CN1357043A - Avirulent xanthomonas-campestris strains producing xanthan - Google Patents

Avirulent xanthomonas-campestris strains producing xanthan Download PDF

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CN1357043A
CN1357043A CN00809327A CN00809327A CN1357043A CN 1357043 A CN1357043 A CN 1357043A CN 00809327 A CN00809327 A CN 00809327A CN 00809327 A CN00809327 A CN 00809327A CN 1357043 A CN1357043 A CN 1357043A
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bacterial strain
xanthomonas
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J·皮尔拉德
J－L·西蒙
P·切瓦勒里奥
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Rhodia Chimie SAS
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    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract

The invention concerns a bacterial strain which has lost its phytopathogenic character by inactivation of at least one virulence gene and preserved its capacity for producing exopolysaccharide.

Description

Produce the Avirulent xanthomonas-campestris strains of xanthan gum
The present invention relates to lose and cause phytopathy character but kept generation exo polysaccharides (exopolysaccharide) basically, especially the novel bacterial strains of xanthan gum ability, particularly bacterial strain, the especially xanthomonas campestris (Xanthomonascampestris) of xanthomonas (Xanthomonas).
The mutation (X.campestris pv.campestris) of causing a disease of xanthomonas campestris bird rape is the phytopathogenic gram negative bacterium of cress, it is used for the industrial production (Martin of xanthan gum, 1994, microbiological research (Res.Microbiol.) 145:9 93-97).
This exo polysaccharides importance economically caused many about the research that participates in this synthetic gene (Martin, 1994, as mentioned above).
Pathogenic many determinatives are described (Dow and Daniels, 1994, the bacterium pathogenesis in the Plants and Animals (In bacterial pathogenesis of plants andanimals), JL Dangl compiles, Springer Verlag, Heidelberg).Wherein there is pair plant tissue to have the extracellular enzyme of hydrolytic activity.When being responsible for the excretory system inactivation of these enzymes outputs, the bacterial strain of xanthomonas campestris has just lost its phytopathogenic phenotype, and this is relevant with the symptom that weakens very much in the plant (Dow and Daniels, 1994, as mentioned above).In the described pathogenic determinative, exo polysaccharides as if the effect that demonstrates in early days of disease (Dow and Daniels, 1994, as mentioned above; People such as Katzen, 1998, bacteriology magazine (J.Bacteriol.) 180:1607-1617).Similarly, the xanthomonas campestris bird rape hrpXc gene described in the mutation (people such as Kamoun of causing a disease, molecule phytomicroorganism interaction (Mol.Plant MicrobeInteract) 5:22-33) participation suppresses the defensive raction of compatible host plant, because the sudden change of this gene can cause distinctive necrotic reaction (anaphylaxis, HR).The avirulence gene that multiple xanthomonas campestris causes a disease described in the mutation also participates in the pathogenic of bacterium because contain corresponding to the plant with the resistant gene that causes the HR reaction can discern they (Dow and Daniels, 1994, as mentioned above; People such as Yang, 1995, molecule phytomicroorganism interaction 8:627-631).(Dow and Daniels in participating in other pathogenic gene of xanthomonas, 1994, as mentioned above), two genes in the pathogenic mutation of xanthomonas campestris bird rape have been described, their sudden change will cause its pathogenic weakening, and do not change the accumulation level (people such as Osbourn, 1990, molecule phytomicroorganism interaction 3:280-285) of extracellular enzyme and exo polysaccharides.Other pathogenic determinative comprises many group adjusting extracellular enzymes and exo polysaccharides synthetic separate gene, and wherein have: rpfA to H gene, their sudden change cause the reduction that exo polysaccharides produces; The rpfN gene is these enzymes and exo polysaccharides synthetic repressor gene (repressor); Clp gene, its sudden change cause pathogenic decline and exo polysaccharides product reduction (Dow and Daniels, 1994, as mentioned above).At last, other pathogenic determinative comprises the hrp gene.
Pathogenic and with the resistance host relevant anaphylaxis relevant with compatible plant of hrp (anaphylaxis is with pathogenic) gene pairs is vital (Alfano and Collmer, 1997, bacteriology magazine 179:5655-5662).In the multiple phytopathogenic bacterium of erwinia, Rhodopseudomonas, Ralstonia genus and xanthomonas, these genes have been cloned, and describing its characteristic in varying degrees, they are conservative relatively (Zurek and Bukowski, 1998, Acta Microbiologica Polonica, 47:227-241; Alfano and Collmer, as mentioned above), especially (people such as Huguet, 1998, molecular microbiology (Molec.Microbiol) 29:1379-1390 in xanthomonas campestris capsicum spot disease is caused a disease mutation (X.campestrispv vesicatoria); People such as Fenselau, 1992, the molecule phytomicroorganism interacts, 5:390-396; Bonas, 1994, as mentioned above).And will be wherein the most conservative be renamed as the hrc gene (people such as Bogdanove, 1996, molecular microbiology, 20:681-683).Synthetic (the Zurek et Bukowski that their expression regulation, the composition that causes the proteinic generation of host response, special excretory system (" III type ") and pericentral siphon dextran are arranged in the function of described up to now hrp gene, 1998, Acta MicrobiologicaPolonica, 47:227-241; Mudgett et Staskawicz, 1998, microbiological current viewpoint (Current Opinion in Microbiology) 1:109-114; Lindgren, 1997, comment (Annu.Rev.Phytopathol.) 35:129-152 pathogenic academic year; Alfano and Collmer, 1997, as mentioned above; Bonas, 1994, as mentioned above).Cloned one group of xanthomonas campestris bird rape cause a disease hrp gene in the mutation (people such as Arlat, 1991, the molecule phytomicroorganism interacts, 4:593-601), but not order-checking.Also be reported that outward appearance, think to cause that by transposon bacterial strain that these genes are undergone mutation has the generation of normal exo polysaccharides according to bacterium colony on the flat board.But do not announce in these bacterial strains that more the xanthan gum of accurate quantification produces ability.
