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Research in Microbiology 2015We previously showed that SlyA of Dickeya dadantii 3937 plays an important role in virulence toward plants, and that the ΔslyA mutant is hypermotile, whereas flagellum...
We previously showed that SlyA of Dickeya dadantii 3937 plays an important role in virulence toward plants, and that the ΔslyA mutant is hypermotile, whereas flagellum synthesis and flagellin production are indistinguishable from the wild type. Here we show that motility factors, including the distance of continuous directed movement, time for that movement and speed, were significantly higher in the ΔslyA mutant than in the wild type. Remarkably, transcription levels of motA and motB, that are involved in flagellar rotation, were elevated in the ΔslyA mutant, suggesting that the mutant's hypermotility was due to an increase in flagellar rotation. In low (10 μM) magnesium medium that activates the PhoP-PhoQ system, growth and virulence of the ΔslyA mutant were much lower than for the wild type; expression of motA, motB, mgtA, pelA, pelB, pelC, pelD, pelE, pelI, indA, tolC, sodC, acsA and hrpN were also reduced in the mutant. Interestingly, motA, motB, pelD, pelE, pelI, sodC and indA were also reduced in phoP and phoQ mutants. Because the SlyA protein directly binds to the promoter region of PhoP, SlyA regulates virulence by controlling multiple pathogenicity-related genes directly and/or at least by controlling PhoP in D. dadantii 3937 when magnesium is low.
Topics: Bacterial Proteins; Enterobacteriaceae; Flagella; Gene Expression Regulation, Bacterial; Magnesium Sulfate; Mutation; Stress, Physiological; Transcription, Genetic; Virulence
PubMed: 26027774
DOI: 10.1016/j.resmic.2015.05.004 -
MBio Apr 2015Recent studies strongly suggest that the gene expression sustaining both normal and pathogenic bacterial growth is governed by the structural dynamics of the chromosome....
UNLABELLED
Recent studies strongly suggest that the gene expression sustaining both normal and pathogenic bacterial growth is governed by the structural dynamics of the chromosome. However, the mechanistic device coordinating the chromosomal configuration with selective expression of the adaptive traits remains largely unknown. We used a holistic approach exploring the inherent relationships between the physicochemical properties of the DNA and the expression of adaptive traits, including virulence factors, in the pathogen Dickeya dadantii (formerly Erwinia chrysanthemi). In the transcriptomes obtained under adverse conditions encountered during bacterial infection, we explored the patterns of chromosomal DNA sequence organization, supercoil dynamics, and gene expression densities, together with the long-range regulatory impacts of the abundant DNA architectural proteins implicated in pathogenicity control. By integrating these data, we identified transient chromosomal domains of coherent gene expression featuring distinct couplings between DNA thermodynamic stability, supercoil dynamics, and virulence traits.
IMPORTANCE
We infer that the organization of transient chromosomal domains serving specific functions acts as a fundamental device for versatile adjustment of the pathogen to environmental stress. We believe that the identification of chromosomal "stress-response" domains harboring distinct virulence traits and mediating the cellular adaptive behavior provides a breakthrough in understanding the control mechanisms of bacterial pathogenicity.
Topics: Bacterial Proteins; Chromosomes, Bacterial; DNA, Bacterial; DNA, Superhelical; Dickeya chrysanthemi; Gene Expression Regulation, Bacterial; Genes, Bacterial; Lamiales; Stress, Physiological; Transcriptome; Virulence; Virulence Factors
PubMed: 25922390
DOI: 10.1128/mBio.00353-15 -
Environmental Microbiology Nov 2015The CpxAR two-component system is present in many Proteobacteria. It controls expression of genes required to maintain envelope integrity in response to environmental...
