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Plant Physiology Nov 2016Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in...
Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens.
Topics: Arabidopsis; Cell Death; Discriminant Analysis; Disease Resistance; Intracellular Space; Least-Squares Analysis; Light; Mitochondria; Models, Biological; NAD; NADPH Oxidases; Nucleotides; Oxidative Stress; Pathogen-Associated Molecular Pattern Molecules; Phenotype; Plant Diseases; Plant Growth Regulators; Plant Immunity; Plant Leaves; Pyridines; Reactive Oxygen Species; Respiratory Burst; Salicylic Acid
PubMed: 27621425
DOI: 10.1104/pp.16.00780 -
Applied and Environmental Microbiology Nov 2016Modification of teichoic acid through the incorporation of d-alanine confers resistance in Gram-positive bacteria to antimicrobial peptides (AMPs). This process involves...
UNLABELLED
Modification of teichoic acid through the incorporation of d-alanine confers resistance in Gram-positive bacteria to antimicrobial peptides (AMPs). This process involves the products of the dltXABCD genes. These genes are widespread in Gram-positive bacteria, and they are also found in a few Gram-negative bacteria. Notably, these genes are present in all soft-rot enterobacteria (Pectobacterium and Dickeya) whose dltDXBAC operons have been sequenced. We studied the function and regulation of these genes in Dickeya dadantii dltB expression was induced in the presence of the AMP polymyxin. It was not regulated by PhoP, which controls the expression of some genes involved in AMP resistance, but was regulated by ArcA, which has been identified as an activator of genes involved in AMP resistance. However, arcA was not the regulator responsible for polymyxin induction of these genes in this bacterium, which underlines the complexity of the mechanisms controlling AMP resistance in D. dadantii Two other genes involved in resistance to AMPs have also been characterized, phoS and phoH dltB, phoS, phoH, and arcA but not dltD mutants were more sensitive to polymyxin than the wild-type strain. Decreased fitness of the dltB, phoS, and phoH mutants in chicory leaves indicates that their products are important for resistance to plant AMPs.
IMPORTANCE
Gram-negative bacteria can modify their lipopolysaccharides (LPSs) to resist antimicrobial peptides (AMPs). Soft-rot enterobacteria (Dickeya and Pectobacterium spp.) possess homologues of the dlt genes in their genomes which, in Gram-positive bacteria, are involved in resistance to AMPs. In this study, we show that these genes confer resistance to AMPs, probably by modifying LPSs, and that they are required for the fitness of the bacteria during plant infection. Two other new genes involved in resistance were also analyzed. These results show that bacterial resistance to AMPs can occur in bacteria through many different mechanisms that need to be characterized.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Proteins; Cichorium intybus; Drug Resistance, Bacterial; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Mutation; Plant Leaves; Polymyxins; Repressor Proteins
PubMed: 27565623
DOI: 10.1128/AEM.01757-16 -
Research in Microbiology May 2016Dickeya dadantii is a pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to production of pectate lyases (Pels) that can...
Regulation of pel genes, major virulence factors in the plant pathogen bacterium Dickeya dadantii, is mediated by cooperative binding of the nucleoid-associated protein H-NS.
Dickeya dadantii is a pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to production of pectate lyases (Pels) that can destroy plant cell walls. Previously, we found that nucleoid-associated protein (NAP) H-NS is a key regulator of pel gene expression. The primary binding sites of this NAP have been determined here by footprinting experiments on the pelD gene, encoding an essential virulence factor. Quantitative analysis of DNAse I footprints and surface plasmon resonance imagery experiments further revealed that high-affinity binding sites initiate cooperative binding to establish the nucleoprotein structure required for gene expression silencing. Mutations in the primary binding sites resulted in reduction or loss of repression by H-NS. Overall, these data suggest that H-NS represses pelD, and by inference, other pel genes, by a cooperative binding mechanism, through oligomerization of H-NS molecules.
Topics: Bacterial Proteins; Binding Sites; DNA Footprinting; DNA, Bacterial; DNA-Binding Proteins; Gammaproteobacteria; Gene Expression Regulation, Bacterial; Mutation; Polysaccharide-Lyases; Protein Binding; Surface Plasmon Resonance; Virulence Factors
PubMed: 26912324
DOI: 10.1016/j.resmic.2016.02.001 -
Scientific Reports Jan 2016Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. They are required for virulence of...
Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. They are required for virulence of zoo- and phyto-pathogens. The glucose backbone of OPGs is substituted by various kinds of molecules depending on the species, O-succinyl residues being the most widely distributed. In our model, Dickeya dadantii, a phytopathogenic bacteria causing soft rot disease in a wide range of plant species, the backbone of OPGs is substituted by O-succinyl residues in media of high osmolarity and by O-acetyl residues whatever the osmolarity. The opgC gene encoding a transmembrane protein required for the succinylation of the OPGs in D. dadantii was found after an in silico search of a gene encoding a protein with the main characteristics recovered in the two previously characterized OpgC of E. coli and R. sphaeroides, i.e. 10 transmembrane segments and one acyl-transferase domain. Characterization of the opgC gene revealed that high osmolarity expression of the succinyl transferase is controlled by both the EnvZ-OmpR and RcsCDB phosphorelay systems. The loss of O-succinyl residue did not affect the virulence of D. dadantii, suggesting that only the glucose backbone of OPGs is required for virulence.
Topics: Bacterial Proteins; Enterobacteriaceae; Escherichia coli; Gene Order; Genetic Complementation Test; Genome, Bacterial; Glucans; Osmolar Concentration; Osmoregulation; Periplasm; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Virulence
PubMed: 26790533
DOI: 10.1038/srep19619 -
Frontiers in Plant Science 2015The necrotrophic bacteria Dickeya dadantii is the causal agent of soft-rot disease in a broad range of hosts. The model plant Nicotiana benthamiana, commonly used as...