In addition, the sudden change that produces in these bacterial strains is very unstable in nature, is not suitable for the industrial use of production xanthan gum.Particularly, employed transposon contains the gene (people such as Simon of the transposase of encoding, 1989, gene (Gene) 80:161-169), do not get rid of the excision incident estimated approximately with the frequency generation transposon of per generation 10-6 to 10-3 (people such as Berg, 1989, Berg and Howe write, removable DNA (Mobible DNA), AAM, Washington, the 879-926 page or leaf; Craig, intestinal bacteria and Salmonellas (Escherichia coli andSalmonella), Neidhardt writes, ASM press, Washington, 2339-2362 page or leaf).In addition, employed transposon contains the resistant gene of antibiotic neomycin and kantlex.At last, the transposon that is inserted in these strain gene groups constitutes the nonhomologous dna element, because it is not the element of this strain gene group self.
Though, do not have in Europe at present to apply special laws and regulations on the management because of the phytopathy character that causes of the pathogenic mutation of xanthomonas campestris bird rape, but because relevant with environment, the risk of agronomy purpose cultivation is very desirable near using non-phytopathogenic xanthomonas campestris to reduce may to pollute.The such bacterial strain that uses conventional random mutagenesis technology to select to be used to produce is a tedious process, non-ly causes phytopathy but keeps the high flux screening of the bacterial strain of throughput characteristic (promptly not having secondary mutation) because it must comprise separating.
In addition, use the xanthan gum that produces transformation (as US 5,514,791 is described) or (Theilleux 1998 through strict regulation to have improved the genetic modification bacterial strain of productivity, the permanent regulations of Dictionnaire permanent bio é thique et biotechnologies[Bioethics and biotechnology], ed L é gislatives[legislature compiles], the 1595-1648 page or leaf).Latter's requirement especially has the structure of harm to what produce in the bacterial strain to plant, must adopt the strict diffusion measure that prevents in the production area.Therefore, necessary cost will have negative economic consequences.
Therefore, require to be used for industrial xanthomonas campestris bacterial strain and stably lack and cause phytopathy character, but keep the throughput characteristic of its xanthan gum.In addition, owing to rules and in order to simplify by separating the refuse treatment that xanthan gum is brought in the biomass, it is useful using the bacterial strain of the heterologous gene that does not contain the antibiotics resistance of encoding.At last, according to France and Europe legislation, preferably using by making up the bacterial strain that obtains from body clone (autocloning), promptly is that it does not contain self heredity any DNA element in addition.
The inventor's research can make up the xanthomonas campestris bacterial strain with the characteristic that requires.
Surprisingly, the present invention shows, has influence on sizable fragment of several thousand bases of virulence related gene by disappearance, obtain stably non-phytopathogenic bacterium, but it still can the production xanthan gum.
More surprisingly, the xanthan gum that bacterial strain that the present invention transformed produces can both be compared with the xanthan gum that wild type strain produced that produces this structure aspect all of quality and quantity.
A theme of the present invention is to lose the bacterial isolates that causes phytopathy character and keep producing the exo polysaccharides ability by at least one virulence gene of inactivation.
According to the present invention, by at least one gene of disappearance hrp or hrc gene group, at least two genes advantageously, preferred at least three genes, 5 to 9 genes of preferred hrp or hrc gene group can advantageously obtain stably non-phytopathogenic bacterial isolates.
Wording " stably lack cause phytopathy character " is meant that through at least 20 generations, advantageously at least 30 generations, the cell cycle in preferred at least 40 generations is still keeping this characteristic later.
Cause phytopathy character and can being advantageously used in the industrial bacterium of exo polysaccharides having lost, especially will mention with the subordinate: erwinia (Erwinia), Rhodopseudomonas (Pseudomonas), Ralstonia and xanthomonas.
A theme of the present invention stably lacks the xanthomonas bacterial strain that causes phytopathy character and basic maintenance generation exo polysaccharides ability especially in essence.
Wording " non-in essence phytopathogenic " is meant at least after inoculating 15 days by the middle arteries and veins of infringement blade, the host cress, does not especially spread damage and/or withered on the leaf of wild cabbage (Brassica oleracera).
Advantageously, the xanthomonas bacterial strain is an xanthomonas campestris, the especially bird rape mutation of causing a disease.
The inactivation of described gene is preferably by 1kb at least in disappearance hrp or the hrc gene group, preferred 3kb at least, and 5kb at least advantageously, the 9kb in preferred hrp or the hrc gene group, and be 40kb and obtain.
In a preferred embodiment, hrpA1 to hrpC2 gene by the pathogenic phytopathogenic wild type strain of mutation of disappearance xanthomonas campestris bird rape obtains according to non-in essence phytopathogenic xanthomonas bacterial strain, especially xanthomonas campestris bacterial strain of the present invention.
What xanthan gum that xanthomonas bacterial strain of the present invention is produced and wild-type produced is essentially identical, and in other words, it has essentially identical molecular weight distribution, also has the degree of modification, the especially acetylize and the propionylization (pyruvylation) of same degree.
Theme of the present invention also has a kind of above method that defines bacterial strain for preparing, and it is characterized in that being being undertaken by the plasmid with all or part of disappearance that contains hrp or hrc gene that homologous recombination obtains.
Theme of the present invention also has a kind of preparation bacterium exo polysaccharides, especially the method for xanthan gum, it is characterized in that bacterial isolates defined above, suitably, the xanthomonas bacterial strain, preferred xanthomonas campestris bacterial strain is cultivated under the condition that allows generation exo polysaccharides in fermention medium.
The following examples will illustrate the structure corresponding to the xanthomonas campestris bacterial strain of feature of the present invention.
In these embodiments, use makes up by the pathogenic mutation bacterial strain of the screening xanthomonas campestris bird rape that xanthan gum obtained.
Much less, according to general knowledge in the described technical field and following given explanation, especially the sequence of reference section branch report when bacterial strain belongs to xanthomonas campestris, xanthomonas and do not belong to together in produce exo polysaccharides, obtainable other bacterial isolates of those skilled in the art also can be used as and produces the non-starting materials that sets out that causes the phytopathy bacterial strain
For understanding embodiment, will be with reference to the accompanying drawings, wherein:
-Fig. 1 diagramming makes up the strategy of the xanthomonas campestris RPA-BIOCAT826 bacterial strain derivative that carries hrp genetically deficient.
