Xanthan Pyruvilation Is Essential for the Virulence of Xanthomonas campestris pv. campestris.

Xanthan, the main exopolysaccharide (EPS) synthesized by Xanthomonas spp., contributes to bacterial stress tolerance and enhances attachment to plant surfaces by helping in biofilm formation. Therefore, xanthan is essential for successful colonization and growth in planta and has also been proposed to be involved in the promotion of pathogenesis by calcium ion chelation and, hence, in the suppression of the plant defense responses in which this cation acts as a signal. The aim of this work was to study the relationship between xanthan structure and its role as a virulence factor. We analyzed four Xanthomonas campestris pv. campestris mutants that synthesize structural variants of xanthan. We found that the lack of acetyl groups that decorate the internal mannose residues, ketal-pyruvate groups, and external mannose residues affects bacterial adhesion and biofilm architecture. In addition, the mutants that synthesized EPS without pyruvilation or without the external mannose residues did not develop disease symptoms in Arabidopsis thaliana. We also observed that the presence of the external mannose residues and, hence, pyruvilation is required for xanthan to suppress callose deposition as well as to interfere with stomatal defense. In conclusion, pyruvilation of xanthan seems to be essential for Xanthomonas campestris pv. campestris virulence.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris.

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

Hybrid modeling of xanthan gum bioproduction in batch bioreactor.

This work is focused on hybrid modeling of xanthan gum bioproduction process by Xanthomonas campestris pv. mangiferaeindicae. Experiments were carried out to evaluate the effects of stirred speed and superficial gas velocity on the kinetics of cell growth, lactose consumption and xanthan gum production in a batch bioreactor using cheese whey as substrate. A hybrid model was employed to simulate the bio-process making use of an artificial neural network (ANN) as a kinetic parameter estimator for the phenomenological model. The hybrid modeling of the process provided a satisfactory fitting quality of the experimental data, since this approach makes possible the incorporation of the effects of operational variables on model parameters. The applicability of the validated model was investigated, using the model as a process simulator to evaluate the effects of initial cell and lactose concentration in the xanthan gum production.

A comparison between shaker and bioreactor performance based on the kinetic parameters of xanthan gum production.

Xanthan gum production was studied using sugarcane broth as the raw material and batch fermentation by Xanthomonas campestris pv. campestris NRRL B-1459. The purpose of this study was to optimize the variables of sucrose, yeast extract, and ammonium nitrate concentrations and to determine the kinetic parameters of this bioreaction under optimized conditions. The effects of yeast extract and ammonium nitrate concentrations for a given sucrose concentration (12.1-37.8 g L(-1)) were evaluated by central composite design to maximize the conversion efficiency. In a bioreactor, the maximum conversion efficiency was achieved using 27.0 g L(-1) sucrose, 2.7 g L(-1) yeast extract, and 0.9 g L(-1) NH(4)NO(3). This point was assayed in a shaker and in a bioreactor to compare bioreaction parameters. These parameters were estimated by the unstructured kinetic model of Weiss and Ollis (Biotechnol Bioeng 22:859-873, 1980) to determinate the yields (Y (P/S)), the maximum growth specific rate (mu (max)), and the saturation cellular concentration (X*). The parameters of the model (mu (max), X*, m, lambda, alpha, and beta) were obtained by nonlinear regression. For production of xanthan gum in a shaker, the values of mu (max) and Y (P/S) obtained were 0.119 h(-1) and 0.34 g g(-1), respectively, while in a bioreactor, they were 0.411 h(-1) and 0.63 g g(-1), respectively.

Effectiveness factor in biological external convection: study in high viscosity systems.

A study regarding the influence of mixing on the efficiency of alginate and xanthan synthesis was carried out. The effectiveness of these two systems in terms of a dimensionless reaction convection number (N(RC)), which is a function of the power input per unit volume, was analysed and compared with low viscosity systems. The results revealed that a decrease in the power input caused a reduction in the growth rate as well as in production rate. It was observed that the effectiveness factor (eta) both for alginate production and xanthan synthesis resulted weakly dependent on the biomass content. A good correlation between effectiveness and N(RC) was obtained for the two tested models. N(RC) number could be appropriately employed in correlating the effectiveness factor for processes with viscosities from 0.001 to more than 1 kg m(-1) s(-1). Due to the parallelism with the conventional internal diffusion approach (Thiele modulus), the advantages for applying N(RC), in particular to correlate the efficiency in systems limited both for external convection and internal diffusional resistance, are shown.