The CpxAR two-component system is present in many Proteobacteria. It controls expression of genes required to maintain envelope integrity in response to environmental injury. Consequently, this two-component system was shown to be required for virulence of several zoo-pathogens, but it has never been investigated in phyto-pathogens. In this paper, we investigate the role of the CpxAR two-component system in vitro and in vivo in Dickeya dadantii, an enterobacterial phytopathogen that causes soft-rot disease in a large variety of plant species. cpxA null mutant displays a constitutively phosphorylated CpxR phenotype as shown by direct analysis of phosphorylation of CpxR by a Phos-Tag retardation gel approach. Virulence in plants is completely abolished in cpxA or cpxR mutants of D. dadantii. In planta, CpxAR is only activated at an early stage of the infection process as shown by Phos-Tag and gene fusion analyses. To our knowledge, this is the first time that the timing of CpxAR phosphorelay activation has been investigated during the infection process by direct monitoring of response regulator phosphorylation.
Topics: Bacterial Proteins; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Phosphorylation; Plant Diseases; Plants; Protein Kinases; Pyridines; Virulence
PubMed: 25856505
DOI: 10.1111/1462-2920.12874 -
The Plant Journal : For Cell and... Apr 2015Transcriptome analysis of bacterial pathogens is a powerful approach to identify and study the expression patterns of genes during host infection. However, analysis of...
Transcriptome analysis of bacterial pathogens is a powerful approach to identify and study the expression patterns of genes during host infection. However, analysis of the early stages of bacterial virulence at the genome scale is lacking with respect to understanding of plant-pathogen interactions and diseases, especially during foliar infection. This is mainly due to both the low ratio of bacterial cells to plant material at the beginning of infection, and the high contamination by chloroplastic material. Here we describe a reliable and straightforward method for bacterial cell purification from infected leaf tissues, effective even if only a small amount of bacteria is present relative to plant material. The efficiency of this method for transcriptomic analysis was validated by analysing the expression profiles of the phytopathogenic enterobacterium Dickeya dadantii, a soft rot disease-causing agent, during the first hours of infection of the model host plant Arabidopsis thaliana. Transcriptome profiles of epiphytic bacteria and bacteria colonizing host tissues were compared, allowing identification of approximately 100 differentially expressed genes. Requiring no specific equipment, cost-friendly and easily transferable to other pathosystems, this method should be of great interest for many other plant-bacteria interaction studies.
Topics: Arabidopsis; Enterobacteriaceae; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Plant Diseases; Virulence
PubMed: 25740271
DOI: 10.1111/tpj.12812 -
Scientific Reports Mar 2015In the track of new biopesticides, four genes namely cytA, cytB, cytC and cytD encoding proteins homologous to Bacillus thuringiensis (Bt) Cyt toxins have been...
In the track of new biopesticides, four genes namely cytA, cytB, cytC and cytD encoding proteins homologous to Bacillus thuringiensis (Bt) Cyt toxins have been identified in the plant pathogenic bacteria Dickeya dadantii genome. Here we show that three Cyt-like δ-endotoxins from D. dadantii (CytA, CytB and CytC) are toxic to the pathogen of the pea aphid Acyrthosiphon pisum in terms of both mortality and growth rate. The phylogenetic analysis of the comprehensive set of Cyt toxins available in genomic databases shows that the whole family is of limited taxonomic occurrence, though in quite diverse microbial taxa. From a structure-function perspective the 3D structure of CytC and its backbone dynamics in solution have been determined by NMR. CytC adopts a cytolysin fold, structurally classified as a Cyt2-like protein. Moreover, the identification of a putative lipid binding pocket in CytC structure, which has been probably maintained in most members of the Cyt-toxin family, could support the importance of this lipid binding cavity for the mechanism of action of the whole family. This integrative approach provided significant insights into the evolutionary and functional history of D. dadantii Cyt toxins, which appears to be interesting leads for biopesticides.
Topics: Amino Acid Sequence; Bacterial Proteins; Endotoxins; Enterobacteriaceae; Models, Molecular; Molecular Sequence Data; Multigene Family; Nuclear Magnetic Resonance, Biomolecular; Phylogeny; Protein Conformation; Sequence Alignment; Solutions
PubMed: 25740111
DOI: 10.1038/srep08791 -
Journal of Innate Immunity 2015Endosymbiosis is common in insects thriving in nutritionally unbalanced habitats. The cereal weevil, Sitophilus oryzae, houses Sodalis pierantonius, a Gram-negative...