Fenselau and Bonas (1995, the molecule phytomicroorganism interacts, 8 (6): 845-854) and people such as Fenselau, (1992, the molecule phytomicroorganism interacts, 5:390-396) having described the cause a disease composition of hrp gene in the mutation of xanthomonas campestris capsicum spot disease, is partly to obtain in Genebank, and its login is numbered U33548.Title according to their plasmid of clone is described the homologous region of cloning from the RPA-BIOCAT826 bacterial strain.People such as Arlat (the molecule phytomicroorganism interacts, and 1991,4:593-601) announce the restriction map in hrp district in the pathogenic mutation of xanthomonas campestris bird rape, and in embodiment 1 to 4, provided the result who is finished.Through dual homologous recombination the Δ hrpA1-C2 disappearance that the pRPA-BCAT140 plasmid described in the embodiment carries is incorporated in the genome.
-Fig. 2 represents to use two genomic dnas of having integrated the derivative of Δ hrpA1-C2 disappearance of following described HRPB5 probe and RPA-BIOCAT826 bacterial strain and this bacterial strain, by the resulting hybridization signal of southern blotting technique method.The position of molecular weight size mark band is by the miles of relative movement comparison on the gel of ethidium bromide staining obtains with shifting in the past.These sizes are represented with kilobase (kb).
-Fig. 3 represents to use five genomic dnas of having integrated the derivative of Δ hrpA1-C2 disappearance of following described HRPC2 probe and RPA-BIOCAT826 bacterial strain and this bacterial strain, by the resulting hybridization signal of southern blotting technique method.The position of molecular weight size mark band is to obtain by the miles of relative movement comparison on the gel of ethidium bromide staining before shifting.These sizes are represented with kb.
Material and method
Unless otherwise indicated, employed technology all is conventional molecular biology known in those skilled in the art and microbiological technology, people's (molecular biology present age method (Current Protocol in Molecular Biology) in 1987 such as Ausubel for example, John Wiley andSons, New York; People such as Maniatis, 1982, molecular cloning: laboratory manual (MolecularCloning:a laboratory manual), cold spring harbor laboratory, New York) and people (protein science present age method (Current Protocol in Protein Science) John Wiley ﹠amp in 1997 such as Coligan; Sons company) described.
1. original strain
(Melle factory RTAM), is selected according to its white form outward appearance rather than common yellow appearance the RPA-BIOCAT826 bacterial strain from the preservation center of Rhodia Chimie.RPA-BIOCAT1016,1017,1019 and 1021 bacterial strains are deposited in CBS, separately be numbered CBS101940, CBS 101941, CBS 101942, CBS 101943 and CBS 101944.
2.MSX substratum
The MSX substratum of cultivating xanthomonas contains: the 0.2g/l yeast extract; 1.2g/lNH 4NO 37.3g/l K 2HPO 40.25g/l MgSO 4.7H 2O, 1g/l glucose and 15g/l bacterium are used for nutrient agar with agar (Bacto-Agar); 10g/l glucose is used for liquid nutrient medium.With sal epsom with glucose is sterilized respectively and add temporarily.Before the sterilization, equilibrate to pH7.2 with being diluted to 10% sulfuric acid pH with substratum.
Genomic dna is (OD660 is less than 0.4) by liquid culture preparation fresh among the MSX.With the 40ml culture centrifugal after, with 11.9ml TE damping fluid suspension cell precipitation (molecular biology present age method, John Wiley and Sons, New York), add 630 μ l 10%SDS (sodium lauryl sulphate), and then add the Proteinase K of 63 μ l 20mg/ml.37 ℃ hatch 1 hour after, add the NaCl solution of 2.1ml 5M, add 1.7ml then and be dissolved in 10%CTAB in the 0.7M NaCl solution, whole mixture was hatched 10 minutes at 65 ℃.After the extracting for the first time of isopyknic chloroform/primary isoamyl alcohol (24: 1) mixture, use isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1) mixture to carry out the extracting second time again, will add the Virahol of 0.6 volume in the supernatant.After centrifugal (10000 rev/mins 5 minutes), the precipitation that obtains with 70% washing with alcohol after drying, is dissolved among the TE of 2ml at least again, and then adds the Ribonuclease in Aqueous Solution of 25 μ l 5mg/ml.37 ℃ hatch 1 hour after, carry out extracting with phenol/chloroform/primary isoamyl alcohol, by 3M sodium-acetate and 2.5 volume of ethanol that add 0.1 volume the DNA in the supernatant is precipitated out.14 000 rev/mins of centrifugal precipitations that obtained in 5 minutes with 70% washing with alcohol, after the drying, are suspended among the TE of 0.5ml at least again.
Embodiment 1
The clone in the hrpC2 district of RPA-BIOCAT826
Use primer XcC2.3 (SEQ ID No.1) and XcC2.4 (SEQ ID No.2) initial by pcr amplification purpose fragment from the genomic dna of RPA-BIOCAT826.The genomic dna of extracting RPA-BIOCAT826 bacterial strain, and in the PCR reaction, use, wherein contain the 100ng genomic dna, each primer of 40pmol, 0.2mM dNTP and 1.25U Pwo polysaccharase (BoehringerMannheim), final volume are the damping fluid of 50 μ l this kind of enzyme.95 ℃ hatch 5 minutes after, mixture is at first through 30 circulations, each circulation comprises that 94 ℃ hatched 1 minute, then following 1 minute of temperature 63 ℃-48 ℃ (0.5 ℃ of each circulation change), and 72 ℃ 1 minute.Ensuing 15 circulations comprise that 94 ℃ hatched 1 minute, 48 ℃ of 1 minute and 72 ℃ 1 minute.Be at last 72 ℃ 10 minutes.Size is carried out purifying near the amplified production of 1.2kb by the Qiaex test kit (Quiagen) that moves and next use on sepharose.Then it is cloned in the pZERO-1 carrier of opening with EcoRV (Invitrogen BV).Behind transformed into escherichia coli (E.coli) the bacterial strain JM110, select to contain the clone who has integrated the segmental plasmid of 1.2kb.With this plasmid called after pRPA-BCAT91 and with the insertion fragment that it contains check order (GenomeExpress, Grenoble, France).With resulting sequence (SEQ ID No.3) and xanthomonas campestris capsicum spot disease cause a disease mutation the hrpC2 gene order (people such as Fenselau, 1992, the molecule phytomicroorganism interacts, and 5:390-396) compares.With the sequence of the 1188bp that represents the 61%hrpC2 gene 87% consistence is arranged.The aminoacid sequence of being inferred by nucleotide sequence demonstrates 92% consistent per-cent with the corresponding section of the HrpC2 protein sequence of the pathogenic mutation of xanthomonas campestris capsicum spot disease.
