Evolution of the metabolic and regulatory networks associated with oxygen availability in two phytopathogenic enterobacteria

<p>Abstract</p> <p>Background</p> <p><it>Dickeya dadantii </it>and <it>Pectobacterium atrosepticum </it>are phytopathogenic enterobacteria capable of facultative anaerobic growth in a wide range of O₂concentrations found in plant and natural environments. The transcriptional response to O₂remains under-explored for these and other phytopathogenic enterobacteria although it has been well characterized for animal-associated genera including <it>Escherichia coli </it>and <it>Salmonella enterica</it>. Knowledge of the extent of conservation of the transcriptional response across orthologous genes in more distantly related species is useful to identify rates and patterns of regulon evolution. Evolutionary events such as loss and acquisition of genes by lateral transfer events along each evolutionary branch results in lineage-specific genes, some of which may have been subsequently incorporated into the O₂-responsive stimulon. Here we present a comparison of transcriptional profiles measured using densely tiled oligonucleotide arrays for two phytopathogens, <it>Dickeya dadantii </it>3937 and <it>Pectobacterium atrosepticum </it>SCRI1043, grown to mid-log phase in MOPS minimal medium (0.1% glucose) with and without O₂.</p> <p>Results</p> <p>More than 7% of the genes of each phytopathogen are differentially expressed with greater than 3-fold changes under anaerobic conditions. In addition to anaerobic metabolism genes, the O₂responsive stimulon includes a variety of virulence and pathogenicity-genes. Few of these genes overlap with orthologous genes in the anaerobic stimulon of <it>E. coli</it>. We define these as the conserved core, in which the transcriptional pattern as well as genetic architecture are well preserved. This conserved core includes previously described anaerobic metabolic pathways such as fermentation. Other components of the anaerobic stimulon show variation in genetic content, genome architecture and regulation. Notably formate metabolism, nitrate/nitrite metabolism, and fermentative butanediol production, differ between <it>E. coli </it>and the phytopathogens. Surprisingly, the overlap of the anaerobic stimulon between the phytopathogens is also relatively small considering that they are closely related, occupy similar niches and employ similar strategies to cause disease. There are cases of interesting divergences in the pattern of transcription of genes between <it>Dickeya </it>and <it>Pectobacterium </it>for virulence-associated subsystems including the type VI secretion system (T6SS), suggesting that fine-tuning of the stimulon impacts interaction with plants or competing microbes.</p> <p>Conclusions</p> <p>The small number of genes (an even smaller number if we consider operons) comprising the conserved core transcriptional response to O₂limitation demonstrates the extent of regulatory divergence prevalent in the Enterobacteriaceae. Our orthology-driven comparative transcriptomics approach indicates that the adaptive response in the eneterobacteria is a result of interaction of core (regulators) and lineage-specific (structural and regulatory) genes. Our subsystems based approach reveals that similar phenotypic outcomes are sometimes achieved by each organism using different genes and regulatory strategies.</p

Proteomanalysen von Blattperoxisomen aus Spinacia oleracea L. and Arabidopsis thaliana (L.) Heynh.

Im Rahmen der vorliegenden Arbeit wurde eine Proteomanalyse von pflanzlichen Peroxisomen begonnen, um unser Verständnis der Funktionen dieser ubiquitären eukaryotischen Zellorganellen zu erweitern. Ein überlieferte Methode zur Isolierung von Blattperoxisomen aus Spinacia oleracea L. wurde durch Ergänzung eines zweiten Saccharose-Dichtegradienten verbessert und führte zur Reduktion der Kontamination von Blattperoxisomen durch Chloroplasten und Mitochondrien auf ein Minimum, während ein neuartigen Typ Proplastiden-ähnlicher Organellen weiterhin detektierbar war. Alternativ wurde für vergleichende Proteomanalysen von gestressten Pflanzen eine Methode von minimalem Zeitumfang entwickelt. Um die Information der ersten pflanzlichen Genomsequenz, der von Arabidopsis thaliana L., für Proteomanalysen auszunutzen, wurde eine neue effiziente Methode zur Anreicherung von Blattperoxisomen entwickelt.Die Methode der zwei-dimensionalen Gelelektrophorese wurde für lösliche Matrixproteine bezüglich der Auswahl und Konzentration chaotroper Reagenzien, Detergenzien und Alkohole optimiert, um maximale Auflösung und Proteinausbeute zu erzielen. Die löslichen Matrixproteine gereinigter Blattperoxisomen wurden durch zwei-dimensionale Gelelektrophorese gefolgt von Massenspektrometrie (MALDI and ESI-MS/MS) charakterisiert. Über etliche bekannte Proteine pflanzlicher Peroxisomen hinaus, beispielsweise die in Photorespiration, Fettsäure-beta-Oxidation und den Metabolism reaktiver Sauerstoffspezies involvierten Enzyme, wurde einige neue Proteine identifiziert, und zwar eine kurzkettige Alkoholdehydrogenase, ein Naphthoat-Synthase-ähnliches Protein, eine Enoyl-CoA-Hydratase/Isomerase und ein kleines Hitzeschockprotein. Alle vier unbekannten Proteine besitzen ein peroxisomales Zielsteuerungssignal des Typs 1 oder 2, welches in homologen pflanzlichen cDNAs konserviert ist. Die subzelluläre Lokalisation der neuen Proteine wurde durch vergleichende 2D-Gelelektrophorese verifiziert. Die Gene von Arabidopsis, die für das Naphthoat-Synthase-ähnliche Protein und das kleine Hitzeschockprotein kodieren, wurde über Reverse Transkriptase Polymerase-Kettenreaktion von Kälte-behandelten Blättern kloniert und erlauben weitere Untersuchungen zur subzellulären Zielsteuerung sowie funktionelle Analysen.Post-translationale Modifikationen wie Proteinphosphorylierungen scheinen eine verbreitete Eigenschaft mehrerer Peroxisomenproteine wie Katalase, Hydroxypyruvat-Reduktase, Ascorbat-Peroxisdase und Isocitrat-Lyase zu sein. Einige neue Proteine bzw. Isoformen von Blattperoxisomen scheinen ausserdem unter spezifischen Stressbedingungen wie durch oxidativen Stress und unter Hochlicht induziert zu werden und eine wichtige aber bislang völlig unbekannte Rolle in der pflanzlichen Stresstoleranz zu spielen

