The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis

The complete genome sequence of the Xanthomonas campestris pv. campestris strain B100 was established. It consisted of a chromosome of 5,079,003bp, with 4471 protein-coding genes and 62 RNA genes. Comparative genomics showed that the genes required for the synthesis of xanthan and xanthan precursors were highly conserved among three sequenced X. campestris pv. campestris genomes, but differed noticeably when compared to the remaining four Xanthomonas genomes available. For the xanthan biosynthesis genes gumB and gumK earlier translational starts were proposed, while gumI and gumL turned out to be unique with no homologues beyond the Xanthomonas genomes sequenced. From the genomic data the biosynthesis pathways for the production of the exopolysaccharide xanthan could be elucidated. The first step of this process is the uptake of sugars serving as carbon and energy sources wherefore genes for 15 carbohydrate import systems could be identified. Metabolic pathways playing a role for xanthan biosynthesis could be deduced from the annotated genome. These reconstructed pathways concerned the storage and metabolization of the imported sugars. The recognized sugar utilization pathways included the Entner-Doudoroff and the pentose phosphate pathway as well as the Embden-Meyerhof pathway (glycolysis). The reconstruction indicated that the nucleotide sugar precursors for xanthan can be converted from intermediates of the pentose phosphate pathway, some of which are also intermediates of glycolysis or the Entner-Doudoroff pathway. Xanthan biosynthesis requires in particular the nucleotide sugars UDP-glucose, UDP-glucuronate, and GDP-mannose, from which xanthan repeat units are built under the control of the gum genes. The updated genome annotation data allowed reconsidering and refining the mechanistic model for xanthan biosynthesis.     

Expression of the gum operon directing xanthan biosynthesis in Xanthomonas campestris and its regulation in planta

The gum gene cluster of Xanthomonas campestris pv. campestris comprises 12 genes whose products are involved in the biosynthesis of the extracellular polysaccharide xanthan. These genes are expressed primarily as an operon from a promoter upstream of the first gene, gumB. Although the regulation of xanthan synthesis in vitro has been well studied, nothing is known of its regulation in planta. A reporter plasmid was constructed in which the promoter region of the gum operon was fused to gusA. In liquid cultures, the expression of the gumgusA reporter was correlated closely with the production of xanthan, although a low basal level of beta-glucuronidase activity was seen in the absence of added carbon sources when xanthan production was very low. The expression of the gumgusA fusion also was subject to positive regulation by rpfF, which is responsible for the synthesis of the diffusible signal factor (DSF). The expression of the gumgusA fusion in bacteria recovered from inoculated turnip leaves was maximal at the later phases of growth and was subject to regulation by rpfF. These results provide indirect support for the operation of the DSF regulatory system in bacteria in planta.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey.

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Isolation of a Xanthomonas campestris strain with elevated beta-galactosidase activity for direct use of lactose in xanthan gum production

AIMS: To isolate a Xanthomonas campestris strain that can use lactose directly for xanthan gum production. METHODS AND RESULTS: The presence of indigenous beta-galactosidase gene in the wild-type Xc17 was detected by PCR and Southern hybridization. Treatment of Xc17 with nitrous acid resulted in the isolation of Xc17L with a 3.5-fold elevation of beta-galactosidase activity capable of growing in lactose-based medium. Xc17L is stable for at least 100 generations in terms of beta-galactosidase expression. The amounts of xanthan produced by Xc17L in lactose-based medium are comparable to those in glucose-based medium. CONCLUSIONS: Xc17L is potentially useful for xanthan production from whey, a waste containing lactose. SIGNIFICANCE AND IMPACT OF THE STUDY: A lactose-utilizing strain of X. campestris strain can be constructed without incorporation of any exotic DNA or antibiotic resistance gene and therefore concern of a gene-modified organism and fear of a spread of an antibiotic-resistant gene are avoided.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis.

The nucleotide sequence of a 3.4-kb EcoRI-PstI DNA fragment of Xanthomonas campestris pv. campestris revealed two open reading frames, which were designated xanA and xanB. The genes xanA and xanB encode proteins of 448 amino acids (molecular weight of 48,919) and 466 amino acids (molecular weight of 50,873), respectively. These genes were identified by analyzing insertion mutants which were known to be involved in xanthan production. Specific tests for the activities of enzymes involved in the biosynthesis of UDP-glucose and GDP-mannose indicated that the xanA gene product was involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate. The deduced amino acid sequence of xanB showed a significant degree of homology (59%) to the phosphomannose isomerase of Pseudomonas aeruginosa, a key enzyme in the biosynthesis of alginate. Moreover, biochemical analysis and complementation experiments with the Escherichia coli manA fragment revealed that xanB encoded a bifunctional enzyme, phosphomannose isomerase-GDP-mannose pyrophosphorylase.     

