1480 bp fragment was isolated by digestion of pGum02-19 with EcoRI (#1979) and BamHI (#3459) and cloned in pBBRlMCS-5 previously digested with the same enzymes to yield pBBR-promC. Digestion of pGum02-19 with HindIII in the MCS and EcoRI (#1979) produced a 1233 bp fragment, which was cloned in pBBR1MCS-5 to yield plasmid pBBR5-B.  
         [0074]    New Zealand white female rabbits were immunized using GumC prepared as described above. A primary injection of 500 μg of the protein with complete Freund&#39;s adjuvant was given to the rabbits, followed by three injections of 250 μg of the protein with incomplete adjuvant on alternate weeks. BALB/c female mice were immunized using GumB prepared as described above. A primary injection of 100 μg of the protein with complete Freund&#39;s adjuvant was given to the mice, followed by three injections of 50 μg of the protein with incomplete adjuvant once a week. Polyclonal antibodies were prepared as described by Harlow &amp; Lane ((1999)  Using antibodies: a laboratory manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and antisera were stored at −70° C. To obtain GumC-specific antibodies, the serum was adsorbed with both  E. coli  BL21(pET22b+) and Xc1231 acetone powders (Harlow &amp; Lane, supra).  
         [0075]    Protein extracts. Plasmids were introduced into the parental strain PRM-1 by electroporation. The resulting strains were grown in YM medium at 28° C. and 250 rpm to middle-logarithmic phase. Cells were harvested by centrifugation and the fresh-weight determined. The pellet was washed twice with 10 mM Tris/HCl, 10 mM EDTA (pH 8.0) to remove exopolysaccharide and resuspended in the same buffer at a concentration of 100 mg/ml. After addition of 100 μl Buffer A (10 mM Tris/HCl, 10 mM EDTA (pH 8.0), 1.5% SDS) to 50 μl of each sample, the mixture was incubated at room temperature for 10 min followed by incubation at 100° C. for 12 min. Cell lysate was centrifuged at 14000×g (Eppendorf 5415 C) for 5 min and the supernatant collected was designated as total protein extract. Protein concentration of each lysate was determined by the method of Markwell ((1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.  Anal Biochem  87(1), 206-10) in the presence of SDS, using BSA as a standard.  
         [0076]    SDS-PAGE and inmunodetection. Cell lysates (30 μg per lane) were mixed with sample buffer (125 mM Tris/HCl, pH6.8; 4% SDS, 20 mM DTT, 0.05% bromophenol blue, 20% glycerol) and boiled for 2 min. Proteins were separated by SDS-10% polyacrylamide gel according to the method of Schagger and von Jagow ((1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.  Analytical Biochemistry  166(2), 368-79). Electroblotting was performed using a semi-dry transfer system (Hoefer Semiphor unit) onto Immobilon-P membranes (PVDF, Millipore). The transfer was performed in a buffer containing 10 mM CAPS (pH11), 10% (v/v) methanol for 30 min at 2.5 mA/cm 2  of gel surface area. Once the electrotransfer was complete, the blots were stained with 0.5% Ponceau-S red to assess the quality of the transfer and washed with Milli-Q®-grade water. The blots were blocked overnight at 4° C. with 5% nonfat milk powder in TBST (150 mM NaCl, 10 mM Tris/HCl pH8, 0.05% Tween-20) (Harlow &amp; Lane, supra) and then incubated with anti-GumB (1:3000) or anti-GumC (1:5000) antibodies in 3% nonfat milk powder in TBST at room temperature for 3 h. Alkaline phosphatase-conjugated goat anti-mouse IgG or anti-rabbit IgG (Sigma) were used for detection, respectively, as described by the manufacturer. The blots were washed three times with TBST and were developed in a solution containing nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP, Promega). Commercial protein markers MW-SDS-70L (Sigma) were used to calibrate SDS-PAGE.  
         [0077]    Blot quantification. The intensities of GumB and GumC protein bands were determined by scanning the NBT/BCIP developed filters with a UVP Densitometer (Ultra Violet Products) and quantified with GelWorks ID Analysis software (NonLinear Dynamics Ltd). Each filter contained a reference lane of a PRM-1(pBBR-prom) extract to establish the level of chromosomally encoded GumB and GumC in the wild type cells. Relative amounts of GumB and GumC were observed. See FIGS. 2A and 2B.  
       Example 6  
     Procedure—Molecular Length or Weight Determination Using Atomic Force Microscopy  
       [0078]    The direct visualization technique called Atomic force Microscopy (AFM) or Scanning Probe Microscope (SPM) was used to image the lengths of xanthan molecules from  X. campestris  strains with (XWCM1/pBBR5-BC) and without (XWCM1) multiple copies of gumB and gumC. The procedure used to perform AFM is detailed below. We observed that the average molecular contour length of xanthan molecules produced by a strain with multiple copies of gumB and gumC was much longer than that of the parental strain.  
         [0079]    A 0.1 wt % of gum solution was prepared by mixing 0.1 g of gum in 100 gram distilled water for ˜3 hours. A 1-ppm stock solution was prepared by diluting 20 μl of the 0.1 wt % solution into a 20 g 0.1M ammonium acetate solution. 20 μl of the 1 ppm stock solution was sprayed onto freshly cleaved mica disc(s) (˜1 cm 2 ). These mica sample disc(s) were then placed in a heated (˜60° C.) vacuum chamber for ˜one hour to remove excess water. The dried mica disc(s) were then scanned using the Tapping Mode of the AFM. The molecular contour length of all AFM images was measured with the software provided by Digital Instruments.  
         [0080]    Contour lengths of population of xanthan molecules were measured. The results of this study are summarized in Table 4. (Molecules in each size class are less than or equal to the length indicated; the number of molecules indicated in a size class do not include the molecules counted in a smaller size class.) These results demonstrated that xanthan molecules from  X. campestris  strain with multiple copies of gumB and gumC were significantly larger then xanthan molecules from control strain. The atomic force microscopy (AFM) or scanning probe microscopy (SPM) was performed with a commercial instrument (Nanoscope IIIa, Digital Instruments, Santa Barbara, Calif.) using a silicon nitride cantilever tip.  
                                                                                                                                               TABLE 4                           AFM Measurement of Xanthan Molecules Contour Length            XWCM1   XWCM1/pBBR5-BC            Length*   Molecules   Frequency   Distribution   Length   Molecules   Frequency   Distribution            (μm)   (count)   (%)   No. Avg.   Wt. Avg.   (μm)   (count)   (%)   No. Avg.   Wt. Avg.                    0.5   225   51.5   ≦3 μm =   ≦3 μm =   0.5   150   28.4   ≦3 μm =   ≦3 μm =       1   130   29.7   99.8%   98.7%   1   163   30.9   90.9%   70.9%       1.5   40   9.2           1.5   82   15.5       2   25   5.7           2   44   8.3       2.5   13   3.0           2.5   29   5.5       3   3   0.7           3   12   2.3       3.5   0   0.0   &gt;3 μm =   &gt;3 μm =   3.5   12   2.3   &gt;3 μm =   &gt;3 μm =       4   0   0.0   0.2%   1.3%   4   13   2.5   9.1%   29.1%       4.5   0   0.0           4.5   7   1.3       5   1   0.2           5   4   0.8       5.5   0   0.0           5.5   4   0.8       6   0   0.0           6   0   0.0       6.5   0   0.0           6.5   3   0.6       7   0   0.0           7   2   0.4       7.5   0   0.0           7.5   0   0.0       8   0   0.0           8   0   0.0       8.5   0   0.0           8.5   1   0.2       9   0   0.0           9   1   0.2       9.5   0   0.0           9.5   1   0.2       10   0   0.0           10   0   0.0       Total   437               Total   528                  
 