Embodiment 2
The clone in RPA-BIOCAT826 HrpA district
Use is cloned this district corresponding to the probe of the pathogenic mutation bacterial strain equivalent regions of xanthomonas campestris capsicum spot disease by the portion gene group library of screening RPA-BIOCAT826 bacterial strain.In the plasmid of pL3o by name, can obtain this zone, this plasmid contains and comprises that the cause a disease EcoRV of the hrpB8 of mutation and hrpA1 gene 6.6kb of xanthomonas campestris capsicum spot disease inserts fragment (people such as Fenselau, 1992, the molecule phytomicroorganism interacts, 5:390-396).
Use primer XcvA15 (SEQ ID No.4) and each 40pmol of XcvA18 (SEQ ID No.5), pL3o plasmid template (40ng), 0.2mM dNTP and 1.25U Pwo polysaccharase (BoehringerMannheim), final volume is the damping fluid of this kind of enzyme of 50 μ l, prepares the HRPA1 probe by PCR.95 ℃ hatch 5 minutes after, mixture is through 30 circulations, each circulation comprises that 94 ℃ hatched for 30 seconds, then 55 ℃ following 1 minute, and 72 ℃ 1.5 minutes.Last 72 ℃ hatch 10 minutes after, earlier on sepharose, and then use Qiaex test kit (Quiagen) to carry out purifying the 664bp product of amplification.
With the EcoRI of 100 units with the BIOCAT826 strain gene group DNA of about 10 μ g 37 ℃ of digestion 16 hours.Use conventional southern blotting technique technology then, purpose is the segmental size of measuring with above-mentioned HRPA1 probe hybridization of EcoRI.With above-mentioned EcoRI digest after moving on the sepharose, transfer on the Hybond N+ film (Amersham), the HRPA1 probe of phosphorus 32 marks that use obtains with Ready-To-Go test kit (Pharmacia Biotech) according to the explanation of manufacturers is at water-based hybridization solution (0.55%SDS; 6%SSC; 0.25% skim-milk) in, hybridized 19 hours for 55 ℃, and with 0.2SSC and 0.1%SDS solution 55 ℃ of washings down, with film-80 ℃ of following radioautograph 19 hours.The development of film shows that the size of hybridization signal is near 7.3kb.
Therefore, with the EcoRI of 1000 units with the RPA BIOCAT826 strain gene group DNA of 100 μ g 37 ℃ of digestion after 20 hours, the portion gene group library that produces this bacterial strain.After moving on the sepharose, size is downcut to 8kb fragment The corresponding area at 7kb, in dialysis tubing (available from the Spectra/Por film of Spectrum Medical Industries company), DNA is extracted from glue by electroelution.Behind the ethanol sedimentation, with DNA with cut through the EcoRI enzyme after be connected in final volume 10 μ l with shrimp alkaline phosphotase (U.S.'s biological chemistry (United States Biochemicals)) dephosphorylized pBlueScript II SK carrier (Stratagene).To connect compound 16 ℃ hatch 14 hours after, the mixture with 1/10 is by electroporation transformed into escherichia coli DH5alpha cell.Use the HRPA1 probe and transfer to bacterium colony on the nylon membrane and hybridize and analyze about 3 000 transformant.12 bacterium colonies that produce positive hybridization signal are carried out purifying on the LB substratum that contains 100 μ g/ml penbritins.The plasmid of 12 purifying bacterium colonies of extracting uses the HRPA1 probe, analyzes the EcoRI digest of these plasmids by the southern blotting technique method, and purpose is the segmental existence of confirming with this probe hybridization of about 7.3kb.After using plurality of enzymes to carry out restriction endonuclease analysis, on the SacII fragment subclone of the SacII fragment of 2.7kb and the 1.6kb pBlueScript II SK carrier after cut, produce pRPA-BCAT135 and pRPA-BCAT134 carrier respectively with the SacII enzyme.These two carriers are carried out part order-checking (GenomeEopress, Grenoble), the result shows the open reading-frame (ORF) (SEQ ID No.6) that has 1818bp, the HrpA1 albumen of the peptide sequence of inferring and the pathogenic mutation of xanthomonas campestris capsicum spot disease (people such as Fenselau, 1992, the molecule phytomicroorganism interacts, and 5:390-396) has 85% consistence.
Embodiment 3
From RPA BIOCAT826, contain the structure of the bacterial strain of Δ hrpA1-C2 disappearance
By being cloned into, the fragment (with reference to Fig. 1) of the fragment of pRPA-BCAT134 and pRPA-BCAT91 comes external structure Δ hrpA1-C2 disappearance among the plasmid pJQ200SK (Quandt and Hynes, 1993, gene (Gene) 127:15-21).Open the pRPA-BCAT91 plasmid with NcoI, use polymerase I (Klenow fragment) then in the presence of 25 μ M dNTP, handled 15 minutes for 30 ℃.After carrying out extracting with phenol/chloroform/primary isoamyl alcohol, use ethanol sedimentation again.Sample dissolution in 40 μ l water, is handled sample for 37 ℃ with 20 XbaI of unit, next use 50 ℃ of processing of 20 ApoI of unit again.Isolate the fragment of about 1.2kb then with glue, and reclaim with Quiex II test kit (Quiagen).The RsaI-SacII fragment of pBCAT134 of the about 1.3kb of purifying uses the same method.These two fragments are connected in the pBlueScript II SK carrier of opening with SacII and XbaI, produce the pRPA-BCAT139 plasmid.Then, the SacI-XbaI fragment of about 2.5kb that can extracting from this plasmid goes out to carry Δ hrpA1-C2 disappearance is so that be cloned among the plasmid pJQ200KS that opens with SacI and XbaI enzyme.The plasmid called after pRPA-BCAT140 that obtains.This plasmid is reproducible not in xanthomonas campestris, the gentamicin resistance marker that it carries can be used for selecting having integrated by homologous recombination the xanthomonas campestris clone of this plasmid, and its entrained sacB forward selective marker can be used for selecting taking place the clone that removed the gentamicin resistance marker after the homologous recombination incident for the second time.
