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

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Abstract

The complete genome sequence of the Xanthomonas campestris pv. campestris strain B100 was established. It consisted of a chromosome of 5,079,003 bp, 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.

Introduction

Xanthomonas species are members of the γ subdivision of the Gram-negative Proteobacteria, which have adopted a plant-associated and usually plant pathogenic lifestyle. Xanthomonads are pathogens of diverse groups of cultivated plants, among them important crops like rice and citrus plants. Based on their specific host ranges, strains of Xanthomonas campestris were differentiated into over 140 pathovars (Swings, 1993). The number of pathovars was reduced when Xanthomonas strains were reclassified to 20 species (Vauterin et al., 1995). In the course of this reclassification many strains previously accepted as X. campestris pathovars became classified as members of distinct species. The largest and most heterogeneous of the new species was X. axonopodis, to which amongst others strains of X. campestris pv. vesicatoria type A strains were reclassified. X. campestris pv. campestris is pathogenic to cruciferous plants, where it causes the “black rot” disease by invading the vascular system of the host plants. The infected crucifers include cultivated Brassicaceae like cabbage and cauliflower as well as the model plant Arabidopsis thaliana. Besides its importance as a phytopathogen, X. campestris pv. campestris is known as the producer of the acid exopolysaccharide xanthan. Xanthan is a heteropolysaccharide with a cellulose-like backbone and trisaccharide side-chains of two mannose and one glucuronate residues that are attached to every second glucose moiety of the main chain (Jansson et al., 1975). There were inconsistent observations concerning the role of xanthan in pathogenicity in the past, and recent data suggesting that xanthan is not required for pathogenicity but contributes to the epiphytic survival of the Xanthomonads (Dunger et al., 2007) have now been challenged for X. axonopodis pv. citri (Rigano et al., 2007). Xanthan is commercially produced by fermentation and employed as a thickening agent and emulsifier in the nutritional, pharmaceutical, and oil drilling industries (Becker et al., 1998). In the last years the xanthan production by commercial providers increased significantly, so that it now exceeds 86,000 tons per annum (Seisun, 2006, Sutherland, 1998). Substantial efforts have been put into understanding the xanthan synthesis, the xanthan solution properties and its downstream recovery as well as the kinetics of X. campestris pv. campestris growth under xanthan production conditions (Garcia-Ochoa et al., 2000). A model established recently describes the kinetics of xanthan production in a batch reactor (Letisse et al., 2003). Further optimization of the xanthan production process would benefit significantly from utilizing genome data of X. campestris pv. campestris model or production strains.

So far genomes of five Xanthomonas strains have been sequenced. These include the X. campestris pv. campestris strains ATCC 33913 (da Silva et al., 2002) and 8004 (Qian et al., 2005), which cause the “black rot” disease on Brassicaceae. X. campestris pv. vesicatoria strain 85-10 (Thieme et al., 2005) is the causative agent of the bacterial spot disease on pepper (Capsicum spp.). The X. oryzae pv. oryzae strains KACC10331 (Lee et al., 2005) and MAFF 311018 (Ochiai et al., 2005) are both pathogens of rice (Oryza sativa) where they cause bacterial blight. The X. axonopodis pv. citri strain 306 (da Silva et al., 2002) causes citrus canker, which affects most commercial citrus cultivars. All genomes comprised circular chromosomes of 4,940,217 base pairs (bp) (X. oryzae pv. oryzae strain MAFF 311018) to 5,178,466 bp (X. campestris pv. vesicatoria strain 85-10), with comparably high G+C contents of 63.7–65.0%. Existence of plasmids was restricted to X. axonopodis pv. citri strain 306 and X. campestris pv. vesicatoria strain 85-10, while all strains carried multiple insertion sequence (IS) elements. An overview on the general genome features of X. campestris strains is given in Table 1. So far the functional analysis of the available genome data was focused on pathogenicity features (da Silva et al., 2002, Lee et al., 2005, Qian et al., 2005; Ochiai et al., 2005; Thieme et al., 2005). Phytopathogenesis was also the focus of genome comparisons related to Xanthomonas (da Silva et al., 2002, Moreira et al., 2005, Qian et al., 2005). Although generally mentioned in the genome publications, synthesis of xanthan and related cell-surface carbohydrates was up to now only marginally analyzed at the genomic level.

