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ABSTRACT: In the economically important phytopathogen, Pectobacterium atrosepticum, expression of plant cell wall degrading enzymes and other virulence determinants is controlled in a cell density-dependent fashion, termed quorum sensing (QS). Canonical QS systems in Gram-negative bacteria contain a LuxI-type protein, synthesizing a signalling molecule, and a LuxR-type regulator, responding to the signalling molecule above threshold concentrations. In P. atrosepticum, the central LuxR-type repressor of virulence, VirR, has been identified and its impacts on virulence characterized. Here we define the broader VirR regulon using chromatin immunoprecipitation (ChIP) and in planta microarrays. Ninety-four direct VirR targets were identified by ChIP microarrays and a consensus VirR binding site was determined. Purified VirR was used in DNA gel shift assays on target promoters and VirR : promoter binding was disrupted by exogenous addition of the signalling molecule, N-(3-oxohexanoyl)-l-homoserine lactone (OHHL). VirR autorepressed, and directly activated the transcription of rsmA in the absence of OHHL. Finally, we showed that VirR directly regulated the production of siderophores and controlled swimming motility. This is the first report characterizing the direct targets of VirR and provides clear evidence that this LuxR-type protein can act in vivo as both an activator and repressor of transcription in the absence of its cognate signalling molecule.
Environmental Microbiology 06/2012; · 5.84 Impact Factor
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Shihui Yang,
Quan Peng,
Qiu Zhang,
Lifang Zou,
Yan Li,
Christelle Robert,
Leighton Pritchard, Hui Liu,
Raymond Hovey,
Qi Wang,
Paul Birch,
Ian K Toth,
Ching-Hong Yang
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ABSTRACT: BACKGROUND: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. METHODOLOGY/PRINCIPAL FINDINGS: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens.
PLoS ONE 01/2010; 5(10):e13472. · 4.09 Impact Factor
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ABSTRACT: The objective of the project reported in the present chapter was the reverse engineering of gene regulatory networks related to quorum sensing in the plant pathogen Pectobacterium atrosepticum from micorarray gene expression profiles, obtained from the wild-type and eight knockout strains. To this end, we have applied various recent methods from multivariate statistics and machine learning: graphical Gaussian models, sparse Bayesian regression, LASSO (least absolute shrinkage and selection operator), Bayesian networks, and nested effects models. We have investigated the degree of similarity between the predictions obtained with the different approaches, and we have assessed the consistency of the reconstructed networks in terms of global topological network properties, based on the node degree distribution. The chapter concludes with a biological evaluation of the predicted network structures.
Methods in molecular biology (Clifton, N.J.) 01/2010; 673:253-81.
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ABSTRACT: Microarray comparative genomic hybridisation (aCGH) provides an estimate of the relative abundance of genomic DNA (gDNA) taken from comparator and reference organisms by hybridisation to a microarray containing probes that represent sequences from the reference organism. The experimental method is used in a number of biological applications, including the detection of human chromosomal aberrations, and in comparative genomic analysis of bacterial strains, but optimisation of the analysis is desirable in each problem domain.We present a method for analysis of bacterial aCGH data that encodes spatial information from the reference genome in a hidden Markov model. This technique is the first such method to be validated in comparisons of sequenced bacteria that diverge at the strain and at the genus level: Pectobacterium atrosepticum SCRI1043 (Pba1043) and Dickeya dadantii 3937 (Dda3937); and Lactococcus lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1363. In all cases our method is found to outperform common and widely used aCGH analysis methods that do not incorporate spatial information. This analysis is applied to comparisons between commercially important plant pathogenic soft-rotting enterobacteria (SRE) Pba1043, P. atrosepticum SCRI1039, P. carotovorum 193, and Dda3937.Our analysis indicates that it should not be assumed that hybridisation strength is a reliable proxy for sequence identity in aCGH experiments, and robustly extends the applicability of aCGH to bacterial comparisons at the genus level. Our results in the SRE further provide evidence for a dynamic, plastic 'accessory' genome, revealing major genomic islands encoding gene products that provide insight into, and may play a direct role in determining, variation amongst the SRE in terms of their environmental survival, host range and aetiology, such as phytotoxin synthesis, multidrug resistance, and nitrogen fixation.