By engaging the pRPA-BCAT140 plasmid is incorporated in the RPA BIOCAT826 bacterial strain.For this reason, the culture 40 μ l of DH5alpha bacterial strain exponential phase of growth of pRPA-BCAT140 will be contained in the MSX substratum, the culture 40 μ l (people such as Ditta of containing HB101 bacterial strain exponential phase of growth of pRK2013 plasmid, 1980, Proc.Natl.Acad.Sci, USA 77:7347-7351) and the RPA-BIOCAT826 strain culture 40 μ l of exponential phase of growth, on the MSK nutrient agar, mix.30 ℃ hatch 24 hours after, the xanthomonas campestris of having integrated the pRPA-BCAT140 plasmid is cloned on the MSX nutrient agar that contains 15 μ g/ml gentamicins continuous purification twice.About 1 square centimeter 8 clones of MSX nutrient agar surface plating that contain 5% sucrose.30 ℃ hatch 72 hours after, separate bacterium colony by twice successive purifying on the MSX nutrient agar.Then, be the clone (according to the clone of this experiment 90 to 100%) differentiate 45 gentamicin sensitivities, with the cultivation of on the MSX nutrient agar that contains 15 μ g/ml gentamicins, going down to posterity of about 300 bacterium colonies.Then with its genome of EcoRI-BamHI digestion and HRPA1 probe carry out the southern blotting technique method analyze about 40 like this.Nearly 25% clone show different with wild-type RPA-BIOCAT826 bacterial strain and with the signal of the integration unanimity of Δ hrpA1-C2 disappearance.Selected 5 clones to be used for the experiment of back: RPA BIOCAT bacterial strain 1016,1017,1019,1021 and 1022.
Embodiment 4
Identify from RPA-BIOCAT826 and the southern blotting technique method that contains the bacterial strain of Δ hrpA1-C2 disappearance
The genomic dna by analyzing EcoRI, BamHI and EcoRI-BamHI digestion and the hybridization collection of illustrative plates of HRP3 ' A1, HRPB5 and HRPC2 probe come Analysis and Identification RPA BIOCAT bacterial strain 1016,1017,1019,1021 and 1022.
HRP3 ' A1 probe is by the 1.6kb SacII fragment of pRPA-BCAT134 plasmid is moved on sepharose, and then uses the Qiaex test kit to carry out purifying to obtain.
The HRPC2 probe is by the 1.2kb EcoRI-XbaI fragment of pRPA-BCAT91 plasmid is moved on sepharose, and then uses the Qiaex test kit to carry out purifying to obtain.
The HRPB5 probe is by the 1.5kb BamHI fragment of pRPA-BCAT129 plasmid is moved on sepharose, and then uses the Qiaex test kit to carry out purifying to obtain.Especially the segmental order-checking of this insertion shows has an open reading-frame (ORF) (SEQ ID No.7), the HrpB5 albumen of the peptide sequence of inferring and the pathogenic mutation of xanthomonas campestris capsicum spot disease (people such as Fenselau, 1995, the molecule phytomicroorganism interacts, and 8:845-854) has 77% consistence.By size is cloned in the pBlueScript II SK carrier at the BamHI genomic DNA fragment of 1.3 to 1.9kb RPA BIOCAT826 bacterial strain, and obtain the pRPA-BCAT129 plasmid with HRPB probe screening bacterium colony according to the similarity method described in the embodiment 2.Use primer RST2 and RST3 (people such as Leite, 1994, applied environment microbiology (Appl.Environ.Microbiol.) 60:1068-1077) to obtain the HRPB probe by PCR with plasmid template pB10g (U.Bonas, individual's contact).Plasmid pB10g quite contains the xanthomonas campestris capsicum spot disease hrpB zone of mutation and the segmental pBlueScript KS of the 7.3kb BamHI plasmid of the hrpA1 gene (people such as Fenselau of causing a disease with wherein having cloned, 1995, the molecule phytomicroorganism interacts, 8:845-854).Use each primer of 40pmol, 50ng pB10g, 0.2mM dNTP and 1.25U Pwo polysaccharase (Boehringer Mannheim) carry out the PCR reaction in final volume is the damping fluid of 50 μ l this kind of enzyme.95 ℃ hatch 5 minutes after, mixture is at first through 24 circulations, each circulation comprises that 95 ℃ hatched for 30 seconds, then in 70 ℃-63 ℃ (0.3 ℃ of each circulation change) following 40 seconds of temperature, and 72 ℃ 1 minute, ensuing 6 circulations comprise that 95 ℃ hatched for 30 seconds, 63 ℃ of 40 second and 72 ℃ 1 minute, be at last 72 ℃ 5 minutes.The amplified production of about 840bp is carried out purifying with use Qiaex test kit (Quiagen) on sepharose.
According to the explanation that provides, use " Megaprime dna marker system " test kit (Amersham) label probe to carry out the analysis of southern blotting technique method.According to the explanation that is provided, the digest of genomic dna after moving on the sepharose, is transferred on the Hybond N+ film (Amersham), by 0.5M phosphate buffered saline buffer and 7%SDS (115ml 1M Na 2HPO 4, 84.6ml[is left out] and MNaH 2PO 4, 200ml water and 28 gram SDS) hatch in the hybridization solution formed.The probe of mark was hatched 5 minutes at 100 ℃, at room temperature hatched again 5 minutes, be diluted in then in the 12ml hybridization solution, hatched 5 minutes for 100 ℃.Next mixture is contacted 6 to 20 hours with film at 65 ℃, film is being contained 0.1M phosphate buffered saline buffer (the 42.3ml 1MNa of 1%SDS 2HPO 4, 57.7ml1M NaH 2PO 4, 900ml water and 10 gram SDS) and middle the washing after 10 to 15 minutes, expose.
Show that with the resulting result of HRPB5 probe (Fig. 2) for the RPA-BIOCAT826 bacterial strain, the hybridization signal that uses EcoRI digestion is at about 4.8kb, the signal that digests with BamHI digestion and EcoRI-BamHI is at 1.6kb.People such as these results and Arlat (the molecule phytomicroorganism interacts, and 1991,4:593-601) location of resulting collection of illustrative plates and hrpB5 gene described above is consistent.Do not demonstrate the hybridization signal with HRPB5 in the RPA-BIOCAT bacterial strain of being studied, this with the genome of these RPA BIOCAT bacterial strains 1016,1017,1019,1021 and 1022 in to have integrated Δ hrpA1-C2 disappearance be consistent (Fig. 2 has only shown the resulting results of hybridization of RPA-BIOCAT bacterial strain).