Identification of Xanthomonas campestris pv. campestris galactose utilization genes from transcriptome data

A 70 mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Xanthomonas campestris pv. campestris B100, a plant-pathogenic bacterium that is industrially employed to produce the exopolysaccharide xanthan gum which has many applications as a stabilizing, thickening, gelling, and emulsifying agent in food, pharmaceutical, and cosmetic industries. As an application example, global changes of gene expression were monitored during growth of X. campestris pv. campestris B100 on two different carbon sources. Exponential growing bacterial cultures were incubated either for 1h or permanently in minimal medium supplemented with 1% galactose in comparison to growth in minimal medium supplemented with 1% glucose. Six genes were identified that were significantly increased in gene expression under both growth conditions. These genes were located in three distinguished chromosomal regions in operon-like gene clusters. Genes from these clusters encode secreted glycosidases, which were predicted to be specific for galactose-containing carbohydrates, as well as transport proteins probably located in the outer and inner cell membrane. Finally genes from one cluster code for cytoplasmic enzymes of a metabolic pathway specific for the breakdown of galactose to intermediates of glycolysis.     

Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris.

Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N.E. Harding, J.M. Cleary, D.K. Caba?as, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tetr) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain.     

Genome Sequence of Xanthomonas campestris JX, an Industrially Productive Strain for Xanthan Gum

Xanthomonas campestris JX, a soil bacterium, is an industrially productive strain for xanthan gum. Here we present a 5.0-Mb assembly of its genome sequence. We have annotated 12 coding sequences (CDSs) responsible for xanthan gum biosynthesis, 346 CDSs encoding carbohydrate metabolism, and 69 CDSs related to virulence, defense, and plant disease.     

Xanthomonas campestris pv. campestris secretes the endoglucanases ENGXCA and ENGXCB: construction of an endoglucanase-deficient mutant for industrial xanthan production

Xanthomonas campestris pv. campestris secretes at least two cellulose-degrading endoglucanases. One of these endoglucanases is encoded by the engXCA gene of X. c. pv. campestris 8400 that was previously characterized by Gough et al. [Gene (1990) 89: 53-59]. An additional endoglucanase encoded by the engXCB gene was identified in X. c. pv. campestris 8400 and FC2. The engXCB gene product that was grouped into the endoglucanase family E contains a putative N-terminal signal peptide, suggesting a secretion by the type II secretion system. The ENGXCB protein contributed approximately 8% to the cellulase activity in xanthan preparations. Deletion of engXCA and engXCB resulted in a fivefold reduction of the cellulose-degrading activity in xanthan preparations. The cellulase activity determined in xanthan preparations of the engXCA-engXCB mutant was only slightly higher than the activity found in preparations that were subjected to heat treatment. Mutations in engXCA and engXCB did not affect the growth rate and xanthan production of X. c. pv. campestris FC2 under several cultivation conditions. The engXCA-engXCB deletion mutant is markerless, which makes this mutant a valuable strain for xanthan production and approaches aimed at inactivating further genes encoding extracellular enzymes.     

Disruption of Xylella fastidiosa CVC gumB and gumF genes affects biofilm formation without a detectable influence on exopolysaccharide production

Xylella fastidiosa causes citrus variegated chlorosis (CVC), a destructive disease of citrus. Xylella fastidiosa forms a biofilm inside plants and insect vectors. Biofilms are complex structures involving X. fastidiosa cells and an extracellular matrix which blocks water and nutrient transport in diseased plants. It is hypothesized that the matrix might be composed of an extracellular polysaccharide (EPS), coded by a cluster of nine genes closely related to the xanthan gum operon of Xanthomonas campestris pv. campestris. To understand the role of X. fastidiosa gum genes on biofilm formation and EPS biosynthesis, we produced gumB and gumF mutants. Xylella fastidiosa mutants were obtained by insertional duplication mutagenesis and recovered after triply cloning the cells. Xylella fastidiosa gumB and gumF mutants exhibited normal cell characteristics; typical colony morphology and EPS biosynthesis were not altered. It was of note that X. fastidiosa mutants showed a reduced capacity to form biofilm when BCYE was used as the sustaining medium, a difference not observed with PW medium. Unlike X. campestris pv. campestris, the expression of the X. fastidiosa gumB or gumF genes was not regulated by glucose.     