[Xanthan production by Xanthomonas campestris in a non-conventional culture medium]. / Producción de xantano por Xanthomonas campestris en un medio de cultivo no convencional.

Among 3 varieties of Xanthomonas campestris, the variety ocumo (X. campestris pv. ocumo), showed the greatest capacity for producing xanthan. This bacteria grows appropriately and produces this polysaccharide in a wide diversity of carbohydrate sources. However, this strain does not produce xanthan when the carbohydrate comes from lignocellulosic materials. The glucose syrup FAVEPRO was the carbon source that showed the best yield (23 g/l) with the greatest viscosity (7000 cps) of xanthan. The optimum production conditions in 1 L erlenmeyer flasks, with a working volume of 0.2 L and in a 14 L (stirred tank type bioreactor) with a working volume of 10 L, were the following: total sugar 5%, urea 0.05%, di-potassium hydrogen phosphate 0.5%, pH 7.5, inoculum 10%, temperature 30 degrees C, agitation 250-1000 rpm and aereation 0.3-1.0 vvm. This strain of X. campestris pv. ocumo was able to produce xanthan (10 g/l) in a culture medium based on a previously treated agricultural waste, called soluble acid extract of cassava bark. The viscosity of this medium increased up to 1500 cps.

Produccion de xantano por Xanthomonas campestris en un medio de cultivo no convencional / Xanthan production by Xanthomonas campestris in no-conventional culture medium

Se determinó que X. campestris vp. Ocumo, exhibió mayor capacidad para crecer y producir xantano que las otras dos variedades de X. campestris analizadas. Utiliza una amplia diversidad de fuentes de carbohidratos. Sin embargo, la cepa no produce el exopolisacárido cuando la fuente de carbohidrato proviente de materiales ricos en lignocelulosa. El Jarabe Glucosado FAVEPRO (JGF) fue la fuente de carbono con la que se logró el mayor rendimiento de xantano (23g/l) con la mayor viscosidad (7000 cps). Las condiciones óptimas de producción determinadas en fiolas de 1 L (volumen de trabajo 10 L) fueron las siguientes: 5 por ciento de azúcares totales, 0,05 por ciento de urea como fuente de nitrógeno, 0.5 por ciento de fosfato dipotásico, pH 7.5, 10 por ciento de inóculo, temperatura 30 grados Celsius, agitación entre 250-1000 rpm y aireación entre 0.3-1.0 vvm. X. campestris vp. Ocumo, fue también capaz de producir xantano (10g/l), y aumentar la viscosidad (hasta 1500 cps) de un medio basado en el extracto ácido soluble de corteza de yuca molida (EACY).

[Isolation of xanthan from Xanthomonas campesteris B-1459 in mechanically agitated fermentors]. / Obtención de xantano a partir de Xanthomonas campestris B-1459 en fermentadores con agitación mecánica.

Xanthan production from Xanthomonas campestris was studied by a mechanically shaken fermentor. Influence of glucose concentration, aeration of culture media, rheology of broths and pH control was evaluated. Different aeration conditions based on variation of stirring rates were assayed. Substrate concentration was determined according to the Miller method, and polymer production was performed by the Cadmus method. The higher xanthan levels (i.e. 2.3%) were obtained at 750 rpm, with 1 v/v. min. In such conditions, viscosity ranges about 7000 cPoise and a low level of dissolved oxygen were detected in the culture medium. Xanthan production was influenced by the glucose concentration and the presence of amaranth within the culture medium. In the processes wherein an automatic control of pH was performed, the polymer concentration did not increase regarding to processes involving regular pH evolution.

Effect of parB on plasmid stability and gene expression in Xanthomonas campestris.

