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The γ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) B100 synthesizes the polysaccharide xanthan, a commercially important viscosifier. Since the complete genome of Xcc B100 is available, systems biology tools were applied to obtain a deeper understanding of the metabolism involved in xanthan biosynthesis. A large-scale metabolic network was reconstructed and manually curated. The reconstructed network included 352 genes, 437 biochemical reactions, 10 transport reactions, and 338 internal metabolites. To use this network for flux balance analysis, the biomass composition of Xcc B100 was determined. The comprehensive model obtained was applied for in silico analyses to predict biomass generation and gene essentiality. Predictions were extensively validated by analyzing batch culture performance and by carbon balancing including xanthan production. Single gene deletion mutants causing deficiencies in the central carbohydrate metabolism were constructed to enforce major flux redistributions. The impact of xanthan production was studied in vivo and in silico, comparing the physiology of a gumD mutant, negative in xanthan production, with the original strain. The results indicate a redistribution of resources from xanthan to biomass, rather than a reduction in carbon uptake. With this high quality metabolic model, both systems biology analyses and synthetic biology reengineering of Xcc gained an important tool.

Xanthomonas campestris pathovar campestris (Xcc) is a plant pathogenic bacterium and as such has to adapt to a variety of environments. During the course of disease, Xcc colonizes the Surface of its host, infects the xylem in the early stages, and develops a fully saprophytic life-style, aided by secreted degradative enzymes, in the late stages. To get some insight into this complex regulation, Xcc was cultivated in the presence of low molecular weight host plant extract (

The extracellular proteome of Xanthomonas campestris pv. campestris (Xcc) cultivated in minimal medium was isolated from the cell-free culture supernatant and separated by two-dimensional gel electrophoresis. This technique resolved 97 clearly visible protein spots, which were excised, digested with trypsin and identified on the basis of their peptide mass fingerprints generated by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry. Using this approach 87 different proteins could be distinguished. The Signal P software predicted putative signal peptides for 53% of the extracellular proteins. These proteins are probably transported over the inner membrane and are localized in the periplasm, the outer membrane or secreted into the extracellular space. Among the secreted proteins are 11 degradative enzymes, which are involved in pathogenesis of Xcc. The proteins without obvious secretion signals are known to serve functions in the cytosol. How the cytosolic proteins are delivered to the extracellular space remains unclear.

Burkholderia cenocepacia produces a diffusible fatty acid signal molecule, cis -2-dodecenoic acid (BDSF), that has been implicated in interspecies and interkingdom communication. Here, we show that BDSF also acts as an intraspecies signal in B. cenocepacia to control factors contributing to virulence of this major opportunistic pathogen.

Interspecies signalling through the action of diffusible signal molecules can influence the behaviour of organisms growing in polymicrobial communities. Stenotrophomonas maltophilia and Pseudomonas aeruginosa occur ubiquitously in the environment and can be found together in diverse niches including the rhizosphere of plants and the cystic fibrosis lung. In mixed species biofilms, S. maltophilia substantially influenced the architecture of P. aeruginosa structures, which developed as extended filaments. This effect depended upon the synthesis of the diffusible signal factor (DSF) by S. maltophilia and could be mimicked by the addition of synthetic DSF. This response of P. aeruginosa to DSF required PA1396, a sensor kinase with an input domain of related amino acid sequence to the sensory input domain of RpfC, which is responsible for DSF perception in xanthomonads. Mutation of PA1396 or addition of DSF to P. aeruginosa led to increased levels of a number of proteins with roles in bacterial stress tolerance, including those implicated in resistance to cationic antimicrobial peptides. This effect was associated with increased tolerance to polymyxins. Homologues of PA1396 occur in a number of phytopathogenic and plant-associated pseudomonads, suggesting that modulation of bacterial behaviour through DSF-mediated interspecies signalling with xanthomonads is a phenomenon that occurs widely.

Background Outer membrane vesicles (OMVs) are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli. Results We have demonstrated that Xanthomonas campestris pv. campestris (Xcc) releases OMVs into the culture supernatant during growth. A proteome study identified 31 different proteins that associate with the OMV fraction of which half are virulence-associated. A comparison with the most abundant outer membrane (OM) proteins revealed that some proteins are enriched in the OMV fraction. This may be connected to differences in the LPS composition between the OMVs and the OM. Furthermore, a comparison of the OMV proteomes from two different culture media indicated that the culture conditions have an impact on the protein composition. Interestingly, the proteins that are common to both culture conditions are mainly involved in virulence. Conclusion Outer membrane vesicles released from the OM of Xcc contain membrane- and virulence-associated proteins. Future experiments will prove whether these structures can serve as "vehicles" for the transport of virulence factors into the host membrane.

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.

Three of the most abundant proteins (OmpW, MopB and SodM) of the extracellular proteome of Xanthomonas campestris pv. campestris were analysed in a luminol-based oxidative burst assay to identify novel pathogen-associated molecular patterns (PAMP). Tobacco cell suspension cultures were used as a model system to monitor elicitor induced plant defence reaction. The candidate proteins were isolated from two-dimensional gels prior to application to the oxidative burst assay. The superoxide dismutase (SodM) was the only isolated protein that could elicit a notable hydrogen peroxide (H2O2) production in tobacco cell cultures indicating the initiation of plant defence. An alignment of the SodM sequences from X. campestris pv. campestris and Escherichia coli revealed 55.7% identity and 29% of the sequence were substitutions for amino acids with similar physicochemical properties. By using a commercially available purified E coli derived SodM preparation, it was possible to show that the amino acid sequence of this protein is responsible for the elicitation of an oxidative burst reaction in the tobacco cell culture model. This suggests that the bacterial superoxide dismutase is a novel pathogen-associated molecular pattern. The minimal elicitor active sequence, however, is still elusive. (c) 2006 Elsevier B.V. All rights reserved.

The chemically-coded affinity tag (CCAT) method combines standard electrophoresis protocols with MALDI-TOF-MS analysis to identify and quantify protein abundances in complex samples in one step. This method is designed to fit into the workflow of SDS-PAGE or two-dimensional electrophoresis (2-DE) only requiring basic proteome laboratory equipment. Prior to electrophoresis two protein samples are separately labelled with a heavy or a light version of the CCAT reagent via reduced cysteines in the proteins. Equal amounts are then combined and electrophoretically separated. Proteins can then be excised from the gel to obtain their peptide mass fingerprint by mass spectrometry. This fingerprint enabled not only identification, but also quantification by comparing relative peak intensities of CCAT-labelled peptides. In this article, we display how the CCAT method can be used to analyse two protein samples in one gel and that the peak intensities of labelled peptides reflect the abundance of a protein in it. (C) 2003 Published by Elsevier B.V.