Endosymbiosis is common in insects thriving in nutritionally unbalanced habitats. The cereal weevil, Sitophilus oryzae, houses Sodalis pierantonius, a Gram-negative intracellular symbiotic bacterium (endosymbiont), within a dedicated organ called a bacteriome. Recent data have shown that the bacteriome expresses certain immune genes that result in local symbiont tolerance and control. Here, we address the question of whether and how the bacteriome responds to insect infections involving exogenous bacteria. We have established an infection model by challenging weevil larvae with the Gram-negative bacterium Dickeya dadantii. We showed that D. dadantii infects host tissues and triggers a systemic immune response. Gene transcript analysis indicated that the bacteriome is also immune responsive, but it expresses immune effector genes to a lesser extent than the systemic and intestinal responses. Most genes putatively involved in immune pathways remain weakly expressed in the bacteriome following D. dadantii infection. Moreover, quantitative PCR experiments showed that the endosymbiont load is not affected by insect infection or the resulting bacteriome immune activation. Thus, the contained immune effector gene expression in the bacteriome may prevent potentially harmful effects of the immune response on endosymbionts, whilst efficiently protecting them from bacterial intruders.
Topics: Animals; Gene Expression Regulation, Bacterial; Gram-Negative Bacteria; Symbiosis; Weevils
PubMed: 25632977
DOI: 10.1159/000368928 -
Molecular Plant Pathology Sep 2015Chemotaxis enables bacteria to move towards an optimal environment in response to chemical signals. In the case of plant-pathogenic bacteria, chemotaxis allows pathogens...
Chemotaxis enables bacteria to move towards an optimal environment in response to chemical signals. In the case of plant-pathogenic bacteria, chemotaxis allows pathogens to explore the plant surface for potential entry sites with the ultimate aim to prosper inside plant tissues and to cause disease. Chemoreceptors, which constitute the sensory core of the chemotaxis system, are usually transmembrane proteins which change their conformation when sensing chemicals in the periplasm and transduce the signal through a kinase pathway to the flagellar motor. In the particular case of the soft-rot pathogen Dickeya dadantii 3937, jasmonic acid released in a plant wound has been found to be a strong chemoattractant which drives pathogen entry into the plant apoplast. In order to identify candidate chemoreceptors sensing wound-derived plant compounds, we carried out a bioinformatics search of candidate chemoreceptors in the genome of Dickeya dadantii 3937. The study of the chemotactic response to several compounds and the analysis of the entry process to Arabidopsis leaves of 10 selected mutants in chemoreceptors allowed us to determine the implications of at least two of them (ABF-0020167 and ABF-0046680) in the chemotaxis-driven entry process through plant wounds. Our data suggest that ABF-0020167 and ABF-0046680 may be candidate receptors of jasmonic acid and xylose, respectively.
Topics: Arabidopsis; Enterobacteriaceae; Plant Leaves; Plant Proteins
PubMed: 25487519
DOI: 10.1111/mpp.12227 -
Microbiology (Reading, England) Dec 2014Osmoregulated periplasmic glucans (OPGs) are general constituents of many proteobacteria. OPGs are important factors required for full virulence in many pathogens...
Increased phosphorylation of the RcsB regulator of the RcsCDB phosphorelay in strains of Dickeya dadantii devoid of osmoregulated periplasmic glucans revealed by Phos-tag gel analysis.
Osmoregulated periplasmic glucans (OPGs) are general constituents of many proteobacteria. OPGs are important factors required for full virulence in many pathogens including Dickeya dadantii. D. dadantii causes the soft-rot disease in a wide range of plant species. The pleiotropic phenotype of opg-negative strains includes total loss of virulence and motility, and is linked to the constitutive activation of the RcsCDB phosphorelay, deduced from expression analysis of genes of the RcsCDB regulon. The constitutive activation of the RcsCDB phosphorelay in an opg-negative strain was demonstrated by direct analysis of the phosphorylation level of the RcsB regulator protein in vivo by using a Phos-tag retardation gel approach, and was correlated with the phenotype and the expression of motility genes. Data revealed a low level of RcsB phosphorylated form in the wild-type strain and a slight increase of phosphorylation in opgG mutant strains sufficient to induce the pleiotropic phenotype observed.