       Example 7  
     Evaluation of Seawater Viscosity  
       [0081]    Xanthan produced by strain XWCM-1/pBBR5-BC was evaluated for seawater vi  
         [0082]    scosity (SWV), compared to a commercial xanthan product (Xanvis™). Typical SWV for Xanvis™ xanthan product is in the range of 18 to 22.  
         [0083]    Seawater viscosity was determined using the following procedure. Seawater solution was prepared by dissolving 41.95 g of sea salt (ASTM D1141-52, from Lake Products Co., Inc. Maryland Heights, Mo.) in 1 liter deionized water. 300 ml of seawater solution was transferred to a mixing cup that was attached to a Hamilton-Beach 936-2 mixer (Hamilton-Beach Div., Washington, D.C.). The mixer speed control was set to low and a single fluted disk attached to the mixing shaft. At the low speed setting, the mixer shaft rotates at approximately 4,000-6,000 rpm. 0.86 g of biogum product was slowly added over 15-30 seconds to the mixing cup and allowed to mix for 5 minutes. The mixer speed control was set to high (11,000±1,000 rpm) and the test solution was allowed to mix for approximately 5 minutes. The mixture was allowed to mix for a total of 45 minutes, starting from time of biogum product addition. At the end of the 45 minutes mixing time, 2-3 drops of Bara Defoam (NL Baroid/NL industries, Inc., Houston, Tex.) was added and stirring was continued for an additional 30 seconds.  
         [0084]    The mixing cup was removed from the mixer and immersed in chilled water to lower the fluid&#39;s temperature to 25±0.5° C. In order to insure a homogeneous solution, the solution was re-mixed after cooling for 5 seconds at 11,000±1,000 rpm. The solution was transferred from the mixing cup to 400 ml Pyrex beaker and Fann viscosity (Fann Viscometer, Model 35A) was measured. This was accomplished by mixing at low speed (about 3 rpm). The reading was allowed to stabilize and then the shear stress value was read from dial and recorded as the SWV value at 3 rpm.  
                             TABLE 5                           Quality of XWCM-1/pBBR5-BC xanthan and Xanvis ™ xanthan                    SWV           Sample   DR a                         XWCM-1/pBBR5-BC   29               30           Xanvis ™ xanthan   22                                  
 
