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RNA (New York, N.Y.) Apr 2017Regulation of gene expression by encoded riboswitches is a prevalent theme in bacteria. Of the hundreds of riboswitch families identified, the majority of them remain as...
Regulation of gene expression by encoded riboswitches is a prevalent theme in bacteria. Of the hundreds of riboswitch families identified, the majority of them remain as orphans, without a clear ligand assignment. The orphan family was recently characterized as guanidine-sensing riboswitches. Herein we present a 2.3 Å crystal structure of the guanidine-bound riboswitch from The riboswitch folds into a boot-shaped structure, with a coaxially stacked P1/P2 stem forming the boot, and a 3'-P3 stem-loop forming the heel. Sophisticated base-pairing and cross-helix tertiary contacts give rise to the ligand-binding pocket between the boot and the heel. The guanidine is recognized in its positively charged guanidinium form, in its sp hybridization state, through a network of coplanar hydrogen bonds and by a cation-π stacking contact on top of a conserved guanosine residue. Disruption of these contacts resulted in severe guanidinium-binding defects. These results provide the structural basis for specific guanidine sensing by riboswitches and pave the way for a deeper understanding of guanidine detoxification-a previously unappreciated aspect of bacterial physiology.
Topics: Base Pairing; Binding Sites; Crystallography, X-Ray; Enterobacteriaceae; Guanidine; Hydrogen Bonding; Ligands; Models, Molecular; Nucleic Acid Conformation; RNA, Bacterial; Riboswitch; Static Electricity; Thermodynamics
PubMed: 28096518
DOI: 10.1261/rna.060186.116 -
Analytical Chemistry Feb 2017Targeted mass spectrometry of a surrogate peptide panel is a powerful method to study the dynamics of protein networks, but chromatographic time scheduling remains a...
Targeted mass spectrometry of a surrogate peptide panel is a powerful method to study the dynamics of protein networks, but chromatographic time scheduling remains a major limitation for dissemination and implementation of robust and large multiplexed assays. We unveil a Multiple Reaction Monitoring method (Scout-MRM) where the use of spiked scout peptides triggers complex transition lists, regardless of the retention time of targeted surrogate peptides. The interest of Scout-MRM method regarding the retention time independency, multiplexing capability, reproducibility, and putative interest in facilitating method transfer was illustrated by a 782-peptide-plex relative assay targeting 445 proteins of the phytopathogen Dickeya dadantii during plant infection.
Topics: Cichorium intybus; Chromatography, High Pressure Liquid; Enterobacteriaceae; Mass Spectrometry; Peptides; Plant Leaves; Proteomics
PubMed: 28029036
DOI: 10.1021/acs.analchem.6b03201 -
Frontiers in Plant Science 2016The negative impact of conventional farming on environment and human health make improvements on farming management mandatory. Imaging techniques are implemented in...
The negative impact of conventional farming on environment and human health make improvements on farming management mandatory. Imaging techniques are implemented in remote sensing for monitoring crop fields and plant phenotyping programs. The increasingly large size and complexity of the data obtained by these techniques, makes the implementation of powerful mathematical tools necessary in order to identify informative parameters and to apply them in precision agriculture. Multicolor fluorescence imaging is a useful approach for the study of plant defense responses to stress factors at bench scale. However, it has not been fully applied to plant phenotyping. This work evaluates the possible application of multicolor fluorescence imaging in combination with thermography for the particular case of zucchini plants affected by soft-rot, caused by . Several statistical models -based on logistic regression analysis (LRA) and artificial neural networks (ANN)- were obtained for the experimental system zucchini-, which classify new samples as "healthy" or "infected." The LRA worked best in identifying high dose-infiltrated leaves (in infiltrated and non-infiltrated areas) whereas ANN offered a higher accuracy at identifying low dose-infiltrated areas. To assess the applicability of these results to cucurbits in a more general way, these models were validated for melon infected by the same pathogen, achieving accurate predictions for the infiltrated areas. The values of accuracy achieved are comparable to those found in the literature for classifiers identifying other infections based on data obtained by different techniques. Thus, MCFI in combination with thermography prove useful at providing data at lab scale that can be analyzed by machine learning. This approach could be scaled up to be applied in plant phenotyping.