The experimental analysis of the biosynthesis of xanthan has been carried out mainly with strains that were not subject to genome sequencing so far. The xanthan synthesis is encoded by the gumBCDEFGHIJKLM genes, which are located in a single gene cluster of 12 kb that is mainly expressed as an operon from a promoter upstream of the first gene, gumB (Katzen et al., 1996, Vojnov et al., 2001). Xanthan synthesis is located at the cell membrane, where defined pentasaccharide repeat units of glucose–glucose–mannose–glucuronate–mannose are built from nucleotide sugars at a polyprenol lipid carrier (Ielpi et al., 1993). This is performed by glycosyltransferases encoded by the gum genes D, M, H, K, and I. The outer mannose at the distal position of the side-chain can be pyruvylated; both mannose residues of the repeat units can be acetylated at varying degrees (Stankowski et al., 1993). Completed repeat units are exported and polymerized to form mature xanthan that is subsequently released to the environment. The export and polymerization process is not well understood.

Besides being used to analyze IS elements (Simon et al., 1991) and the TonB-dependent iron uptake (Wiggerich et al., 1997, Wiggerich and Puhler, 2000), X. campestris pv. campestris strain B100 has been employed as a model strain to investigate the biosynthesis of the cell surface polysaccharides. Originally, it was isolated in a screening that aimed at identifying a strain suitable for xanthan production. The biosynthesis of xanthan depends on the nucleotide sugar precursors UDP-glucose, GDP-mannose, and UDP-glucuronate, the latter being generated by oxidation of UDP-glucose (Lin et al., 1995). The genes xanA and xanB encode the bifunctional enzymes phosphoglucomutase/phosphomannomutase and mannose-6-phosphate isomerase/mannose-1-phosphate guanylyltransferase, respectively, which catalyze four reactions and thus most of the steps within the UDP-glucose and GDP-mannose synthesis pathways (Koplin et al., 1992). In addition to the xanAB gene products only the glycolysis enzyme phosphoglucose isomerase (Tung and Kuo, 1999) and the UTP-glucose-1-phosphate uridylyltransferase (Wei et al., 1996) are required. At the nucleotide sugar precursors level, synthesis of xanthan is linked to the synthesis of lipopolysaccharides (LPS) (Koplin et al., 1993, Vorholter et al., 2001). The LPS precursor synthesis genes cluster on the X. campestris pv. campestris chromosome with the xanAB genes (Hotte et al., 1990) and further genes involved in LPS synthesis (Steinmann et al., 1997). All analyses related to this over all 30 kb gene cluster were based on X. campestris pv. campestris strain B100. A preliminary analysis of DNA sequence data obtained from sequencing transposon insertion sites of an X. campestris pv. campestris strain B100 transposon-tagged shotgun library revealed unexpected differences to the genome of X. campestris pv. campestris strain ATCC 33913 (Vorholter et al., 2003). As a consequence it was worthwhile to complete the nucleotide sequence of the X. campestris pv. campestris B100, to compare the Xanthomonas genes related to xanthan synthesis, and to use this genome data as a basis to reconstruct the metabolic pathways leading to the synthesis of xanthan.