         [0085]    References  
         [0086]    1. Kidby, D., Sandford, P., Herman, A., and Cadmus, M. (1977) Maintenance procedures for the curtailment of genetic instability:  Xanthomonas campestris  NRRL B-1459 . Applied and Environmental Microbiology  33(4), 840-5  
         [0087]    2. Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X. W., Finlay, D. R., Guiney, D., and Helinski, D. R. (1985) Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression.  Plasmid  13(2), 149-53  
         [0088]    3. Simon, R., Priefer U. and Puhler A. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria.  Biotechnology  1, 784-791  
         [0089]    4. Harding, N. E., Cleary, J. M., Cabanas, D. K., Rosen, I. G., and Kang, K. S. (1987) Genetic and physical analyses of a cluster of genes essential for xanthan gumBiosynthesis in  Xanthomonas campestris. J Bacteriol  169(6), 2854-61.  
         [0090]    5. Katzen, F., Becker, A., Zorreguieta, A., Puhler, A., and Ielpi, L. (1996) Promoter analysis of the  Xanthomonas campestris  pv.  campestris  gum operon directing biosynthesis of the xanthan polysaccharide.  J Bacteriol  178(14), 4313-8.  
         [0091]    6. Capage, M. R., D. H. Doherty, M. R. Betlach, and R. W. Vanderslice. (1987) Recombinant-DNA mediated production of xanthan gum. International patent WO87/05938.  
         [0092]    7. Becker, A., Niehaus, K., and Puhler, A. (1995) Low-molecular-weight succinoglycan is predominantly produced by  Rhizobium meliloti  strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain.  Molecular Microbiology  16(2), 191-203  
         [0093]    8. Schafer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G., and Puhler, A. (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of  Corynebacterium glutarnicum. Gene  145(1), 69-73  
         [0094]    9. Yanisch_Perron, C., Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.  Gene  33(1), 103-19  
         [0095]    10. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M., and Peterson, K. M. (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.  Gene  166(1), 175-6  
         [0096]    11. Harding, N. E., Cleary, J. M., Cabanas, D. K., Rosen, I. G., and Kang, K. S. (1987) Genetic and physical analyses of a cluster of genes essential for xanthan gumBiosynthesis in  Xanthomonas campestris. Journal of Bacteriology  169(6), 2854-61  
         [0097]    12. Harding N. E., R. S., Raimondi A., Cleary J. M and Ielpi L. (1993) Identification, genetic and biochemical analysis of genes involved in synthesis sugar nucleotide precursors of xanthan gum.  J. Gen. Microbiol  139, 447-457  
         [0098]    13. Sambrook, J., and Russell, D. W. (2001)  Molecular cloning: a laboratory manual,  3rd Ed., Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press, 2001.  
     
       
       
         1 
         
           
             5  
           
           
             1  
             7604  
             DNA  
             Xanthomomas campestris  
             
               CDS  
               (2715)...(4133)  
               gumC  
             
           
            1 

accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc     60 

aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg    120 

aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca catttccccg    180 

aaaagtgcca cctggcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg    240 

atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact    300 

tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct    360 

tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc    420 

gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata gatatagatc    480 

tcactacgcg gctgctcaaa cctgggcaga acgtaagccg cgagagcgcc aacaaccgct    540 

tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa    600 

tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga    660 

tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg    720 

cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt aacatcgttg    780 

ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc    840 

ttgctgcttg gatgcccgag gcatagactg tacaaaaaaa cagtcataac aagccatgaa    900 

aaccgccact gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga    960 

gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca actgggttcg   1020 

tgccttcatc cgtttccacg gtgtgcgtcc atgggcaaat attatacgca aggcgacaag   1080 

gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc   1140 

agaatgctta atgaattaca acagttttta tgcatgcgcc caatacgcaa accgcctctc   1200 

cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg   1260 

ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta   1320 

cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca   1380 

ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa   1440 

aagctgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttgc atgcctgcag   1500 

gtcgactcta gtggtcgtcg gttcgaatcc ggctaccccg accaaacaac aggcctacgt   1560 

cgcaagacgt gggccttttt gttgcgtcgc aacatgtcag ttcgatggca ttccaggcta   1620 

tgccactatg cgcaacggca tattgcaagg cggcatatgc aagtcctgta cgcaattatt   1680 

tcgcggttca ggctgctaca agtcgggatc agcaggcgtc cgtaagtgcc cggaaacgct   1740 

agagttcgta tgctgagaat gacgacccag gtcacgttct cttaacgtcg aggcgacgaa   1800 

cttgaatcaa taggccaacg ccgtcaaaaa aatggcgtgt tgtgccttgc gatgtgttcg   1860 

ttctatgcca tagtgcactg caacacgcga ttcaacgttg gtcccggcac gcgtcgggat   1920 

gcaacttcct gtcgtacgtt cgtgctggcg cctgagccgg ttgaatgctg cgcgaggtcc   1980 

tgtcccaccc aacagaggca gccagctaca cgc atg aag aaa ctg atc gga cga    2034 
                                     Met Lys Lys Leu Ile Gly Arg 
                                      1               5 