PubMed: 27994607
DOI: 10.3389/fpls.2016.01790 -
Molecular Plant Pathology Feb 2018Soft-rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II...
Soft-rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions. We focused on the production of AtrbohD-dependent reactive oxygen species (ROS), callose deposition and cell death as indicators of these reactions. We observed a significant reduction in ROS and callose formation with a bacterial mutant in which genes encoding five pectate lyases (Pels) were disrupted. Treatment of plant leaves with bacterial culture filtrates containing Pels resulted in ROS and callose production, and both reactions were dependent on a functional AtrbohD gene. ROS and callose were produced in response to treatment with a cellular fraction of a T2SS-negative mutant grown in a Pels-inducing medium. Finally, ROS and callose were produced in leaves treated with purified Pels that had also been shown to induce the expression of jasmonic acid-dependent defence genes. Pel catalytic activity is required for the induction of ROS accumulation. In contrast, cell death observed in leaves infected with the wild-type strain appeared to be independent of a functional AtrbohD gene. It was also independent of the bacterial production of pectic enzymes and the type III secretion system (T3SS). In conclusion, the work presented here shows that D. dadantii is recognized by the A. thaliana innate immune system through the action of pectic enzymes secreted by bacteria at the site of infection. This recognition leads to AtrbohD-dependent ROS and callose accumulation, but not cell death.
Topics: Arabidopsis; Gammaproteobacteria; Glucans; Immunity, Innate; Oligosaccharides; Polysaccharide-Lyases; Reactive Oxygen Species; Type III Secretion Systems; Virulence
PubMed: 27925401
DOI: 10.1111/mpp.12522 -
Biochimica Et Biophysica Acta Nov 2016In bacteria, important genes are often controlled at the transcriptional level by several factors, forming a complex and intertwined web of interactions. Yet,...
In bacteria, important genes are often controlled at the transcriptional level by several factors, forming a complex and intertwined web of interactions. Yet, transcriptional regulators are often studied separately and little information is available concerning their interactions. In this work, we dissect the regulation of the major virulence gene pelD in D. dadantii by taking into account the effects of individual binding sites for regulatory proteins FIS and CRP, and the impact of a newly discovered divergent promoter div. Using a combination of biochemistry and genetics approaches we provide an unprecedented level of detail on the multifactorial regulation of bacterial transcription. We show that the growth phase dependent regulation of pelD is under the control of changing composition of higher-order nucleoprotein complexes between FIS, CRP, div and pelD during the growth cycle that allow sequential expression of div and pelD in the early and late exponential growth phases, respectively. This work highlights the importance of "orphan" promoters in gene regulation and that the individual binding sites for a regulator can serve several purposes and have different effects on transcription, adding a new level of complexity to bacterial transcriptional regulation.
Topics: Gammaproteobacteria; Nucleoproteins; Transcription, Genetic; Virulence
PubMed: 27498372
DOI: 10.1016/j.bbagrm.2016.08.001 -
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 -
ACS Chemical Biology Jul 2016Transcriptional activity is exquisitely sensitive to changes in promoter DNA topology. Transcription factors may therefore control gene activity by modulating the...
Transcriptional activity is exquisitely sensitive to changes in promoter DNA topology. Transcription factors may therefore control gene activity by modulating the relative positioning of -10 and -35 promoter elements. The plant pathogen Pectobacterium atrosepticum, which causes soft rot in potatoes, must alter gene expression patterns to ensure growth in planta. In the related soft-rot enterobacterium Dickeya dadantii, PecS functions as a master regulator of virulence gene expression. Here, we report that P. atrosepticum PecS controls gene activity by altering promoter DNA topology in response to pH. While PecS binds the pecS promoter with high affinity regardless of pH, it induces significant DNA distortion only at neutral pH, the pH at which the pecS promoter is repressed in vivo. At pH ∼8, DNA distortions are attenuated, and PecS no longer represses the pecS promoter. A specific histidine (H142) located in a crevice between the dimerization- and DNA-binding regions is required for pH-dependent changes in DNA distortion and repression of gene activity, and mutation of this histidine renders the mutant protein incapable of repressing the pecS promoter. We propose that protonated PecS induces a DNA conformation at neutral pH in which -10 and -35 promoter elements are suboptimally positioned for RNA polymerase binding; on deprotonation of PecS, binding is no longer associated with significant changes in DNA conformation, allowing gene expression. We suggest that this mode of gene regulation leads to differential expression of the PecS regulon in response to alkalinization of the plant apoplast.