Show that with the resulting result of HRPC2 probe (Fig. 3) for the RPA-BIOCAT826 bacterial strain, the hybridization signal that uses EcoRI digestion is at about 5.5kb, with the signal of EcoRI-BamHI digestion at 2.6kb.(the molecule phytomicroorganism interacts people such as these results and Arlat, 1991,4:593-601) resulting collection of illustrative plates, the cause a disease group structure (people such as Fenselau of hrp gene of mutation of xanthomonas campestris capsicum spot disease, 1992, the molecule phytomicroorganism interacts, and 5:390-396) location with hrpB5 gene described above is consistent.By RPA BIOCAT bacterial strain 1016,1017,1019 and 1021 resulting results, the hybridization signal that uses EcoRI digestion between 7 to 8kb, with the signal of EcoRI-BamHI digestion at 4.4kb.Collection of illustrative plates as shown in Figure 1, it is consistent having integrated Δ hrpA1-C2 disappearance in the genome of these results and RPA BIOCAT bacterial strain 1016,1017,1019,1021 and 1022.
At last, use the resulting result of HRP3 ' A1 probe to show, for the RPA-BIOCAT826 bacterial strain, the hybridization signal that uses EcoRI-BamHI digestion is at 7.3kb.Use RPA-BIOCAT bacterial strain 1016,1017,1019 and 1021, this hybridization signal is at 4.4kb.This with these strain gene groups in to have integrated Δ hrpA1-C2 disappearance be consistent.
Embodiment 5:
From RPA-BIOCAT826 and contain the virulence of the bacterial strain of Δ hrpA1-C2 disappearance
On brassica oleracea plants (Brassica oleracera var.captiva eultivar Siria), carry out virulence test, obtain its seed from Clause Semences (av.Lucien Clause, 91221Br é tigny-sur-Orge, France).According to following constant in the controlled environment chamber in this kind of plant of plantation: 25 ℃ of 14 hours, 55% temperature, saturated light intensity (4000W/m); 25 ℃ 10 hours, 60% humidity.Infect for example after planting about 13 days in two leaf stages.Each bacterial strain is all used 8 strain plants, and the toothpick that infects in the middle arteries and veins terminal portions use of first leaf punctures.Be immersed in 2 days cultures (about 10 of the bacterial strain of being studied in the MSK substratum by top with toothpick 8Make it to obtain in the bacterium/ml) to pollute.Negative control comprises makes pepper that xanthomonas campestris capsicum spot disease Plant diseases, that be separated at Clause Semences cause a disease reference strain mixture (B229RI bacterial strain=RPA-BIOCAT381, the B230RII bacterial strain=RPA-BIOCAT382) of mutation take place.Positive control comprises makes wild cabbage generation Plant diseases, the xanthomonas campestris bird rape that is separated at ClauseSemences cause a disease reference strain mixture (2963 bacterial strains=RPA-BIOCAT379, the 63C2AM bacterial strain=RPA-BIOCAT380) of mutation.Observe the symptoms (the yellow damage of V-arrangement), and after infecting 12 and 14 days, measure.Below the correspondence: 0, there is not symptom; 1, have near the infection point and fade; 2, downright bad less than 0.5 square centimeter; 3, downright bad at 0.5 to 1.5 square centimeter; 4, downright bad greater than 1.5 square centimeters; 5, the extensive necrosis of blade is given a mark to each strain plant.With the mark summation with a kind of 8 strain plants of strain infection is the pathogenic mark (table 1) of this bacterial strain.Table 1: the xanthomonas bacterial strain cause the plant characteristic of disease
Bacterial strain + 12 days + 14 days
?BIOCAT?381/382 ?0 ?0
?BIOCAT?379/380 ?32 ?39
?BIOCAT826 ?28 ?34
?BIOCAT?1016 ?4 ?4
?BIOCAT?1017 ?5 ?5
?BIOCAT?1019 ?2 ?3
?BIOCAT?1021 ?1 ?1
?BIOCAT?1022 ?4 ?5
Though the RPA-BIOCAT826 bacterial strain can cause the constantly withered of blade, constructed bacterial strain can only cause that at the most partial necrosis is withered, thereby reflects no pathogenicity.
Embodiment 6 is from RPA-BIOCAT826 and contain the xanthan gum production of the bacterial strain of hrpA1-C2 disappearance
Evaluate the throughput of bacterial strain xanthan gum by measuring the contained solid of 40ml culture that available isopropanol precipitating obtains.In MSX through after 24 hours pre-cultivations, in the 500ml triangular flask, put into 100ml MSX substratum, bacterium (the pre-culture of 0.4ml OD660=0.25) with approximately same amount inoculates, 30 ℃ of shaking culture (200 rev/mins) were taken out 40 gram cultures and are mixed with the 150ml Virahol after 6 days.After the filtration, with 70ml Virahol flushing twice, drying is left at it then and is weighed in the baking box with the precipitation that reclaimed.With three of the RPA-BIOCAT826 bacterial strain independently culture carry out this operation, the result demonstrates has about 10% throughput mutability.Use RPA-BIOCAT826 bacterial strain and its resulting result of Δ hrpA1-C2 derivative as shown in table 2.The xanthan gum throughput of table 2:RPA-BIOCAT826 bacterial strain and its Δ hrpA1-C2 derivative.
Bacterial strain Xanthan gum dry weight (gram) Throughput (gram/gram)
BIOCAT826 ?0.323 ?8.1×10 -3
?BIOCAT?1016 ?0.362 ?9.0×10 -3
?BIOCAT?1017 ?0.366 ?9.1×10 -3
?BIOCAT?1019 ?0.371 ?9.3×10 -3
?BIOCAT?1021 ?0.334 ?8.4×10 -3
?BIOCAT?1022 ?0.329 ?8.2×10 -3
But throughput with in every gram culture with the Virahol extracting to solid gram number represent.