The necrotrophic bacteria Dickeya dadantii is the causal agent of soft-rot disease in a broad range of hosts. The model plant Nicotiana benthamiana, commonly used as experimental host for a very broad range of plant pathogens, is susceptible to infection by D. dadantii. The inoculation with D. dadantii at high dose seems to overcome the plant defense capacity, inducing maceration and death of the tissue, although restricted to the infiltrated area. By contrast, the output of the defense response to low dose inoculation is inhibition of maceration and limitation in the growth, or even eradication, of bacteria. Responses of tissue invaded by bacteria (neighboring the infiltrated areas after 2-3 days post-inoculation) included: (i) inhibition of photosynthesis in terms of photosystem II efficiency; (ii) activation of energy dissipation as non-photochemical quenching in photosystem II, which is related to the activation of plant defense mechanisms; and (iii) accumulation of secondary metabolites in cell walls of the epidermis (lignins) and the apoplast of the mesophyll (phytoalexins). Infiltrated tissues showed an increase in the content of the main hormones regulating stress responses, including abscisic acid, jasmonic acid, and salicylic acid. We propose a mechanism involving the three hormones by which N. benthamiana could activate an efficient defense response against D. dadantii.
PubMed: 26779238
DOI: 10.3389/fpls.2015.01209 -
Frontiers in Microbiology 2015Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system...
Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.
PubMed: 26733980
DOI: 10.3389/fmicb.2015.01442 -
Environmental Microbiology Nov 2015Dickeya dadantii is a globally dispersed phytopathogen which causes diseases on a wide range of host plants. This pathogen utilizes the type III secretion system (T3SS)...
Dickeya dadantii is a globally dispersed phytopathogen which causes diseases on a wide range of host plants. This pathogen utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to degrade the plant cell wall. Although the regulatory small RNA (sRNA) RsmB, cyclic diguanylate monophosphate (c-di-GMP) and flagellar regulator have been reported to affect the regulation of these two virulence factors or multiple cell behaviours such as motility and biofilm formation, the linkage between these regulatory components that coordinate the cell behaviours remain unclear. Here, we revealed a sophisticated regulatory network that connects the sRNA, c-di-GMP signalling and flagellar master regulator FlhDC. We propose multi-tiered regulatory mechanisms that link the FlhDC to the T3SS through three distinct pathways including the FlhDC-FliA-YcgR3937 pathway; the FlhDC-EcpC-RpoN-HrpL pathway; and the FlhDC-rsmB-RsmA-HrpL pathway. Among these, EcpC is the most dominant factor for FlhDC to positively regulate T3SS expression.
Topics: Amino Acid Sequence; Bacterial Proteins; Cyclic GMP; Enterobacteriaceae; Fimbriae Proteins; Flagella; Flagellin; Gene Expression Regulation, Bacterial; Plant Diseases; Polysaccharide-Lyases; Regulatory Sequences, Ribonucleic Acid; Signal Transduction; Transcription Factors; Type III Secretion Systems; Vegetables; Virulence; Virulence Factors
PubMed: 26462993
DOI: 10.1111/1462-2920.13029 -
Brazilian Journal of Microbiology :... 2015One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other...
One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.
Topics: Bacteriophages; Base Sequence; Biological Control Agents; DNA, Bacterial; Dickeya chrysanthemi; Microbial Sensitivity Tests; Molecular Sequence Data; Myoviridae; Plant Diseases; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Siphoviridae; Solanum tuberosum
PubMed: 26413062
DOI: 10.1590/S1517-838246320140498 -
Environmental Microbiology Reports Oct 2015This review emphasizes the biological roles of the osmoregulated periplasmic glucans (OPGs). Osmoregulated periplasmic glucans occur in almost all α-, β- and... (Review)
Review
This review emphasizes the biological roles of the osmoregulated periplasmic glucans (OPGs). Osmoregulated periplasmic glucans occur in almost all α-, β- and γ-Proteobacteria. This polymer of glucose is required for full virulence. The roles of the OPGs are complex and vary depending on the species. Here, we outline the four major roles of the OPGs through four different pathogenic and one symbiotic bacterial models (Dickeya dadantii, Salmonella enterica, Pseudomonas aeruginosa, Brucella abortus and Sinorhizobium meliloti). When periplasmic, the OPGs are a part of the signal transduction pathway and indirectly regulate genes involved in virulence. The OPGs can also be secreted. When outside of the cell, they interact directly with antibiotics to protect the bacterial cell or interact with the host cell to facilitate the invasion process. When OPGs are not found, as in the ε-Proteobacteria, OPG-like oligosaccharides are present. Their presence strengthens the evidence that OPGs play an important role in virulence.
Topics: Alphaproteobacteria; Anti-Bacterial Agents; Gammaproteobacteria; Glucans; Osmoregulation; Periplasm; Signal Transduction; Virulence
PubMed: 26265506
DOI: 10.1111/1758-2229.12325 -
Infection and Immunity Sep 2015The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor....
The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥ 37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.
Topics: Animals; Bacterial Proteins; Blotting, Western; Disease Models, Animal; Evolution, Molecular; Gene Deletion; Glucans; Mice; Operon; Periplasmic Proteins; Reverse Transcriptase Polymerase Chain Reaction; Yersinia pestis; Yersinia pseudotuberculosis
PubMed: 26150539
DOI: 10.1128/IAI.00482-15