Xanthomonas Campestris NRRL B-1459 Research Articles

Optimizing Curdlan Synthesis: Engineering Agrobacterium tumefaciens ATCC31749 for Enhanced Production Using Dextrin as a Carbon Source

A key goal in current research on industrial curdlan production is the expansion of carbon sources for fermentation. In this study, a recombinant bacterial strain, sp-AmyAXCC, capable of fermenting and synthesizing curdlan using dextrin as a carbon source, was produced via heterologous expression of IPTG-inducible α-amylase from Xanthomonas campestris NRRL B-1459 in Agrobacterium tumefaciens ATCC31749. External expression of the enzyme was confirmed by western blotting, and the expression levels of exogenous proteins during the fermentation process were monitored. Additionally, the properties of the curdlan product were characterized using attenuated total reflectance-Fourier transform infrared spectroscopy and X-ray diffraction. The recombinant strain produced curdlan at a titer of 30.40 ± 0.14 g/L, gel strength of 703.5 ± 34.2 g/cm2, and a molecular weight of 3.58 × 106 Da, which is 33% greater than the molecular weight of native curdlan (2.69 × 106 Da). In the batch fermentation of sp-AmyAXCC with 12% dextrin as a carbon source, the titer of curdlan was 66.7 g/L with a yield of 0.56 g/g, and a productivity rate of 0.62 g/L/h at 108 h. The results of this study expand the substrate spectrum for Agrobacterium fermentation in curdlan production and provides guidance for further industrialization of curdlan production.

Draft genome of the xanthan producer Xanthomonas campestris NRRL B-1459 (ATCC 13951)

Xanthomonas campestris NRRL B-1459 was used in pioneering studies related to the biotechnological production of xanthan, the commercially most important polysaccharide of bacterial origin. The analysis of its genome revealed a 5.1Mb chromosome plus the first complete plasmid of an X. campestris strain applied in biotechnology.

Xanthan gum production of Xanthomonas spp. Isolated from different plants

Xanthan gum were produced from the following Xanthomonas strains; standard strain Xanthomonas campestris NRRL B-1459 and isolated strains Xanthomonas arbicola pv. juglandis, Xanthomonas axonopodis pv. vesicatoria, Xanthomonas axonopodis pv. begonia, Xanthomonas axonopodis pv. dieffenbachia. The viscosity features of the xanthan gums obtained were determined at 25–80°C with different pH values and were compared to commercial xanthan gum. Our results indicate that X. arbicola pv. juglandis showed the highest productivity (8.22±1.52 g/L gum). This was followed by X. axonopodis pv. begonia (7.74±1.30 g/L gum), and the control bacterial strain X. campestris NRRL B-1459 (7.46±0.28 g/L gum). X. axonopodis pv. vesicatoria showed the lowest productivity (6.40±0.55 g/L gum). No xanthan gum could be obtained from X. axonopodis pv. dieffenbachia. Xanthan gum produced by X. axonopodis pv. vesicatoria showed the highest viscosity value (428 mPa·sec at 1% solution) in all Xanthomonas strains isolated from plants.

Optimisation of xanthan gum production by palm date ( Phoenix dactylifera L.) juice by-products using response surface methodology

The present study was undertaken investigate and optimise the possibility of xanthan gum production by Xanthomonas campestris NRRL B-1459 in batch experiments on date palm juice by-products. Using an experimental Response Surface Methodology complemented with a Central Composite Orthogonal Design, three major independent variables (date juice carbon source, nitrogen source and temperature) were evaluated for their individual and interactive effects on biomass and xanthan gum production. The optimal conditions selected were: 84.68 g/l for carbon source, 2.7 g/l for nitrogen source, and 30.1 °C for temperature. The experimental value obtained for xanthan production under these conditions was about 43.35 g/l, which was close to the 42.96 g/l value predicted by the model. Higher yields of biomass production could be obtained at 46.68 g/l for carbon source, 4.58 g/l for nitrogen source and 30 °C for temperature. The maximum value obtained for biomass production was 3.35 g/l, which was higher than the 2.98 g/l value predicted by the model. The xanthan formed was subjected to HPLC and TLC analyses and its molecular weight as well as pyruvate content were identified. The findings indicated that this polysaccharide contained glucose, glucoronic acid and mannose. Overall, the date palm juice by-products presented in the current study seem to exhibit promising properties that can open new pathways for the production of efficient and cost-effective xanthan gum.

Visualizing the infection process of Xanthomonas campestris in cabbage using green fluorescent protein

The plant pathogen, Xanthomonas campestris NRRL B-1459 was chromosomally tagged with gfp, and the transformant, which was subjected to Southern hybridization showed the presence of gfp in the chromosome. The virulence-related gene of the transformant was not affected by the insertion of gfp. After inoculation into cabbage plants, the infection process was visually studied in planta. Using a fluorescence microscope, the migration and distribution of gfp-labelled bacteria was visualized in real time. As the gfp-labelled cells were easily visualized from the beginning of infection, we observed a time delay of 2 days between distribution of the Xanthomonas cells in cabbage plant and the appearance of visible necrosis.

Preservation of Xanthomonas campestris on agar slopes: effects on xanthan production

Xanthomonas campestris NRRL B-1459 and a variant E2, when preserved on agar slopes (transferred monthly) over 11 months did not deteriorate in their ability to produce xanthan in quantity and quality, as determined by culture in 500-ml baffled flasks. Variations between 8 and 14% (with respect to the average) in the final xanthan concentration were observed for the E2 and B-1459 strains, respectively. A wide range of final viscosities was obtained; these were consistent with the changes in gum concentration. Differences were more likely associated with differences in fermentation kinetics rather than being inherent to the strains. The rheological quality of both polysacharides was relatively constant throughout the time of culture maintenance. Preservation of these bacteria on agar slopes was an adequate method, in contrast to previous reports. In the period studied, strain E2 produced higher gum titres and slightly lower gum quality compared to strain B-1459.