＜110〉RHODIA CHIMIE＜120〉＜130〉BFF 99/0315＜140〉FR 9907963＜141〉1999-06-22＜160〉7＜170〉PatentIn Ver.2.1＜210〉1＜211〉20＜212〉DNA＜213〉＜220〉＜223〉：＜400〉1aaattcgtca agggtgatgc 20＜210〉2＜211〉20＜212〉DNA＜213〉＜220〉＜223〉：＜400〉2gttccacctg gtcgacaagc 20＜210〉3＜211〉1189＜212〉DNA＜213〉＜400〉3aaattcgtca agggtgatgc gatcgccggc ctggtgatca ccatggtcaa catcttggcc 60ggcatcgtgg taggcgtgac ctaccacggc atgagcgcgg gcgaggccgc caaccgcttt 120gcgatcctgt cggtaggcga tgcgatggtg tcgcagatcg cctcgctgct gatctcggtg 180gcggccggcg tcatgatcac ccgcgtcgcc aacgagaatg aaaccaagat cagctcgctc 240gggctcgaca tcggccgcca gctcaccagc aacgcacgtg ccttgatggc agcgagtgtg 300ctgctggcct gctttgcgtt cgtgccggga tttccggcgc tgctgttcct gctgctggca 360gcggcggtcg gtgccggggg ctatacgatc tggcgcaagc aacgcgacac cagcgggagc 420gatcagcccg cactgccatc aaccagccgc aaaggtgcca aaggcgatgc gccgcacatc 480cgcaagagcg ccccggattt cgcctcgccc ttgtcgatgc ggctttcgcc gcaactggct 540gcacggctcg acccggcgct gctggatcag gcgatcgaaa gcgagcggag gcaattggtc 600gagctgctgg gattgccgtt cccggggatc gcgatatggc agagcgaatc cctgcagggc 660ctgcagtacg aagtgttgat ccacgatgtg ccggaaaccc gcagcgcgtt gagcgatacg 720gcggacatgc agaaagcgct ggcccaacaa gccatcgcac cgttgcatgc acgcgcgcat 780ctgttcgtcg gcatccagga gacgcagtgg atgctggaac aggtgggcgc ggactatccc 840gggctggttg cagaggtcaa caaggccatg ccagcccaac gcatcgccga tgtgttgcgg 900cgactgctgg aagaacgcat cccggtgcgc aacatcaaga gcatcctgga gagcctggtg 960gtgtggggac cgaaggaaaa ggatctgctg atgctgaccg agtatgtgcg ctgcgatctc 1020ggccgctatc ttgcgcacac cgcgaccgca ggcaccggac agctgcctgc ggtgatgctc 1080gaccacgccg tggaacagtt gatccggcag tcgattcgcg ccacaccggc cggcaatttc 1140ctggcgctgc caccggagca ggccaatcag cttgtcgacc aggtggaaa 1189＜210〉4＜211〉20＜212〉DNA＜213〉＜220〉＜223〉：＜400〉4gatccaacag ctggacaacc 20＜210〉5＜211〉20＜212〉DNA＜213〉＜220〉＜223〉：＜400〉5aacggaatct tcgacaggcc 20＜210〉6＜211〉1818＜212〉DNA＜213〉＜400〉6atggcatacg cctgtcctcc agttcaccgc catcgacgcg cgccgttggc cgctgccttg 60ttgcttggct tgctgccgct gctgccgccg catgccaacg ccgcgtcggt gccgtggcac 120tcgcgcagct tcaaatacgt tgccgaccgc aaggatctca aggaggtgct gcgcgacctg 180tccgccagcc aatccatcac cacctggatt tcaccggagg tgaccggcac gctcagtggc 240aaattcgaag ccactccgca gaagtttctc gacgatctat cgggcacgtt cggttttgtc 300tggtattacg atggctcggt gctcagaatc tggggcgcga acgagaccaa gaatgcgacc 360ttgagtttgg gcgctgcatc gacgagtgcg ctgcgcgatg cgcttgcgcg catgcggctg 420gacgatccgc gctttccggt ccgttatgac gagacagcgc acctggcggt ggtgtcgggc 480ccgccgggtt atgtggatac cgtcgcggcg atcgccaagc aggtcgagca ggtcgcgcgc 540caacgcgacg ccaccgaagt gcaggtgttt cagctgcatt atgcgcaggc ggccgaccac 600accacccgca tcggtggtca agacatccag gtgccgggca tggccagcct gttgcgcaac 660atatacggcg tgcgtggcgc gcccactgcg gcgctgcccg ggccaggcgc gaatttcggg 720cgtgtgcaac cgatcggcgg tggctcgtcc aataccttcg gcaacagcgg tcagcgccag 780agtggcggca gcggcattct cggtttgcct gcgtcgtggt tcggcgctgg gtcgccgtcc 840gagcgggtgc cggtcagtcc gccgttgccg ggcagtggca atagcgccaa tgcgccggcc 900agcgtgtggc cggagatgag ccaggccaga cgcgatgcgc cgctggcggt ggacgccggc 960agcggcggtg agctggcatc cgacgcgccg gtgatcgaag ccgacccgcg caccaacggc 1020attctcattc gcgaccgccc cgagcggatg gccgcctatg gcacgttgat ccagcagctc 1080gacaaccgtc ccaagctgct gcagatcgat gccaccatca tcgagatccg cgacggcgcc 1140ctgcaggatc tcggcgtgga ctggcggttc cacagccggc gtgtggatgt gcagaccggc 1200gacgggcgtg gtggccagct tggctacgat ggcagcttga gcggtgcagc agccgccggt 1260gcagccgcgc cgttgggcgg gacgttgacc gctgtcctgg gcgatgcagg gcgttacctg 1320atgacgcgcg tctcggcgct cgagcagacc aacaaggcca agatcgtctc caccccgcag 1380gtggcgacgc tggacaacgt ggaagcggtg atggaccaca agcaacaggc attcgtgcgt 1440gtcagcggtt atgcatccgc cgacctctac aacctgtccg cgggtgtatc gctacgcgta 1500ttgccaagtg tggtgccggg gtcgccaaat ggtcagatgc gcctggatgt gcgtatcgaa 1560gacgggcagt tgggcgccaa taccgtcgat ggcattcccg tcatcacctc cagcgagatc 1620accacgcagg ccttcgtcaa cgagggccag agcctgctga tcgccggtta tgcttccgac 1680accgatcaga cagatctgaa caacgtcccc gggctgtcca ggattccatt ggtcggcaac 1740ctgttcaagc atcgccagca gagcgggtcg cggttgcagc ggttgttcct gctgaccccg 1800catatcgtct cgccctga 1818＜210〉7＜211〉702＜212〉DNA＜213〉＜400〉7atgcgtcttt ggctgaggtc cacaccggaa gcggtcggcc ttgactgcga ggtcatccca 60cgcgaggcat tggcctgtgt gctggaactg gacgcagcgg gtgcacaggt gcacgcgcgt 120tgcgcgcagg cgctggcgga cgcccagacg cgtgcgcagg cgctgctcga cgctgcccaa 180cggcaggccg aggccatcct tcaggatgcc cacgacaggg ccgagcgcag tgcacgcctg 240ggctatgccg ccgggctgcg ccgtcagctc gacgcgtgga acgagcgcgg cgtgcggcat 300gccttcgcgg cccaggacgc cgcacggcgc gcccgcgagc gcctggccga gatcgtcgcg 360cacgcctgcg agcaggttct gcacgggcac gatcctgcgg cgctgtacgc gcgcgccgca 420caggcgctgg acggcgccct ggacgaggcg aacgccctgc aggtgagcgt gcatcccgat 480gcgctggacg atgcacggcg cgccttcgat gcggccgcag cggccggcgg atggagcatg 540ccggtggaac tgtgcggtga tacgactctg gccttgggtg cctgcgtgtg cgaatgggat 600accggcgtgt tcgagaccga tctgcgtgat cagctgcgca gtctccggcg cgtcattcgc 660cgcgtgttgg ccacgccgca ggcggtgccg gatgcttgct ga 702

Claims (19)

1. bacterial isolates, it has been lost by at least one virulence gene of inactivation and has caused phytopathy character, and keeps the ability that produces exo polysaccharides.