Improvement in bioreactor productivities using free radicals: HOCl-induced overproduction of xanthan gum from Xanthomonas campestris and its mechanism

Free-radical induction has been employed as a novel strategy to improve bioreactor productivity and, more specifically, the quality and productivity of xanthan gum from Xanthomonas campestris cultures. A 210% increase in xanthan yield and a 20% increase in viscosity (quality) resulted from HOCl (oxidant) treatment. The acetate mass fraction in xanthan gum decreased by 42% and its pyruvate mass fraction increased by 63% as a result of HOCl treatment. The growth rate was almost unaffected by HOCl treatment. A hypothesis to explain the mechanism of xanthan gum overproduction by free-radical induction has been formulated. The significant aspects of the hypothesis, such as SoxS protein binding to the promoter region of the gum gene and the consequent increase in mRNA concentrations, have been experimentally verified.     

A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity.

A DNA fragment from Xanthomonas campestris pv. campestris that partially restored the carbohydrate fermentation pattern of a cya crp Escherichia coli strain was cloned and expressed in E. coli. The nucleotide sequence of this fragment revealed the presence of a 700-base-pair open reading frame that coded for a protein highly similar to the catabolite activation factor (CAP) of E. coli (accordingly named CLP for CAP-like protein). An X. campestris pv. campestris clp mutant was constructed by reverse genetics. This strain was not affected in the utilization of various carbon sources but had strongly reduced pathogenicity. Production of xanthan gum, pigment, and extracellular enzymes was either increased or decreased, suggesting that CLP plays a role in the regulation of phytopathogenicity.     

Cloning of a locus involved in pathogenicity and production of extracellular polysaccharide and protease in Xanthomonas campestris pv. campestris

Abstract A Tn5-induced mutant of Xanthomonas campestris pv campestris deficient in protease and extracellular polysaccharide production, and non-pathogenic to turnip seedlings, was isolated. Recombinant clones which restored all three characteristics concomitantly were obtained from a wild-type genomic library. One of the plasmid clones, pIJ3090, showed no hybridization to previously isolated clones involved In protease production. The mutant was not complemented by other known clones involved in pathogenicity, in xanthan gum production or in protease production.     

野油菜黄单胞菌野油菜致病变种8004菌株wxcA基因与EPS的产量有关

在以前的工作中,采用转座子Tn5 gusA5对野油菜黄单胞菌野油菜致病变种(Xcc)8004菌株进行诱变,获得一批胞外多糖(EPS)合成减少的突变体,对这些突变体的Tn5 gusA5的插入位点进行分析后,发现有两株突变体是wxcA基因不同插入位点的突变体。以前认为wxcA基因与脂多糖(LPS)的O-抗原合成有关而与EPS的合成无关。为明确wxc4基因的功能,对8004菌株的wxcA基因进行缺失,获得的△wxcA突变体的EPS产量与野生型菌株相比,减少了50％,并且一段PCR合成的包含wxcA基因的DNA片段能反式互补△wxcA突变体,恢复突变体的EPS产量。这证实了8004菌株的wxcA基因与EPS的合成产量有关。     

Crude Exopolysaccharides (EPS) from Xanthomonas campestris pv. manihotis, Xanthomonas campestris pv. Campestris and Commercial Xanthan Gum as Inducers of Protection in Coffee Plants against Hemileia vastatrix

Foliar-applied exopolysaccharides, obtained from bacterial cells of either Xanthomonas campestris pv. manihotis (EPS-Xcm) or Xanthomonas campestris pv. campestris (EPS-Xcc), isolate NRRL B-1459, were tested for their ability to induce local and systemic protection against coffee leaf rust caused by Hemileia vastatrix. Both preparations of EPS were effective in inducing local and systemic protection when applied 72 h before challenge with the pathogen. Protection was also observed when plants were treated with different concentrations of a commercially available preparation of xanthan gum (CXG).
Systemic protection was induced by EPS-Xcm, EPS-Xcc and CXG even after the removal of the treated leaves immediately before the challenge. Local protection lasted at least 5 weeks, when EPS-Xcm was applied at the concentration of 100 μg equivalents of glucose/ml. Fluorescent microscopic studies of pathogen development in protected and control leaves indicated that neither the germination, appressoria formation nor the number of infection sites were affected by treatment with EPS-Xcm.