ctc tgc caa ggc ctc agc ctg gct ctg ctc tgc tcg atg tcg ctg ggc     2082 
Leu Cys Gln Gly Leu Ser Leu Ala Leu Leu Cys Ser Met Ser Leu Gly 
         10                  15                  20 

gct tgc agc acc ggc ccg gag atg gcg tct tcg ctg ccg cat ccg gac     2130 
Ala Cys Ser Thr Gly Pro Glu Met Ala Ser Ser Leu Pro His Pro Asp 
     25                  30                  35 

ccg ctg gca atg tcc acg gtg cag ccc gaa tac cgt ctt gcg ccg ggc     2178 
Pro Leu Ala Met Ser Thr Val Gln Pro Glu Tyr Arg Leu Ala Pro Gly 
 40                  45                  50                  55 

gat ctg ttg ctg gtg aag gtg ttt cag atc gac gat ctg gag cgg cag     2226 
Asp Leu Leu Leu Val Lys Val Phe Gln Ile Asp Asp Leu Glu Arg Gln 
                 60                  65                  70 

gtc cgc atc gac cag aac ggt cac atc tca ctg ccg ttg att ggc gac     2274 
Val Arg Ile Asp Gln Asn Gly His Ile Ser Leu Pro Leu Ile Gly Asp 
             75                  80                  85 

gtc aag gcc gcc ggt ctg ggc gtt ggc gaa ctg gaa aag ctg gtc gcc     2322 
Val Lys Ala Ala Gly Leu Gly Val Gly Glu Leu Glu Lys Leu Val Ala 
         90                  95                 100 

gat cgg tat cgc gca ggc tac ctg cag cag ccg cag att tcg gta ttc     2370 
Asp Arg Tyr Arg Ala Gly Tyr Leu Gln Gln Pro Gln Ile Ser Val Phe 
    105                 110                 115 

gtg cag gag tcc aac ggg cgt cgc gtc acg gtc act ggt gcg gta gac     2418 
Val Gln Glu Ser Asn Gly Arg Arg Val Thr Val Thr Gly Ala Val Asp 
120                 125                 130                 135 

gag ccg ggc atc tac ccg gtg atc ggc gcc aac ctc acc ttg cag cag     2466 
Glu Pro Gly Ile Tyr Pro Val Ile Gly Ala Asn Leu Thr Leu Gln Gln 
                140                 145                 150 

gcg atc gcg cag gcc aag ggt gtc agc acg gtg gca agc cgc ggc aac     2514 
Ala Ile Ala Gln Ala Lys Gly Val Ser Thr Val Ala Ser Arg Gly Asn 
            155                 160                 165 

gtg atc gtg ttc cgc atg gtc aac ggg caa aaa atg att gcg cgg ttc     2562 
Val Ile Val Phe Arg Met Val Asn Gly Gln Lys Met Ile Ala Arg Phe 
        170                 175                 180 

gac ctg acc gag atc gag aag ggg gcc aat ccg gat cct gag att tat     2610 
Asp Leu Thr Glu Ile Glu Lys Gly Ala Asn Pro Asp Pro Glu Ile Tyr 
    185                 190                 195 

ggc ggc gac att gtc gtg gtg tat cgc tcg gat gcg cgc gtg tgg ttg     2658 
Gly Gly Asp Ile Val Val Val Tyr Arg Ser Asp Ala Arg Val Trp Leu 
200                 205                 210                 215 

cgc acc atg ctg gaa ctg acc ccc ttg gtg atg gtg tgg cgc gct tac     2706 
Arg Thr Met Leu Glu Leu Thr Pro Leu Val Met Val Trp Arg Ala Tyr 
                220                 225                 230 

cga tga gt atg aat tca gac aat cgt tcc tct tcg tcg cag cgt cat      2753 
Arg  *    Met Asn Ser Asp Asn Arg Ser Ser Ser Ser Gln Arg His 
                  235                 240                 245 

ggt cat ctg gaa ctg gca gat gtc gac ttg atg gac tac tgg cgc gcc     2801 
Gly His Leu Glu Leu Ala Asp Val Asp Leu Met Asp Tyr Trp Arg Ala 
                250                 255                 260 

ctg gtc tcg cag ctc tgg ctg atc atc ctg atc gcc gtc ggc gcg ctg     2849 
Leu Val Ser Gln Leu Trp Leu Ile Ile Leu Ile Ala Val Gly Ala Leu 
            265                 270                 275 

ttg ctg gca ttc ggc atc acg atg ttg atg ccc gag aag tac cgc gcc     2897 
Leu Leu Ala Phe Gly Ile Thr Met Leu Met Pro Glu Lys Tyr Arg Ala 
        280                 285                 290 