PLoS Computational Biology 09/2009; 5(8):e1000473. · 5.22 Impact Factor
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Hui Liu,
Sarah J Coulthurst,
Leighton Pritchard,
Peter E Hedley,
Michael Ravensdale,
Sonia Humphris,
Tom Burr,
Gunnhild Takle,
May-Bente Brurberg,
Paul R J Birch,
George P C Salmond,
Ian K Toth
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ABSTRACT: Quorum sensing (QS) in vitro controls production of plant cell wall degrading enzymes (PCWDEs) and other virulence factors in the soft rotting enterobacterial plant pathogen Pectobacterium atrosepticum (Pba). Here, we demonstrate the genome-wide regulatory role of QS in vivo during the Pba-potato interaction, using a Pba-specific microarray. We show that 26% of the Pba genome exhibited differential transcription in a QS (expI-) mutant, compared to the wild-type, suggesting that QS may make a greater contribution to pathogenesis than previously thought. We identify novel components of the QS regulon, including the Type I and II secretion systems, which are involved in the secretion of PCWDEs; a novel Type VI secretion system (T6SS) and its predicted substrates Hcp and VgrG; more than 70 known or putative regulators, some of which have been demonstrated to control pathogenesis and, remarkably, the Type III secretion system and associated effector proteins, and coronafacoyl-amide conjugates, both of which play roles in the manipulation of plant defences. We show that the T6SS and a novel potential regulator, VirS, are required for full virulence in Pba, and propose a model placing QS at the apex of a regulatory hierarchy controlling the later stages of disease progression in Pba. Our findings indicate that QS is a master regulator of phytopathogenesis, controlling multiple other regulators that, in turn, co-ordinately regulate genes associated with manipulation of host defences in concert with the destructive arsenal of PCWDEs that manifest the soft rot disease phenotype.
PLoS Pathogens 07/2008; 4(6):e1000093. · 9.13 Impact Factor
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ABSTRACT: Erwinia carotovora subsp. atroseptica is an enterobacterial phytopathogen causing economically significant soft rot disease. Pathogenesis is mediated by multiple secreted virulence factors, many of which are secreted by the type II (Out) secretion system. DsbA catalyzes the introduction of disulfide bonds into periplasmic and secreted proteins. In this study, the extracellular proteome (secretome) of wild type E. carotovora subsp. atroseptica SCRI1043, and dsbA and out mutants, was analyzed by spectral counting mass spectrometry. This revealed that dsbA inactivation had a huge impact on the secretome and identified diverse DsbA- and Out-dependent secreted proteins, representing known, predicted, and novel candidate virulence factors. Further characterization of the dsbA mutant showed that secreted enzyme activities, motility, production of the quorum-sensing signal, and virulence were absent or substantially reduced. The impact of DsbA on secreted virulence factor production was mediated at multiple levels, including impacting on the Out secretion system and the virulence gene regulatory network. Transcriptome analyses revealed that the abundance of a broad, but defined, set of transcripts, including many virulence factors, was altered in the dsbA mutant, identifying a new virulence regulon responsive to extracytoplasmic conditions. In conclusion, DsbA plays a crucial, multifaceted role in the pathogenesis of E. carotovora subsp. atroseptica.
Journal of Biological Chemistry 07/2008; 283(35):23739-53. · 4.77 Impact Factor
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Ramani S Ravirala,
Ravi D Barabote,
David M Wheeler,
Sylvie Reverchon,
Ouwatha Tatum,
Jeremy Malouf, Hui Liu,
Leighton Pritchard,
Peter E Hedley,
Paul R J Birch,
Ian K Toth,
Paxton Payton,
Michael J D San Francisco
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ABSTRACT: Salicylic acid (SA) is an important signaling molecule in local and systemic plant resistance. Following infection by microbial pathogens and the initial oxidative burst in plants, SA accumulation functions in the amplification of defense gene expression. Production of pathogenesis-related proteins and toxic antimicrobial chemicals serves to protect the plant from infection. Successful microbial pathogens utilize a variety of mechanisms to rid themselves of toxic antimicrobial compounds. Important among these mechanisms are multidrug-resistance pumps that bring about the active efflux of toxic compounds from microbial cells. Here, we show that a combination SA and its precursors, t-cinnamic acid and benzoic acid, can activate expression of specific multidrug efflux pump-encoding genes in the plant pathogen Erwinia chrysanthemi and enhance survival of the bacterium in the presence of model as well as plant-derived antimicrobial chemicals. This ability of plant-pathogenic bacteria to co-opt plant defense-signaling molecules to activate multidrug efflux pumps may have evolved to ensure bacterial survival in susceptible host plants.
Molecular Plant-Microbe Interactions 04/2007; 20(3):313-20. · 4.43 Impact Factor