Section snippets

Whole genome shotgun sequencing

DNA shotgun libraries with insert sizes of 1.5 kb, 2 kb, 3 kb and 4–6 kb were constructed in pUC19 vector (Yanisch-Perron et al., 1985) by Qiagen GmbH. Plasmid clones were end sequenced on ABI3700 capillary sequencing machines (ABI) by Qiagen GmbH and on ABI3730xl DNA analyser (ABI) at Bielefeld University. In addition to this, sequences from a transposon-tagged library (Vorholter et al., 2003) and from another 1.5-kb shotgun library constructed in pGEM-T Easy by MWG Biotech AG, were incorporated

General features of the X. campestris pv. campestris strain B100 genome

The genome of X. campestris pv. campestris strain B100 is composed of a circular chromosome of 5,079,002 bp (Fig. 1). A total of 4471 protein-coding sequences (CDS) were predicted within the genome of X. campestris pv. campestris strain B100 (Table 1). Further 62 RNA genes were identified, among them 54 tRNAs and 2 rRNA operons (Fig. 1B). Overall 3148 (70%) of the CDS were assigned to functional categories of the COG database (Tatusov et al., 1997). Among these CDS were 270 that encoded

Acknowledgements

The authors thank Daniela Bartels, Lars Gaigalat, Sascha Mormann, Diana Nakunst, and Jens Plassmeier for checking gene prediction results in the initial phase of the sequencing project. The project was supported by the GenoMik-Plus programme of the German Federal Ministry of Education and Research (BMBF), grant 03138805A, and by the BMBF project BioExPoSys.

References (60)

  • S.F. Altschul et al.

    Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

    Nucleic Acids Res.

    (1997)
  • M. Barreras et al.

    Functional characterization of GumK, a membrane-associated beta-glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis

    Glycobiology

    (2004)
  • D. Bartels et al.

    BACCardI—a tool for the validation of genomic assemblies, assisting genome finishing and intergenome comparison

    Bioinformatics

    (2005)
  • A. Becker et al.

    Xanthan gum biosynthesis and application: a biochemical/genetic perspective

    Appl. Microbiol. Biotechnol.

    (1998)
  • S. Blanvillain et al.

    Plant carbohydrate scavenging through tonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria

    PLoS ONE

    (2007)
  • R.F. Collins et al.

    The 3D structure of a periplasm-spanning platform required for assembly of group 1 capsular polysaccharides in Escherichia coli

    Proc. Natl. Acad. Sci. U.S.A.

    (2007)
  • P.M. Coutinho et al.

    Carbohydrate-active enzymes: an integrated database approach.

  • A.C. da Silva et al.

    Comparison of the genomes of two Xanthomonas pathogens with differing host specificities

    Nature

    (2002)
  • C. Dong et al.

    Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein

    Nature

    (2006)
  • G. Dunger et al.

    Xanthan is not essential for pathogenicity in citrus canker but contributes to Xanthomonas epiphytic survival

    Arch. Microbiol.

    (2007)
  • P.A. Frey

    The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose

    FASEB J.

    (1996)
  • D. Gordon et al.

    Consed: a graphical tool for sequence finishing

    Genome Res.

    (1998)
  • D. Gordon et al.

    Automated finishing with autofinish

    Genome Res.

    (2001)
  • B. Hotte et al.

    Cloning and analysis of a 35.3-kilobase DNA region involved in exopolysaccharide production by Xanthomonas campestris pv. campestris

    J. Bacteriol.

    (1990)
  • L. Ielpi et al.

    Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris

    J. Bacteriol.

    (1993)
  • F. Katzen et al.

    Promoter analysis of the Xanthomonas campestris pv. campestris gum operon directing biosynthesis of the xanthan polysaccharide

    J. Bacteriol.

    (1996)
  • F. Katzen et al.

    Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence

    J. Bacteriol.

    (1998)
  • R. Koplin et al.

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

    J. Bacteriol.

    (1992)
  • R. Koplin et al.

    A 3.9-kb DNA region of Xanthomonas campestris pv. campestris that is necessary for lipopolysaccharide production encodes a set of enzymes involved in the synthesis of dTDP-rhamnose

    J. Bacteriol.

    (1993)
  • H. Kitano et al.

    Using process diagrams for the graphical representation of biological networks

    Nat. Biotechnol.

    (2005)
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