acc agc acc ctg cag atc gaa cgt gac tcg ctc aat gtg gtg aac gtc     2945 
Thr Ser Thr Leu Gln Ile Glu Arg Asp Ser Leu Asn Val Val Asn Val 
    295                 300                 305 

gac aac ctg atg ccg gtg gaa tcg ccg cag gat cgc gat ttc tac cag     2993 
Asp Asn Leu Met Pro Val Glu Ser Pro Gln Asp Arg Asp Phe Tyr Gln 
310                 315                 320                 325 

acc cag tac cag ttg ctg cag agc cgt tcg ctg gcg cgt gcg gtg atc     3041 
Thr Gln Tyr Gln Leu Leu Gln Ser Arg Ser Leu Ala Arg Ala Val Ile 
                330                 335                 340 

cgg gaa gcc aag ctc gat cag gag ccg gcg ttc aag gag cag gtg gag     3089 
Arg Glu Ala Lys Leu Asp Gln Glu Pro Ala Phe Lys Glu Gln Val Glu 
            345                 350                 355 

gag gcg ctg gcc aaa gcc gcc gaa aag aat ccc gag gcg ggt aag tcg     3137 
Glu Ala Leu Ala Lys Ala Ala Glu Lys Asn Pro Glu Ala Gly Lys Ser 
        360                 365                 370 

ctc gat tcg cgg cag gcg atc gtc gag cgc agc ctc acc gat acg ttg     3185 
Leu Asp Ser Arg Gln Ala Ile Val Glu Arg Ser Leu Thr Asp Thr Leu 
    375                 380                 385 

ctc gcc ggg ctg gtg gtc gag ccg atc ctc aac tcg cgc ctg gtg tac     3233 
Leu Ala Gly Leu Val Val Glu Pro Ile Leu Asn Ser Arg Leu Val Tyr 
390                 395                 400                 405 

gtc aat tac gat tcg cca gac ccg gtg ctg gcc gcc aag atc gcc aat     3281 
Val Asn Tyr Asp Ser Pro Asp Pro Val Leu Ala Ala Lys Ile Ala Asn 
                410                 415                 420 

acg tac ccg aag gtg ttc atc gtc agc acc cag gaa cgc cgc atg aag     3329 
Thr Tyr Pro Lys Val Phe Ile Val Ser Thr Gln Glu Arg Arg Met Lys 
            425                 430                 435 

gcg tct tcg ttt gcg aca cag ttt ctg gct gag cgc ctg aag cag ttg     3377 
Ala Ser Ser Phe Ala Thr Gln Phe Leu Ala Glu Arg Leu Lys Gln Leu 
        440                 445                 450 

cgc gag aag gtc gaa gac tct gaa aag gat ctg gtc tcg tat tcg acc     3425 
Arg Glu Lys Val Glu Asp Ser Glu Lys Asp Leu Val Ser Tyr Ser Thr 
    455                 460                 465 

gaa gag cag atc gtg tcg gtt ggc gat gac aag ccc tcg ctg cct gcg     3473 
Glu Glu Gln Ile Val Ser Val Gly Asp Asp Lys Pro Ser Leu Pro Ala 
470                 475                 480                 485 

cag aat ctg acc gat ctc aat gcg ttg ctg gca tcc gca cag gac gcc     3521 
Gln Asn Leu Thr Asp Leu Asn Ala Leu Leu Ala Ser Ala Gln Asp Ala 
                490                 495                 500 

cgg atc aag gcc gag tca gct tgg cgg cag gct tcc agt ggc gat ggc     3569 
Arg Ile Lys Ala Glu Ser Ala Trp Arg Gln Ala Ser Ser Gly Asp Gly 
            505                 510                 515 

atg tca ttg ccg cag gtg ttg agc agc ccg ctg att caa agc ctg cgc     3617 
Met Ser Leu Pro Gln Val Leu Ser Ser Pro Leu Ile Gln Ser Leu Arg 
        520                 525                 530 

agc gag cag gtg cgt ctg acc agc gag tac cag cag aaa ctg tcg acc     3665 
Ser Glu Gln Val Arg Leu Thr Ser Glu Tyr Gln Gln Lys Leu Ser Thr 
    535                 540                 545 

ttc aag ccg gat tac ccg gag atg cag cgc ctc aag gcg cag atc gaa     3713 
Phe Lys Pro Asp Tyr Pro Glu Met Gln Arg Leu Lys Ala Gln Ile Glu 
550                 555                 560                 565 

gag tcg cgt cgt cag atc aat ggc gaa gtc atc aat atc cgt cag tcg     3761 
Glu Ser Arg Arg Gln Ile Asn Gly Glu Val Ile Asn Ile Arg Gln Ser 
                570                 575                 580 

