Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris

Sidhu VK, Vorhölter F-J, Niehaus K, Watt SA. Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris. BMC Microbiology. 2008;8(1):87.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 influence of a modified lipopolysaccharide O-antigen on the biosynthesis of xanthan in Xanthomonas campestris pv. campestris B100

Steffens T, Vorhölter F-J, Giampa M, Hublik G, Pühler A, Niehaus K. The influence of a modified lipopolysaccharide O-antigen on the biosynthesis of xanthan in Xanthomonas campestris pv. campestris B100. BMC Microbiology. 2016;16(1): 93.Background The exopolysaccharide xanthan is a natural product which is extensively used in industry. It is a thickening agent in many fields, from oil recovery to the food sector. Xanthan is produced by the Gram negative bacterium Xanthomonas campestris pv. campestris (Xcc). We analyzed the lipopolysaccharide (LPS) of three mutant strains of the Xcc wild type B100 to distinguish if the xanthan production can be increased when LPS biosynthesis is affected. Results The Xcc B100 O-antigen (OA) is composed of a linear main chain of rhamnose residues with N-acetylfucosamine (FucNAc) side branches at every second rhamnose. It is the major LPS constituent. The O-antigen was missing completely in the mutant strain H21012 (deficient in wxcB), since neither rhamnose nor FucNAc could be detected as part of the LPS by MALDI-TOF-MS, and only a slight amount of rhamnose and no FucNAc was found by GC analysis. The LPS of two other mutants was analyzed, Xcc H28110 (deficient in wxcK) and H20110 (wxcN). In both of them no FucNAc could be detected in the LPS fraction, while the rhamnose moieties were more abundant than in wild type LPS. The measurements were carried out by GC and confirmed by MALDI-TOF-MS analyses that indicated an altered OA in which the branches are missing, while the rhamnan main chain seemed longer than in the wild type. Quantification of xanthan confirmed our hypothesis that a missing OA can lead to an increased production of the extracellular polysaccharide. About 6.3 g xanthan per g biomass were produced by the Xcc mutant H21012 (wxcB), as compared to the wild type production of approximately 5 g xanthan per g biomass. In the two mutant strains with modified OA however, Xcc H28110 (wxcK) and Xcc H20110 (wxcN), the xanthan production of 5.5 g and 5.3 g, respectively, was not significantly increased. Conclusions Mutations affecting LPS biosynthesis can be beneficial for the production of the extracellular polysaccharide xanthan. However, only complete inhibition of the OA resulted in increased xanthan production. The inhibition of the FucNAc side branches did not lead to increased production, but provoked a novel LPS phenotype. The data suggests an elongation of the linear rhamnan main chain of the LPS OA in both the Xcc H28110 (wxcK) and Xcc H20110 (wxcN) mutant strains

Gene discovery by genome-wide CDS re-prediction and microarray-based transcriptional analysis in phytopathogen Xanthomonas campestris

Zhou L, Vorhölter F-J, He Y-Q, et al. Gene discovery by genome-wide CDS re-prediction and microarray-based transcriptional analysis in phytopathogen Xanthomonas campestris. BMC Genomics. 2011;12(1): 359.ABSTRACT: One of the major tasks of the post-genomic era is "reading" genomic sequences in order to extract all the biological information contained in them. Although a wide variety of techniques is used to solve the gene finding problem and a number of prokaryotic gene-finding software are available, gene recognition in bacteria is far from being always straightforward. This study reported a thorough search for new CDS in the two published Xcc genomes. In the first, putative CDSs encoded in the two genomes were re-predicted using three gene finders, resulting in the identification of 2850 putative new CDSs. In the second, similarity searching was conducted and 278 CDSs were found to have homologs in other bacterial species. In the third, oligonucleotide microarray and RT-PCR analysis identified 147 CDSs with detectable mRNA transcripts. Finally, in-frame deletion and subsequent phenotype analysis of confirmed that Xcc_CDS002 encoding a novel SIR2-like domain protein is involved in virulence and Xcc_CDS1553 encoding a ArsR family transcription factor is involved in arsenate resistance. Despite sophisticated approaches available for genome annotation, many cellular transcripts have remained unidentified so far in Xcc genomes. Through a combined strategy involving bioinformatic, postgenomic and genetic approaches, a reliable list of 306 new CDSs was identified and a more thorough understanding of some cellular processes was gained

Complete Genome Sequence of the Barley Pathogen Xanthomonas translucens pv. translucens DSM 18974 T (ATCC 19319 T)

Jaenicke S, Bunk B, Wibberg D, et al. Complete Genome Sequence of the Barley Pathogen Xanthomonas translucens pv. translucens DSM 18974 T (ATCC 19319 T). Genome Announcements. 2016;4(6): e01334-16.We report here the complete 4.7-Mb genome sequence of Xanthomonas translucens pv. translucens DSM 18974T, which causes black chaff disease on barley (Hordeum vulgare). Genome data of this X. translucens type strain will improve our understanding of this bacterial species

