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International Journal of Molecular... May 2021Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic...
Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic effect. The disturbance in the intracellular toxin and antitoxin ratio typically leads to inhibition of bacterial growth or bacterial cell death. Despite the fact that TA modules are widespread in bacteria and archaea, the biological role of these systems is ambiguous. Nevertheless, a number of studies suggests that the TA modules are engaged in such important processes as biofilm formation, stress response or virulence and maintenance of mobile genetic elements. The 3937 strain serves as a model for pathogens causing the soft-rot disease in a wide range of angiosperm plants. Until now, several chromosome-encoded type II TA systems were identified in silico in the genome of this economically important bacterium however so far only one of them was experimentally validated. In this study, we investigated three putative type II TA systems in 3937: , and , which represents a novel toxin/antitoxin superfamily. We provide an experimental proof for their functionality in vivo both in and . Finally, we examined the prevalence of those systems across the Pectobacteriaceae family by a phylogenetic analysis.
Topics: Bacterial Proteins; Bacterial Toxins; Dickeya; Gene Expression Regulation, Bacterial; Plant Diseases; Toxin-Antitoxin Systems; Virulence
PubMed: 34073004
DOI: 10.3390/ijms22115932 -
Plant Disease Apr 2021Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection...
Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect in suspensions diluted to 4.89 × 10, 4.89 × 10, 9.78 × 10, and 9.78 × 10 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect in suspensions diluted up to 1.23 × 10 and 1.17 × 10 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.
Topics: Dickeya; Enterobacteriaceae; Enzyme-Linked Immunosorbent Assay; Ipomoea batatas; Plant Diseases
PubMed: 33689450
DOI: 10.1094/PDIS-07-20-1551-RE -
Scientific Reports Feb 2021Autophagy is a ubiquitous vesicular process for protein and organelle recycling in eukaryotes. In plant, autophagy is reported to play pivotal roles in nutrient...
Autophagy is a ubiquitous vesicular process for protein and organelle recycling in eukaryotes. In plant, autophagy is reported to play pivotal roles in nutrient recycling, adaptation to biotic and abiotic stresses. The role of autophagy in plant immunity remains poorly understood. Several reports showed enhanced susceptibility of different Arabidopsis autophagy mutants (atg) to necrotrophic fungal pathogens. Interaction of necrotrophic bacterial pathogens with autophagy is overlooked. We then investigated such interaction by inoculating the necrotrophic enterobacterium Dickeya dadantii in leaves of the atg2 and atg5 mutants and an ATG8a overexpressing line. Overexpressing ATG8a enhances plant tolerance to D. dadantii. While atg5 mutant displayed similar susceptibility to the WT, the atg2 mutant exhibited accelerated leaf senescence and enhanced susceptibility upon infection. Both phenotypes were reversed when the sid2 mutation, abolishing SA signaling, was introduced in the atg2 mutant. High levels of SA signaling in atg2 mutant resulted in repression of the jasmonic acid (JA) defense pathway known to limit D. dadantii progression in A. thaliana. We provide evidence that in atg2 mutant, the disturbed hormonal balance leading to higher SA signaling is the main factor causing increased susceptibility to the D. dadantii necrotroph by repressing the JA pathway and accelerating developmental senescence.
Topics: Arabidopsis; Arabidopsis Proteins; Autophagy; Dickeya; Gene Expression Regulation, Plant; Mutation; Plant Diseases; Salicylic Acid; Signal Transduction; Up-Regulation
PubMed: 33574453
DOI: 10.1038/s41598-021-83067-6 -
The Journal of Biological Chemistry 2021The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The...
The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The molecular mechanisms of substrate recruitment by T2SS remain elusive but a prevailing view is that the secretion determinants could be of a structural nature. The phytopathogenic γ-proteobacteria, Pectobacterium carotovorum and Dickeya dadantii, secrete similar sets of homologous plant cell wall degrading enzymes, mainly pectinases, by similar T2SSs, called Out. However, the orthologous pectate lyases Pel3 and PelI from these bacteria, which share 67% of sequence identity, are not secreted by the counterpart T2SS of each bacterium, indicating a fine-tuned control of protein recruitment. To identify the related secretion determinants, we first performed a structural characterization and comparison of Pel3 with PelI using X-ray crystallography. Then, to assess the biological relevance of the observed structural variations, we conducted a loop-substitution analysis of Pel3 combined with secretion assays. We showed that there is not one element with a definite secondary structure but several distant and structurally flexible loop regions that are essential for the secretion of Pel3 and that these loop regions act together as a composite secretion signal. Interestingly, depending on the crystal contacts, one of these key secretion determinants undergoes disorder-to-order transitions that could reflect its transient structuration upon the contact with the appropriate T2SS components. We hypothesize that such T2SS-induced structuration of some intrinsically disordered zones of secretion substrates could be part of the recruitment mechanism used by T2SS.
Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Cell Wall; Cloning, Molecular; Crystallography, X-Ray; Dickeya; Escherichia coli; Gene Expression; Genetic Vectors; Isoenzymes; Models, Molecular; Pectobacterium carotovorum; Phylogeny; Plant Cells; Plants; Polysaccharide-Lyases; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Type II Secretion Systems
PubMed: 33465378
DOI: 10.1016/j.jbc.2021.100305 -
Nucleic Acids Research Jan 2021Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host...
Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαβ heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a 'transcriptional domainin' protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.
Topics: Bacterial Proteins; Binding Sites; Cellulase; Cichorium intybus; DNA, Bacterial; DNA, Superhelical; Dickeya; Dimerization; Gene Expression Regulation, Bacterial; Genetic Association Studies; Integration Host Factors; Motion; Peptide Hydrolases; Plasmids; Polygalacturonase; Promoter Regions, Genetic; Recombinant Proteins; Siderophores; Transcription, Genetic; Transcriptome; Virulence
PubMed: 33337488
DOI: 10.1093/nar/gkaa1227 -
Innovation and Application of the Type III Secretion System Inhibitors in Plant Pathogenic Bacteria.Microorganisms Dec 2020Many Gram-negative pathogenic bacteria rely on a functional type III secretion system (T3SS), which injects multiple effector proteins into eukaryotic host cells, for... (Review)
Review
Many Gram-negative pathogenic bacteria rely on a functional type III secretion system (T3SS), which injects multiple effector proteins into eukaryotic host cells, for their pathogenicity. Genetic studies conducted in different host-microbe pathosystems often revealed a sophisticated regulatory mechanism of their T3SSs, suggesting that the expression of T3SS is tightly controlled and constantly monitored by bacteria in response to the ever-changing host environment. Therefore, it is critical to understand the regulation of T3SS in pathogenic bacteria for successful disease management. This review focuses on a model plant pathogen, , and summarizes the current knowledge of its T3SS regulation. We highlight the roles of several T3SS regulators that were recently discovered, including the transcriptional regulators: FlhDC, RpoS, and SlyA; the post-transcriptional regulators: PNPase, Hfq with its dependent sRNA ArcZ, and the RsmA/B system; and the bacterial second messenger cyclic-di-GMP (c-di-GMP). Homologs of these regulatory components have also been characterized in almost all major bacterial plant pathogens like , , spp., spp., and spp. The second half of this review shifts focus to an in-depth discussion of the innovation and development of T3SS inhibitors, small molecules that inhibit T3SSs, in the field of plant pathology. This includes T3SS inhibitors that are derived from plant phenolic compounds, plant coumarins, and salicylidene acylhydrazides. We also discuss their modes of action in bacteria and application for controlling plant diseases.
PubMed: 33317075
DOI: 10.3390/microorganisms8121956 -
Pathogens (Basel, Switzerland) Dec 2020Copper nanoparticles (CuNPs) can offer an alternative to conventional copper bactericides and possibly slow down the development of bacterial resistance. This will...
Copper nanoparticles (CuNPs) can offer an alternative to conventional copper bactericides and possibly slow down the development of bacterial resistance. This will consequently lower the accumulation rate of copper to soil and water and lower the environmental and health burden imposed by copper application. Physical and chemical methods have been reported to synthesize CuNPs but their use as bactericides in plants has been understudied. In this study, two different CuNPs products have been developed, CuNP1 and CuNP2 in two respective concentrations (1500 ppm or 300 ppm). Both products were characterized using Dynamic Light Scattering, Transmission Electron Microscopy, Attenuated Total Reflection measurements, X-ray Photoelectron Spectroscopy, X-ray Diffraction and Scattering, and Laser Doppler Electrophoresis. They were evaluated for their antibacterial efficacy in vitro against the gram-negative species , , , , , pv. , and pv. . Evaluation was based on comparisons with two commercial bactericides: Kocide (copper hydroxide) and Nordox (copper oxide). CuNP1 inhibited the growth of five species, restrained the growth of and had no effect in pv . MICs were significantly lower than those of the commercial formulations. CuNP2 inhibited the growth of and restrained growth of pv. . Again, its overall activity was higher compared to commercial formulations. An extensive in vitro evaluation of CuNPs that show higher potential compared to their conventional counterpart is reported for the first time and suggests that synthesis of stable CuNPs can lead to the development of low-cost sustainable commercial products.