Topics: Bacterial Proteins; Electrophoresis; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Phosphorylation; Protein Processing, Post-Translational
PubMed: 25320363
DOI: 10.1099/mic.0.081273-0 -
Molecular Plant Pathology Jun 2015Dickeya dadantii is a plant-pathogenic enterobacterium responsible for plant soft rot disease in a wide range of hosts, including the model plant Arabidopsis thaliana....
Dickeya dadantii is a plant-pathogenic enterobacterium responsible for plant soft rot disease in a wide range of hosts, including the model plant Arabidopsis thaliana. Iron distribution in infected A. thaliana was investigated at the cellular scale using the Perls'-diaminobenzidine-H2 O2 (PDH) method. Iron visualization during infection reveals a loss of iron from cellular compartments and plant cell walls. During symptom progression, two distinct zones are clearly visible: a macerated zone displaying weak iron content and a healthy zone displaying strong iron content. Immunolabelling of cell wall methylated pectin shows that pectin degradation is correlated with iron release from cell walls, indicating a strong relationship between cell wall integrity and iron in plant tissues. Using a D. dadantii lipopolysaccharide antibody, we show that bacteria are restricted to the infected tissue, and that they accumulate iron in planta. In conclusion, weak iron content is strictly correlated with bacterial cell localization in the infected tissues, indicating a crucial role of this element during the interaction. This is the first report of iron localization at the cellular level during a plant-microbe interaction and shows that PDH is a method of choice in this type of investigation.
Topics: Arabidopsis; Cell Wall; Enterobacteriaceae; Ferritins; Iron; Plant Diseases; Plant Leaves; Protein Transport
PubMed: 25266463
DOI: 10.1111/mpp.12208 -
Molecular Plant-microbe Interactions :... Oct 2014The bacterial soft rot pathogen Dickeya dadantii utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to...
The bacterial soft rot pathogen Dickeya dadantii utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to disintegrate the plant cell wall. A transposon mutagenesis fluorescence-activated cell sorting screen was used to identify mutants with altered promoter activities of the T3SS pilus gene hrpA. Several insertion mutations, resulting in changes in hrpA expression, were mapped to a new locus, opgGH, which encodes the gene cluster responsible for osmoregulated periplasmic glucan (OPG) synthesis proteins. Our data showed that OPG was involved in T3SS and Pel regulation by altering the expression of the regulatory small RNA RsmB. Through genome searching, the mechanism of two novel regulatory components, the RcsCD-RcsB phosphorelay and CsrD on OPG and the rsmB gene, was further investigated. The Rcs phosphorelay and OPG inversely regulated rsmB at transcriptional and post-transcriptional levels, respectively. CsrD exhibited dual functionality in T3SS and Pel regulation by manipulating levels of RsmB RNA and cyclic diguanylate monophosphate (c-di-GMP). CsrD positively regulated the promoter activity of the rsmB gene but negatively controlled RsmB RNA at the post-transcriptional level via OpgGH. In addition, CsrD contains both GGDEF and EAL domains but acted as a c-di-GMP phosphodiesterase. When the expression of the csrD gene was induced, CsrD regulated T3SS expression and Pel production through controlling intracellular c-di-GMP levels.
Topics: Bacterial Proteins; Bacterial Secretion Systems; Cell Wall; Cyclic GMP; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Models, Biological; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Phenotype; Plant Diseases; Plants; Polysaccharide-Lyases; Promoter Regions, Genetic; Protein Structure, Tertiary; Sequence Analysis, DNA; Transcriptional Activation; Virulence; Virulence Factors
PubMed: 25180688
DOI: 10.1094/MPMI-01-14-0026-R