The stabilization locus parB was subcloned into the broad host range plasmid pAP2, which contains the alpha-amylase gene from Bacillus subtilis, and introduced into Xanthomonas campestris pv campestris and X.c.pv manihotis. Analysis of the stability of plasmid pAP2 (parB-) and pAP23 (parB+) showed that the parB locus decreased significantly the plasmid loss rate mainly by X.c.pv campestris. The lower efficiency of stabilization in X.c.pv manihotis was probably due to the incompatibility system between the native plasmids and the newly introduced pAP23. Although parB had conferred higher stability, it determined a lower rate of alpha-amylase activity even by the strain Cm where its stabilization rate was higher.

Evaluación de medios de cultivo para detectar la reacción de hidrólisis del almidón por patovares de Xanthomonas campestris / Evaluation of culture media for detecting the starch hydrolysis reaction in pathovars of Xanthomonas campestris

Se emplearon 60 cepas de diferentes patovares de Xanthomonas campestris para evaluar la reacción de hidrólisis del almidón y compuestos intermedios de degradación del mismo en diferentes medios de cultivo: almidón solubre, almidón de papa, almidón insolubre de maíz, amilopectina de papa, amilopectina de maíz y amilosa de papa. El 74% de las cepas presentó reacción positiva en los 6 medios. De las restantes, los patovares holcicola, pelargonii, pruni y vitians dieron reacción negativa y, las cepas de X. campestris pv. vesicolatoria mostraron resultados variables. Los medios que contienen almidones insolubles de papa y de maíz manifestaron mayores ventajas para determinar la reacción de hidrólisis no solamente por la claridad y tamaño de los halos formados sino también por su utilidad para aislamientos directos. La reacción de hidrólisis del almidón serviría como un carácter complementario para diferenciar especies de Xanthomonas, pero no sería válida para separar a los patovares del grupo de X. campestris debido a la variación suficiente que existe entre ellos pese al alto porcentaje de reacciones positivas detectadas

Evaluación de medios de cultivo para detectar la reacción de hidrólisis del almidón por patovares de Xanthomonas campestris / Evaluation of culture media for detecting the starch hydrolysis reaction in pathovars of Xanthomonas campestris

Se emplearon 60 cepas de diferentes patovares de Xanthomonas campestris para evaluar la reacción de hidrólisis del almidón y compuestos intermedios de degradación del mismo en diferentes medios de cultivo: almidón solubre, almidón de papa, almidón insolubre de maíz, amilopectina de papa, amilopectina de maíz y amilosa de papa. El 74% de las cepas presentó reacción positiva en los 6 medios. De las restantes, los patovares holcicola, pelargonii, pruni y vitians dieron reacción negativa y, las cepas de X. campestris pv. vesicolatoria mostraron resultados variables. Los medios que contienen almidones insolubles de papa y de maíz manifestaron mayores ventajas para determinar la reacción de hidrólisis no solamente por la claridad y tamaño de los halos formados sino también por su utilidad para aislamientos directos. La reacción de hidrólisis del almidón serviría como un carácter complementario para diferenciar especies de Xanthomonas, pero no sería válida para separar a los patovares del grupo de X. campestris debido a la variación suficiente que existe entre ellos pese al alto porcentaje de reacciones positivas detectadas (AU)

[Evaluation of culture media for detecting the starch hydrolysis reaction in pathovars of Xanthomonas campestris]. / Evaluación de medios de cultivo para detectar la reacción de hidrólisis del almidón por patovares de Xanthomonas campestris.

Sixty strains of different pathovars of Xanthomonas campestris have been tested for the evaluation of various starch agars and compounds of starch degradation on six media: soluble starch, potato insoluble starch, corn insoluble starch, potato amylopectin, corn amylopectin and potato amylose. The purpose of the present investigation was the selection of the most suitable medium for the visualization of the starch hydrolysis test, presenting this reaction as a distinct character between pathovars of the Xanthomonas campestris group. From 60 strains tested, 74% gave positive reactions. Pathovars holcicola, pelargonii, pruni and vitians were negative. Regarding X. campestris pv. vesicatoria cultures, results were variable. Potato and corn insoluble starch agars were the most suitable media for the visualization of the starch hydrolysis reaction and at the same time the most appropriate for direct isolation. Differentiation at species level could be practicable, but within the Xanthomonas campestris group, variation amongst pathovars suggest the unsuitability of the test in spite of the high percentage of positive reactions.