PubMed: 33291381
DOI: 10.3390/pathogens9121024 -
Analytical Biochemistry Apr 2021A rapid and sensitive High Performance Liquid Chromatography (HPLC) method with photometric and fluorescence detection is developed for routine analysis of...
A rapid and sensitive High Performance Liquid Chromatography (HPLC) method with photometric and fluorescence detection is developed for routine analysis of 2-Keto-3-deoxy-gluconate (KDG), a catabolite product of pectin and alginate. These polysaccharides are primary-based compounds for biofuel production and for generation of high-value-added products. HPLC is performed, after derivatization of the 2-oxo-acid groups of the metabolite with o-phenylenediamine (oPD), using a linear gradient of trifluoroacetic acid and acetonitrile. Quantification is accomplished with an internal standard method. The gradient is optimized to distinguish KDG from its close structural analogues such as 5-keto-4-deoxyuronate (DKI) and 2,5-diketo-3-deoxygluconate (DKII). The proposed method is simple, highly sensitive and accurate for time course analysis of pectin or alginate degradation.
Topics: Alginates; Dickeya; Gluconates; Pectins
PubMed: 33285123
DOI: 10.1016/j.ab.2020.114061 -
PHAGE (New Rochelle, N.Y.) Dec 2020Bacterial soft rot caused by members of the soft rot afflicts plant production of both vegetable and ornamental crops. Recent outbreaks highlight sp. in the etiology...
Bacterial soft rot caused by members of the soft rot afflicts plant production of both vegetable and ornamental crops. Recent outbreaks highlight sp. in the etiology of this disease in potatoes. Since there is a lack of control strategies for these diseases, alternative approaches have been suggested, including the use of biological control mediated by bacteriophages (phages). However, phages infecting many of these members are still undiscovered or poorly described. Two phages targeting subsp. (NCPPB 4097) were isolated from household organic waste and purified. They were then further characterized using whole-genome sequencing and comparative genomics, transmission electron microscopy, latent period and burst size. Dickeya phage Sucellus displayed Siphovirus morphology and had a genome of 39,826 bp with very limited similarity to any previously described phages. Dickeya phage Amaethon had a Podovirus morphology with a genome comprising 41,436 bp and limited similarity to phages in the genus. The phages exhibited burst sizes of app. 94 and 240 virions per cell with latent periods of 91 and 86 minutes for Sucellus and Amaethon, respectively. While both phages had similar adsorption efficiencies and latent periods, the rise periods for the two phages diverged markedly, highlighting an odd growth pattern. Together, the two phages isolated here expand the known diversity of phages infecting the important plant pathogen . As they both share limited similarity to previously described groups of phages, they likely constitute novel genera within their respective groups.
PubMed: 36147281
DOI: 10.1089/phage.2020.0039 -
Carbohydrate Research Nov 2020The species Dickeya aquatica was established in 2014 after the genomic characterization of the pectinolytic bacteria isolated from water. It was demonstrated that D....
The structure of the O-polysaccharide isolated from pectinolytic gram-negative bacterium Dickeya aquatica IFB0154 is different from the O-polysaccharides of other Dickeya species.
The species Dickeya aquatica was established in 2014 after the genomic characterization of the pectinolytic bacteria isolated from water. It was demonstrated that D. aquatica was able to cause symptoms of soft rot on the fruit of tomato and cucumber. According to earlier works, lipopolysaccharides are regarded as an important virulence factor of Pectobacteriaceae. An O-specific polysaccharide containing d-Fuc and l-Rha was obtained by mild acid hydrolysis of the lipopolysaccharide of D. aquatica IFB0154 (strain Dw044 isolated in Finland). By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear disaccharide of the structure shown below. The rhamnose residue was partially acetylated at O-2 or O-3. OAc (~40%) ↓ →3)-α-d-Fucp-(1 → 4)-α-l-Rhap-(1→ ↑ OAc (~30%) The O-polysaccharides isolated from Dickeya dianthicola IFB0485 and Dickeya zeae IPO946 have a different structure, identical to that previously described for several strains of Dickeya solani and Dickeya dadantii 3937.
Topics: Carbohydrate Sequence; Dickeya; O Antigens; Species Specificity
PubMed: 32911204
DOI: 10.1016/j.carres.2020.108135