Topics: Bacterial Proteins; DNA, Plant; Gene Expression Regulation, Plant; Hydrogen-Ion Concentration; Nucleic Acid Conformation; Pectobacterium; Protein Binding; Solanum tuberosum
PubMed: 27213700
DOI: 10.1021/acschembio.6b00168 -
International Journal of Systematic and... Sep 2016Gram-stain-negative, pectinolytic bacteria were repeatedly isolated from pear trees displaying symptoms of bleeding canker in China. Three strains, JS5T, LN1 and QZH3,...
Gram-stain-negative, pectinolytic bacteria were repeatedly isolated from pear trees displaying symptoms of bleeding canker in China. Three strains, JS5T, LN1 and QZH3, had identical 16S rRNA gene sequences that shared 99 % similarity to the type strain of Dickeya dadantii. Phylogenetic analysis of strains JS5T, LN1 and QZH3 with isolates representing all species of the genus Dickeya and related Pectobacterium species supported their affiliation to Dickeya. Multi-locus sequence typing employing concatenated sequences encoding recA, fusA, gapA, purA, rplB, dnaX and the intergenic spacer illustrated a phylogeny which placed strains JS5T, LN1 and QZH3 as a distinct clade, separate from all other species of the genus Dickeya. Average nucleotide identity values obtained in comparison with all species of the genus Dickeya supported the distinctiveness of strain JS5T within the genus Dickeya. Additionally, all three strains were phenotypically distinguished from other species of the genus Dickeya by failing to hydrolyse casein, and by producing acids from (-)-d-arabinose, (+)melibiose, (+)raffinose, mannitol and myo-inositol, but not from 5-keto-d-gluconate or β-gentiobiose. The name Dickeya fangzhongdai sp. nov. is proposed to accommodate these strains; the type strain is JS5T (=CGMCC 1.15464T=DSM 101947T).
Topics: Bacterial Typing Techniques; China; DNA, Bacterial; Enterobacteriaceae; Fatty Acids; Genes, Bacterial; Multilocus Sequence Typing; Phenotype; Phylogeny; Plant Diseases; Pyrus; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 27045848
DOI: 10.1099/ijsem.0.001060 -
Environmental Microbiology Nov 2016Dickeya species are soft rot disease-causing bacterial plant pathogens and an emerging agricultural threat in Europe. Environmental modulation of gene expression is...
Dickeya species are soft rot disease-causing bacterial plant pathogens and an emerging agricultural threat in Europe. Environmental modulation of gene expression is critical for Dickeya dadantii pathogenesis. While the bacterium uses various environmental cues to distinguish between its habitats, an intricate transcriptional control system coordinating the expression of virulence genes ensures efficient infection. Understanding of this behaviour requires a detailed knowledge of expression patterns under a wide range of environmental conditions, which is currently lacking. To obtain a comprehensive picture of this adaptive response, we devised a strategy to examine the D. dadantii transcriptome in a series of 32 infection-relevant conditions encountered in the hosts. We propose a temporal map of the bacterial response to various stress conditions and show that D. dadantii elicits complex genetic behaviour combining common stress-response genes with distinct sets of genes specifically induced under each particular stress. Comparison of our dataset with an in planta expression profile reveals the combined impact of stress factors and enables us to predict the major stress confronting D. dadantii at a particular stage of infection. We provide a comprehensive catalog of D. dadantii genomic responses to environmentally relevant stimuli, thus facilitating future studies of this important plant pathogen.
Topics: Bacterial Proteins; Enterobacteriaceae; Europe; Gene Expression Regulation, Bacterial; Genomics; Plant Diseases; Plants; Virulence; Virulence Factors
PubMed: 26940633
DOI: 10.1111/1462-2920.13267