EDGAR: A software framework for the comparative analysis of prokaryotic genomes

Blom J, Albaum S, Doppmeier D, et al. EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinformatics. 2009;10(1): 154.Background:The introduction of next generation sequencing approaches has caused a rapid increase in the number of completely sequenced genomes. As one result of this development, it is now feasible to analyze large groups of related genomes in a comparative approach. A main task in comparative genomics is the identification of orthologous genes in different genomes and the classification of genes as core genes or singletons. Results: To support these studies EDGAR – ''Efficient Database framework for comparative Genome Analyses using BLAST score Ratios'' – was developed. EDGAR is designed to automatically perform genome comparisons in a high throughput approach. Comparative analyses for 582 genomes across 75 genus groups taken from the NCBI genomes database were conducted with the software and the results were integrated into an underlying database. To demonstrate a specific application case, we analyzed ten genomes of the bacterial genus Xanthomonas, for which phylogenetic studies were awkward due to divergent taxonomic systems. The resultant phylogeny EDGAR provided was consistent with outcomes from traditional approaches performed recently and moreover, it was possible to root each strain with unprecedented accuracy. Conclusion: EDGAR provides novel analysis features and significantly simplifies the comparative analysis of related genomes. The software supports a quick survey of evolutionary relationships and simplifies the process of obtaining new biological insights into the differential gene content of kindred genomes. Visualization features, like synteny plots or Venn diagrams, are offered to the scientific community through a web-based and therefore platform independent user interface http://edgar.cebitec.uni-bielefeld.de webcite, where the precomputed data sets can be browsed

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae

Gross R, Guzman CA, Sebaihia M, et al. The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae. BMC Genomics. 2008;9(1): 449.Background: Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. Results: In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. Conclusion: The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches

RNA-Seq facilitates a new perspective on signal transduction and gene regulation in important plant pathogens

Vorhölter F-J. RNA-Seq facilitates a new perspective on signal transduction and gene regulation in important plant pathogens. Molecular Microbiology. 2013;88(6):1041-1046.RNA-Seq is opening new doors for the functional understanding of microorganisms. Advances in RNA-Seq technology are allowing investigators to focus their studies on specific functional questions. An interesting example is presented by An etal. (2013) in this issue of Molecular Microbiology. New genes were identified for proteins and ncRNAs when the authors concentrated on the role of the rpf genes, which code for key components of a signal transduction hub in the plant pathogen Xanthomonas campestris pv. campestris. Although rpf gene products were already known to be involved in controlling transcription of many genes, including those encoding several important virulence factors, novel and unexpected properties of this signal transduction system emerged from the RNA-Seq analysis. In addition to identifying new target genes influenced by the rpf genes, the study found that the regulons of RpfC and RpfG, the sensor and response regulator of the master two-component regulatory system, only partially overlapped, indicating that the Rpf signalling system is even more complex than previously appreciated

Analyse und Charakterisierung einer 35 Kilobasen großen Genregion aus Xanthomonas campestris pathovar campestris, deren Gene vorwiegend an der Biosynthese des Lipopolysaccharids beteiligt sind

Vorhölter F-J. Analyse und Charakterisierung einer 35 Kilobasen großen Genregion aus Xanthomonas campestris pathovar campestris, deren Gene vorwiegend an der Biosynthese des Lipopolysaccharids beteiligt sind. Bielefeld; 2001

Lipopolysaccharide biosynthesis in Xanthomonas campestris pv. campestris: a cluster of 15 genes is involved in the biosynthesis of the LPS O-antigen and the LPS core

Vorhölter F-J, Niehaus K, Pühler A. Lipopolysaccharide biosynthesis in Xanthomonas campestris pv. campestris: a cluster of 15 genes is involved in the biosynthesis of the LPS O-antigen and the LPS core. MOLECULAR GENETICS AND GENOMICS. 2001;266(1):79-95.As a result of mutational and DNA sequence analysis, a wxc gene cluster involved in the synthesis of the surface lipopolysaccharide (LPS) was identified in Xanthomonas campestris pv. campestris. This gene cluster comprises 15 genes. It was located on a cloned 35-kb fragment of chromosomal DNA, close, but not directly adjacent, to previously characterized genes for LPS biosynthesis. The G + C content of all but one of the it we genes was atypically low for X. campestris pv. campestris, while the G + C distribution was uniform throughout the cluster. An SDS-PAGE analysis of mutant strains defective in various wxc genes confirmed that genes from this cluster were involved in LPS biosynthesis. The mutant phenotypes allowed the differentiation of three regions within the wxc cluster. Genes from wxc region 1 are necessary for the biosynthesis of the water-soluble LPS O-antigen. Analysis of DNA and deduced amino acid sequences led to the identification of two glycosyltransferases, two components of an ABC transport system, and a possible kinase among the seven putative proteins encoded by genes constituting wxc region 1. The two genes in wxc region 2 were similar to gmd and rmd, which direct the synthesis of the sugar nucleotide GDP-D-rhamnose. Mutations affecting wxc region 2 demonstrated its involvement in the formation of the LPS core. Genes from wxc region 3 showed similarities to genes that code for enzymes that modify nucleotide sugars, and to components of sugar translocation systems that have so far been rarely described in bacteria