ctg aag gcg acc tac gac gcc tcc gtg cat cag gag cag ctg ctc aac     3809 
Leu Lys Ala Thr Tyr Asp Ala Ser Val His Gln Glu Gln Leu Leu Asn 
            585                 590                 595 

gac cgc atc gcc ggt ctg cgg tcc aac gag ctg gat ctg cag agc cgc     3857 
Asp Arg Ile Ala Gly Leu Arg Ser Asn Glu Leu Asp Leu Gln Ser Arg 
        600                 605                 610 

agc atc cgc tac aac atg ctc aag cgc gac gtc gac acc aac cgc cag     3905 
Ser Ile Arg Tyr Asn Met Leu Lys Arg Asp Val Asp Thr Asn Arg Gln 
    615                 620                 625 

ctc tac gat gcg ctc ctg cag cgc tac aag gaa atc ggc gtg gcg agc     3953 
Leu Tyr Asp Ala Leu Leu Gln Arg Tyr Lys Glu Ile Gly Val Ala Ser 
630                 635                 640                 645 

aac gtg ggc gcc aac aac gtg acc atc gtc gat acc gca gac gtg cct     4001 
Asn Val Gly Ala Asn Asn Val Thr Ile Val Asp Thr Ala Asp Val Pro 
                650                 655                 660 

acg tct aag act tcg ccg aaa ctc aaa ttg aac ctc gcg ttg ggc ctg     4049 
Thr Ser Lys Thr Ser Pro Lys Leu Lys Leu Asn Leu Ala Leu Gly Leu 
            665                 670                 675 

atc ttt ggc gta ttc ctg ggc gtg gct gtg gct ctg gtt cgc tac ttc     4097 
Ile Phe Gly Val Phe Leu Gly Val Ala Val Ala Leu Val Arg Tyr Phe 
        680                 685                 690 

ctg cgt ggg cct tct ccg agg tcg cgg ttg aac tga catcgtgatg          4143 
Leu Arg Gly Pro Ser Pro Arg Ser Arg Leu Asn  * 
    695                 700 

ttgcaaaacg atggttaatt gaagtgacaa ctgattcagc gtggaaaagg tgggatcccg   4203 

taaggtgcgg gctccctcgt ttgaaggttt gtctctgttg aaacaaaggg ctgtcgtgcg   4263 

atctggggtc ggtaggtatt accgcggtga tcggacgaca ggatgattga aagctcgcgt   4323 

gcgattcgta tgttcccccg catgcctgca ggtcgactct agagcggccg ccaccgcggt   4383 

ggagctccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac   4443 

aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc   4503 

ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc   4563 

gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa   4623 

tttttgttaa atcagctcat tttttaacca ataggccgac tgcgatgagt ggcagggcgg   4683 

ggcgtaattt ttttaaggca gttattggtg cccttaaacg cctggtgcta cgcctgaata   4743 

agtgataata agcggatgaa tggcagaaat tcgaaagcaa attcgacccg gtcgtcggtt   4803 

cagggcaggg tcgttaaata gccgcttatg tctattgctg gtttaccggt ttattgacta   4863 

ccggaagcag tgtgaccgtg tgcttctcaa atgcctgagg ccagtttgct caggctctcc   4923 

ccgtggaggt aataattgac gatatgatca tttattctgc ctcccagagc ctgataaaaa   4983 

cggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca   5043 

tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgaggc ggctacagcc   5103 

gatagtctgg aacagcgcac ttacgggttg ctgcgcaacc caagtgctac cggcgcggca   5163 

gcgtgacccg tgtcggcggc tccaacggct cgccatcgtc cagaaaacac ggctcatcgg   5223 

gcatcggcag gcgctgctgc ccgcgccgtt cccattcctc cgtttcggtc aaggctggca   5283 

ggtctggttc catgcccgga atgccgggct ggctgggcgg ctcctcgccg gggccggtcg   5343 

gtagttgctg ctcgcccgga tacagggtcg ggatgcggcg caggtcgcca tgccccaaca   5403 

gcgattcgtc ctggtcgtcg tgatcaacca ccacggcggc actgaacacc gacaggcgca   5463 

actggtcgcg gggctggccc cacgccacgc ggtcattgac cacgtaggcc gacacggtgc   5523 

cggggccgtt gagcttcacg acggagatcc agcgctcggc caccaagtcc ttgactgcgt   5583 

attggaccgt ccgcaaagaa cgtccgatga gcttggaaag tgtcttctgg ctgaccacca   5643 

cggcgttctg gtggcccatc tgcgccacga ggtgatgcag cagcattgcc gccgtgggtt   5703 

tcctcgcaat aagcccggcc cacgcctcat gcgctttgcg ttccgtttgc acccagtgac   5763 

cgggcttgtt cttggcttga atgccgattt ctctggactg cgtggccatg cttatctcca   5823 

tgcggtaggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact tttcggcagc   5883 

gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc tttaacctac   5943 

gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtacccccct tttttaagtt   6003 

gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgc ccaaagaagg   6063 

gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggca aaaagtggcc   6123 

cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg gcgctgcccc   6183 

cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg gcttcgcctt   6243 

cgactgcccc cactcgcata ggcttgggtc gttccaggcg cgtcaaggcc aagccgctgc   6303 

gcggtcgctg cgcgagcctt gacccgcctt ccacttggtg tccaaccggc aagcgaagcg   6363 

cgcaggccgc aggccggagg cttttcccca gagaaaatta aaaaaattga tggggcaagg   6423 

ccgcaggccg cgcagttgga gccggtgggt atgtggtcga aggctgggta gccggtgggc   6483 

aatccctgtg gtcaagctcg tgggcaggcg cagcctgtcc atcagcttgt ccagcagggt   6543 

tgtccacggg ccgagcgaag cgagccagcc ggtggccgct cgcggccatc gtccacatat   6603 

ccacgggctg gcaagggagc gcagcgaccg cgcagggcga agcccggaga gcaagcccgt   6663 

agggcgccgc agccgccgta ggcggtcacg actttgcgaa gcaaagtcta gtgagtatac   6723 

tcaagcattg agtggcccgc cggaggcacc gccttgcgct gcccccgtcg agccggttgg   6783 

acaccaaaag ggaggggcag gcatggcggc atacgcgatc atgcgatgca agaagctggc   6843 

gaaaatgggc aacgtggcgg ccagtctcaa gcacgcctac cgcgagcgcg agacgcccaa   6903 

cgctgacgcc agcaggacgc cagagaacga gcactgggcg gccagcagca ccgatgaagc   6963 

gatgggccga ctgcgcgagt tgctgccaga gaagcggcgc aaggacgctg tgttggcggt   7023 

cgagtacgtc atgacggcca gcccggaatg gtggaagtcg gccagccaag aacagcaggc   7083 

ggcgttcttc gagaaggcgc acaagtggct ggcggacaag tacggggcgg atcgcatcgt   7143 

gacggccagc atccaccgtg acgaaaccag cccgcacatg accgcgttcg tggtgccgct   7203 

gacgcaggac ggcaggctgt cggccaagga gttcatcggc aacaaagcgc agatgacccg   7263 

cgaccagacc acgtttgcgg ccgctgtggc cgatctaggg ctgcaacggg gcatcgaggg   7323 

cagcaaggca cgtcacacgc gcattcaggc gttctacgag gccctggagc ggccaccagt   7383 

gggccacgtc accatcagcc cgcaagcggt cgagccacgc gcctatgcac cgcagggatt   7443 

ggccgaaaag ctgggaatct caaagcgcgt tgagacgccg gaagccgtgg ccgaccggct   7503 

gacaaaagcg gttcggcagg ggtatgagcc tgccctacag gccgccgcag gagcgcgtga   7563 

gatgcgcaag aaggccgatc aagcccaaga gacggcccga g                       7604 

 
           
             2  
             232  
             PRT  
             Xanthomomas campestris  
           
            2 

Met Lys Lys Leu Ile Gly Arg Leu Cys Gln Gly Leu Ser Leu Ala Leu 
 1               5                  10                  15 

Leu Cys Ser Met Ser Leu Gly Ala Cys Ser Thr Gly Pro Glu Met Ala 
            20                  25                  30 

Ser Ser Leu Pro His Pro Asp Pro Leu Ala Met Ser Thr Val Gln Pro 
        35                  40                  45 

Glu Tyr Arg Leu Ala Pro Gly Asp Leu Leu Leu Val Lys Val Phe Gln 
    50                  55                  60 

Ile Asp Asp Leu Glu Arg Gln Val Arg Ile Asp Gln Asn Gly His Ile 
65                  70                  75                  80 

Ser Leu Pro Leu Ile Gly Asp Val Lys Ala Ala Gly Leu Gly Val Gly 
                85                  90                  95 

Glu Leu Glu Lys Leu Val Ala Asp Arg Tyr Arg Ala Gly Tyr Leu Gln 
            100                 105                 110 

Gln Pro Gln Ile Ser Val Phe Val Gln Glu Ser Asn Gly Arg Arg Val 
        115                 120                 125 

Thr Val Thr Gly Ala Val Asp Glu Pro Gly Ile Tyr Pro Val Ile Gly 
    130                 135                 140 

Ala Asn Leu Thr Leu Gln Gln Ala Ile Ala Gln Ala Lys Gly Val Ser 
145                 150                 155                 160 

Thr Val Ala Ser Arg Gly Asn Val Ile Val Phe Arg Met Val Asn Gly 
                165                 170                 175 

Gln Lys Met Ile Ala Arg Phe Asp Leu Thr Glu Ile Glu Lys Gly Ala 
            180                 185                 190 

Asn Pro Asp Pro Glu Ile Tyr Gly Gly Asp Ile Val Val Val Tyr Arg 
        195                 200                 205 

Ser Asp Ala Arg Val Trp Leu Arg Thr Met Leu Glu Leu Thr Pro Leu 
    210                 215                 220 