2. claim 1 bacterial isolates required for protection is characterized in that at least one gene by inactivation hrp or hrc gene group, at least two genes advantageously, and preferably at least three genes make it become stably non-phytopathogenic bacterial isolates.
3. claim 1 or 2 bacterial isolateses required for protection is characterized in that making it become stably non-phytopathogenic bacterial isolates by 5 to 9 genes of inactivation hrp or hrc gene group.
4. claim 1 bacterial isolates required for protection is characterized in that it is to lose the xanthomonas bacterial strain that causes phytopathy character but keep generation exo polysaccharides ability by at least one virulence gene of inactivation.
5. claim 4 xanthomonas bacterial strain required for protection is characterized in that it is the bacterial strain of xanthomonas campestris.
6. claim 5 xanthomonas bacterial strain required for protection is characterized in that it is the pathogenic mutation of xanthomonas campestris bird rape.
7. above any claim xanthomonas bacterial strain required for protection; it is characterized in that by 1kb at least in disappearance hrp or the hrc gene group; preferred 3kb at least, advantageously the DNA district of 5kb obtains the inactivation of described gene at least, and this bacterial strain keeps producing the ability of exo polysaccharides.
8. above any right is described to require xanthomonas bacterial strain required for protection, it is characterized in that it has comprised the disappearance in the DNA district of 40kb at the most.
9. claim 8 xanthomonas bacterial strain required for protection is characterized in that it is to obtain to all or part of of hrpC2 gene by disappearance hrpA1.
10. non-in essence phytopathogenic xanthomonas bacterial strain is characterized in that it comprises in hrp or the hrc gene group 1kb at least, preferred 3kb at least, and the disappearance in the DNA zone of 5kb at least advantageously, and it has kept the ability that produces exo polysaccharides.
11. non-in essence phytopathogenic xanthomonas bacterial strain is characterized in that it is that all or part of disappearance by the hrpA1-C2 gene obtains.
12. non-in essence phytopathogenic xanthomonas campestris bacterial strain, it is selected from BIOCAT 1016, BIOCAT 1017, BIOCAT 1019, BIOCAT 1021 and BIOCAT 1022 bacterial strains, and wherein these bacterial strains are deposited in CBS with numbering CBS 101940, CBS 101941, CBS 101942, CBS 101943 and CBS 101944 respectively.
13. any of claim 4 to 13 xanthomonas bacterial strain required for protection is characterized in that exo polysaccharides is an xanthan gum.
14.pRPA-BCAT plasmid is used to produce claim 9 to 13 bacterial strain required for protection.
15. prepare the method for any bacterial strain required for protection of claim 7 to 13, it is characterized in that carrying out homologous recombination by the plasmid with all or part of disappearance that contains hrp or hrc gene obtains this bacterial strain.
16. preparation bacterium exo polysaccharides; especially the method for xanthan gum; it is characterized in that any bacterial isolates required for protection with claim 1 to 13; suitably; the bacterial isolates of xanthomonas; the bacterial isolates of preferred xanthomonas campestris is cultivated under the condition that allows generation exo polysaccharides in fermention medium.
17. a nucleic acid is characterized in that it comprises nucleotide sequence SEQ ID No.3.
18. a nucleic acid is characterized in that it comprises nucleotide sequence SEQ ID No.6.
19. a nucleic acid is characterized in that it comprises nucleotide sequence SEQ ID No.7.
CN00809327A 1999-06-22 2000-06-21 Avirulent xanthomonas-campestris strains producing xanthan Pending CN1357043A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054470A1 (en) * 2003-12-01 2005-06-16 Ji-Liang Tang A gene encoding phosphoenolpyruvate synthase for plant protection
CN101993841A (en) * 2010-02-11 2011-03-30 淄博中轩生化有限公司 Xanthomonas sp.SN-58 and method for preparing xanthan gum by using xanthomonas sp.SN-58
CN105505824A (en) * 2016-01-06 2016-04-20 江南大学 Method for preparing xanthan gum through xanthomonas campestris fermentation and application of xanthan gum

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US7727747B2 (en) 2006-11-14 2010-06-01 Bemidji State University Foundation Solid or semi-solid state fermentation of xanthan on potato or potato waste

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054470A1 (en) * 2003-12-01 2005-06-16 Ji-Liang Tang A gene encoding phosphoenolpyruvate synthase for plant protection
CN101993841A (en) * 2010-02-11 2011-03-30 淄博中轩生化有限公司 Xanthomonas sp.SN-58 and method for preparing xanthan gum by using xanthomonas sp.SN-58
CN105505824A (en) * 2016-01-06 2016-04-20 江南大学 Method for preparing xanthan gum through xanthomonas campestris fermentation and application of xanthan gum
CN105505824B (en) * 2016-01-06 2019-02-22 江南大学 A kind of method and its application preparing xanthan gum with xanthomonas campestris fermentation

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