Ralstonia solanacearum Extracellular Polysaccharide Is a Specific Elicitor of Defense Responses in Wilt-Resistant Tomato Plants

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps 2 mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps 2 mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps 2 mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest tha

Identification and Characterization of a Stress-Inducible and a Constitutive Small Heat-Shock Protein Targeted to the Matrix of Plant Peroxisomes

Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL>) and a functional PTS2 (RLx(5)HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one- and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions

Sequencing of K60, Type Strain of the Major Plant Pathogen Ralstonia solanacearum

International audienc

Comparative Transcriptome Analysis Reveals Cool Virulence Factors of <i>Ralstonia solanacearum</i> Race 3 Biovar 2

<div><p>While most strains of the plant pathogenic bacterium <i>Ralstonia solanacearum</i> are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of <i>R</i>. <i>solanacearum</i> R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature <i>in planta</i>. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking <i>lecM</i>, <i>aidA</i>, or <i>aidC</i> were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The <i>lecM</i> and <i>aidC</i> mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the <i>lecM</i> mutant was defective in biofilm formation <i>in vitro</i>. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.</p></div

UW551ΔlecM and UW551ΔaidC had reduced survival in potato tubers at 4°C.

<p>Potato tubers were injected with different <i>R</i>. <i>solanacearum</i> strains, and bacterial cell numbers were counted by grinding and dilution plating tubers at different times after inoculation. The experiment was repeated three times, with three tubers per strain per time point. At 6, 9 and 12 weeks after inoculation, the population sizes of UW551ΔlecM and UW551ΔaidC in tubers were significantly lower than those of the wild type parent strain (<i>P</i><0.05, ANOVA).</p

Transcriptome Changes Associated with Anaerobic Growth in <i>Yersinia intermedia</i> (ATCC29909)

<div><p>Background</p><p>The yersiniae (Enterobacteriaceae) occupy a variety of niches, including some in human and flea hosts. Metabolic adaptations of the yersiniae, which contribute to their success in these specialized environments, remain largely unknown. We report results of an investigation of the transcriptome under aerobic and anaerobic conditions for <i>Y. intermedia</i>, a non-pathogenic member of the genus that has been used as a research surrogate for <i>Y. pestis</i>. <i>Y. intermedia</i> shares characteristics of pathogenic yersiniae, but is not known to cause disease in humans. Oxygen restriction is an important environmental stimulus experienced by many bacteria during their life-cycles and greatly influences their survival in specific environments. How oxygen availability affects physiology in the yersiniae is of importance in their life cycles but has not been extensively characterized.</p> <p>Methodology/Principal Findings</p><p>Tiled oligonucleotide arrays based on a draft genome sequence of <i>Y. intermedia</i> were used in transcript profiling experiments to identify genes that change expression in response to oxygen availability during growth in minimal media with glucose. The expression of more than 400 genes, constituting about 10% of the genome, was significantly altered due to oxygen-limitation in early log phase under these conditions. Broad functional categorization indicated that, in addition to genes involved in central metabolism, genes involved in adaptation to stress and genes likely involved with host interactions were affected by oxygen-availability. Notable among these, were genes encoding functions for motility, chemotaxis and biosynthesis of cobalamin, which were up-regulated and those for iron/heme utilization, methionine metabolism and urease, which were down-regulated.</p> <p>Conclusions/Significance</p><p>This is the first transcriptome analysis of a non-pathogenic <i>Yersinia</i><i>spp.</i> and one of few elucidating the global response to oxygen limitation for any of the yersiniae. Thus this study lays the foundation for further experimental characterization of oxygen-responsive genes and pathways in this ecologically diverse genus.</p> </div