Val Met Val Trp Arg Ala Tyr Arg 
225                 230 

 
           
             3  
             472  
             PRT  
             Xanthomomas campestris  
           
            3 

Met Asn Ser Asp Asn Arg Ser Ser Ser Ser Gln Arg His Gly His Leu 
 1               5                  10                  15 

Glu Leu Ala Asp Val Asp Leu Met Asp Tyr Trp Arg Ala Leu Val Ser 
            20                  25                  30 

Gln Leu Trp Leu Ile Ile Leu Ile Ala Val Gly Ala Leu Leu Leu Ala 
        35                  40                  45 

Phe Gly Ile Thr Met Leu Met Pro Glu Lys Tyr Arg Ala Thr Ser Thr 
    50                  55                  60 

Leu Gln Ile Glu Arg Asp Ser Leu Asn Val Val Asn Val Asp Asn Leu 
65                  70                  75                  80 

Met Pro Val Glu Ser Pro Gln Asp Arg Asp Phe Tyr Gln Thr Gln Tyr 
                85                  90                  95 

Gln Leu Leu Gln Ser Arg Ser Leu Ala Arg Ala Val Ile Arg Glu Ala 
            100                 105                 110 

Lys Leu Asp Gln Glu Pro Ala Phe Lys Glu Gln Val Glu Glu Ala Leu 
        115                 120                 125 

Ala Lys Ala Ala Glu Lys Asn Pro Glu Ala Gly Lys Ser Leu Asp Ser 
    130                 135                 140 

Arg Gln Ala Ile Val Glu Arg Ser Leu Thr Asp Thr Leu Leu Ala Gly 
145                 150                 155                 160 

Leu Val Val Glu Pro Ile Leu Asn Ser Arg Leu Val Tyr Val Asn Tyr 
                165                 170                 175 

Asp Ser Pro Asp Pro Val Leu Ala Ala Lys Ile Ala Asn Thr Tyr Pro 
            180                 185                 190 

Lys Val Phe Ile Val Ser Thr Gln Glu Arg Arg Met Lys Ala Ser Ser 
        195                 200                 205 

Phe Ala Thr Gln Phe Leu Ala Glu Arg Leu Lys Gln Leu Arg Glu Lys 
    210                 215                 220 

Val Glu Asp Ser Glu Lys Asp Leu Val Ser Tyr Ser Thr Glu Glu Gln 
225                 230                 235                 240 

Ile Val Ser Val Gly Asp Asp Lys Pro Ser Leu Pro Ala Gln Asn Leu 
                245                 250                 255 

Thr Asp Leu Asn Ala Leu Leu Ala Ser Ala Gln Asp Ala Arg Ile Lys 
            260                 265                 270 

Ala Glu Ser Ala Trp Arg Gln Ala Ser Ser Gly Asp Gly Met Ser Leu 
        275                 280                 285 

Pro Gln Val Leu Ser Ser Pro Leu Ile Gln Ser Leu Arg Ser Glu Gln 
    290                 295                 300 

Val Arg Leu Thr Ser Glu Tyr Gln Gln Lys Leu Ser Thr Phe Lys Pro 
305                 310                 315                 320 

Asp Tyr Pro Glu Met Gln Arg Leu Lys Ala Gln Ile Glu Glu Ser Arg 
                325                 330                 335 

Arg Gln Ile Asn Gly Glu Val Ile Asn Ile Arg Gln Ser Leu Lys Ala 
            340                 345                 350 

Thr Tyr Asp Ala Ser Val His Gln Glu Gln Leu Leu Asn Asp Arg Ile 
        355                 360                 365 

Ala Gly Leu Arg Ser Asn Glu Leu Asp Leu Gln Ser Arg Ser Ile Arg 
    370                 375                 380 

Tyr Asn Met Leu Lys Arg Asp Val Asp Thr Asn Arg Gln Leu Tyr Asp 
385                 390                 395                 400 

Ala Leu Leu Gln Arg Tyr Lys Glu Ile Gly Val Ala Ser Asn Val Gly 
                405                 410                 415 

Ala Asn Asn Val Thr Ile Val Asp Thr Ala Asp Val Pro Thr Ser Lys 
            420                 425                 430 

Thr Ser Pro Lys Leu Lys Leu Asn Leu Ala Leu Gly Leu Ile Phe Gly 
        435                 440                 445 

Val Phe Leu Gly Val Ala Val Ala Leu Val Arg Tyr Phe Leu Arg Gly 
    450                 455                 460 

Pro Ser Pro Arg Ser Arg Leu Asn 
465                 470 

 
           
             4  
             31  
             DNA  
             Xanthomomas campestris  
           
            4 

ggaattccat atgttgatgc ccgagaagta c                                    31 

 
           
             5  
             33  
             DNA  
             Xanthomomas campestris  
           
            5 

cgggatcctc aaaagatcag gcccaacgcg agg                                  33