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.

Xanthan production by Xanthomonas campestris NRRL B-1459 and interfacial approach by zeta potential measurement

Dissociation of cell growth and metabolite production allows the creation of continuous reactors where conversion yields of carbon to product are very high. This is especially true for products such as xanthans, where physical parameters change rapidly, especially viscosity. At pH 5, cell growth and biomass production are increased. At pH 7, the opposite situation is observed, xanthan production is increased. Moreover, production is promoted at 30°C, while 25°C is the optimum temperature for cell growth. The composition of the medium (nitrogen source, citrate concentration, d-glucose concentration) has also been studied. Two-stage fermentation is an effective method, yielding up to 90% in the bioconversion of 1% d-glucose to xanthan. The xanthan/cell ratio is ∼10. The major difficulty in carrying out continuous production is the anionic characteristics of the gum.

Xanthan production by Xanthomonas campestris NRRL B-1459 and interfacial approach by zeta potential measurement

Dissociation of cell growth and metabolite production allows the creation of continuous reactors where conversion yields of carbon to product are very high. This is especially true for products such as xanthans, where physical parameters change rapidly, especially viscosity. At pH 5, cell growth and biomass production are increased. At pH 7, the opposite situation is observed, xanthan production is increased. Moreover, production is promoted at 30°C, while 25°C is the optimum temperature for cell growth. The composition of the medium (nitrogen source, citrate concentration, d -glucose concentration) has also been studied. Two-stage fermentation is an effective method, yielding up to 90% in the bioconversion of 1% d -glucose to xanthan. The xanthan/cell ratio is ∼10. The major difficulty in carrying out continuous production is the anionic characteristics of the gum.

Maintenance procedures for the curtailment of genetic instability: Xanthomonas campestris NRRL B-1459

Characteristics are described of small-colony variants of Xanthomonas campestris NRRL B-1459 which are frequently encountered when routine culture maintenance procedures are employed. In contrast to the parental type, smallcolony variants were shown to be resistant to a number of antibiotics, to acridine orange, and to phage which are virulent for the parent colony type. Sensitivity to ultraviolet radiation was similar in both colony types. A simple method for preservation of viable cells is described. The suitability of the method for providing reproducible inocula free from variant cell types is examined.

Colonial variation in Xanthomonas campestris NRRL B-1459 and characterization of the polysaccharide from a variant strain

Stock cultures of Xanthomonas campestris NRRL B-1459 require special attention to maintenance and propagation to assure consistent production in good yields of the extracellular polysaccharide xanthan. Under customary conditions of propagative maintenance on agar slants, variant colony types develop that are smaller in size than the normal type. The rate of regression of the normal to the variant forms was diminished when the D-glucose content of the stock medium was sufficient to avoid depletion during storage and when transfer to fresh medium was reduced to 14-day intervals. Under conditions for polysaccharide production, the normal large-colony type gives crude culture liquors after 48 h of 7000 centipoise (cp) viscosity; the predominant variant form gives only 4000 cp. On the basis of 2.1% initial D-glucose, biopolymer yields for the normal and variant strains were 62 and 43%, respectively. Polysaccharide produced by the variant (small-colony type) differs adversely in solution properties from that of the parent strain (large-colony type); it differs also in its lower content of pyruvic acid and O-acetyl substituents. The molar ratios of constituent sugars (D-glucose, D-mannose, and D-glucuronic acid), however, were identical in polysaccharides with the normal and variant strains. Exclusion of colonial variants from fermentations is prerequisite to production of xanthan optimum in properties and yield.

Studies on dilute solutions and dispersion of the polysaccharide from Xanthomonas campestris NRRL B-1459

Some properties of the extracellular polysaccharide produced by the bacterium Xanthomonas campestris NRRL B-1459 were measured in partially purified culture fluids and in liquid systems prepared from thoroughly purified and lyophilized material. High intrinsic viscosities resulted from dispersions and solutions of polysaccharide B-1459 in 0.01 m ammonium acetate and 4 m urea. Heating a dispersion of B-1459 in 4 m urea yielded a true macromolecular solution in which the polysaccharide had an apparent molecular weight of approximately 2 × 10 6. Lightscattering measurements on culture fluids of B-1459 made 4 m in urea gave molecular weights of 13 × 10 6 and 50 × 10 6. Various solution-properties and factors that affect the viscosity of dispersions prepared from lyophilized polysaccharide are presented.

Biological Assays for Two Mycotoxins Produced by Fusarium tricinctum

A survey was made to detect microorganisms useful for assaying butenolide [4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone] and T-2 toxin [4beta, 15-diacetoxy-8alpha-(3-methylbutyryloxy)-12,13-epoxytricothec -9-en-3alpha-ol]. These mycotoxins produced by strains of Fusarium tricinctum have been implicated in mycotoxicosis of livestock. Although butenolide proved to be a very weak antibiotic, assay discs containing 100 mug of this toxin inhibited Sprillum serpens NRRL B-2052, Vibrio tyrogenus NRRL B-1033, and Xanthomonas campestris NRRL B-1459. T-2 toxin had no effect on 54 bacterial strains but inhibited 6 of 11 fungi. Growth of Rhodotorula rubra NRRL Y-7222 and Penicillium digitatum NRRL 1202 was retarded by assay discs containing 4 mug of T-2 toxin. Solutions with less than 1 mug of T-2 per ml toxin were readily detected by a pea seed germination test. Germination was reduced more than 50% when seeds imbibed solutions of 0.5 mug of T-2 toxin per ml. Butenolide had no effect on pea seed germination at concentrations as high as 200 mug/ml.