-
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 -
Natural Product Research Dec 2023The GC and GC/MS analysis of the essential oil samples obtained separately from whole aerial parts and the roots of resulted in the identification of a total of 40...
A comparative study on chemical composition and antibacterial activity of essential oils from the aerial parts and roots of (Siebold & Zucc.) Wedd. growing in the Central Himalayan region.
The GC and GC/MS analysis of the essential oil samples obtained separately from whole aerial parts and the roots of resulted in the identification of a total of 40 compounds from the whole aerial parts and 45 compounds from the roots representing 89.7% and 82.9%, respectively, of the total essential oil composition. The essential oil from aerial parts is dominated by oxygenated sesquiterpenes (66.4%) with elemol (55.1%), germacrene D-4-ol (4.2%), linalool (3.5%) and phytol (3.4%) as the major constituents while the root essential oil is dominated by oxygenated monoterpenes (30.4%) with -myrtanol (16.3%), -myrtanol (11.6%), nopinone (7.8%), cadin-4-en-10-ol (4.7%), and -elemol (3.4%) as major constituents. The essential oil from whole aerial parts showed bactericidal effect at 250 µL/mL concentration (MBC) against and while the root essential oil exhibited bactericidal effect at 125 µL/mL against , and .
PubMed: 38073501
DOI: 10.1080/14786419.2023.2289187 -
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 -
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 -
Applied and Environmental Microbiology Mar 2020While rhamnolipids of the type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected...
While rhamnolipids of the type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected known and identified new genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in and A likely horizontal gene transfer event into is the only identified exception. The phylogeny of the RhlA homologs from and species is consistent with the organism phylogeny, and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the do not follow the organisms' phylogeny but form their own branch. Furthermore, in many and from the , an isolated homolog can be found in the genome. The RhlAs from PA01, LMG 05825, LMG 20103, PG1, LMG 19182, sp. strain R57-5, Ech586, and PRI-2C were expressed in and tested for HAA production. Indeed, except for the RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) highlights the congener specificity of the RhlA proteins. The congener length varies from 4 to 18 carbon atoms, with the main congeners consisting of different combinations of saturated or monounsaturated C, C, and C fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity. The RhlA specificity explains the observed differences in 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.
Topics: Acyltransferases; Bacteria; Bacterial Proteins; Carboxylic Acids; Glycolipids
PubMed: 31924623
DOI: 10.1128/AEM.02317-19 -
An uncommon [K(Mg)] metal ion triad imparts stability and selectivity to the Guanidine-I riboswitch.RNA (New York, N.Y.) Oct 2021The widespread -I riboswitch class exemplifies divergent riboswitch evolution. To analyze how natural selection has diversified its versatile RNA fold, we determined the...
The widespread -I riboswitch class exemplifies divergent riboswitch evolution. To analyze how natural selection has diversified its versatile RNA fold, we determined the X-ray crystal structure of the -I subtype-1 (Guanidine-I) riboswitch aptamer domain. Differing from the previously reported structures of orthologs from and , our structure reveals a chelated K ion adjacent to two Mg ions in the guanidine-binding pocket. Thermal melting analysis shows that K chelation, which induces localized conformational changes in the binding pocket, improves guanidinium-RNA interactions. Analysis of ribosome structures suggests that the [K(Mg)] ion triad is uncommon. It is, however, reminiscent of metal ion clusters found in the active sites of ribozymes and DNA polymerases. Previous structural characterization of -I subtype-2 RNAs, which bind the effector ligands ppGpp and PRPP, indicate that in those paralogs, an adenine responsible for K chelation in the Guanidine-I riboswitch is replaced by a pyrimidine. This mutation results in a water molecule and Mg ion binding in place of the K ion. Thus, our structural analysis demonstrates how ion and solvent chelation tune divergent ligand specificity and affinity among -I riboswitches.
Topics: Aptamers, Nucleotide; Base Pairing; Base Sequence; Biological Evolution; Burkholderia; Chelating Agents; Clostridiales; Crystallography, X-Ray; Dickeya; Guanidines; Magnesium; Models, Molecular; Mutation; Nucleic Acid Conformation; Potassium; Ribosomes; Riboswitch; Water
PubMed: 34257148
DOI: 10.1261/rna.078824.121 -
ACS Applied Bio Materials Apr 2020(-)-Epigallocatechin gallate (EGCG) is a polyphenolic compound that shows a number of health-promoting effects, especially a broad antimicrobial activity. Virus-derived...
(-)-Epigallocatechin gallate (EGCG) is a polyphenolic compound that shows a number of health-promoting effects, especially a broad antimicrobial activity. Virus-derived nanoparticles (VNPs) represent a promising drug carrier since they possess properties like biodegradability and their surface and interior are highly modifiable. Turnip mosaic virus (TuMV) VNPs offer an attractive number of conjugation sites on the external surface. EGCG-TuMV VNPs were synthesized by Mannich condensation, and their antimicrobial activities against the model bacteria , , and were tested. EGCG-TuMV VNPs did not only maintain TuMV structure but also showed an enhanced antimicrobial activity over that found with free EGCG for all of the bacteria tested. Biofilm formation by was also inhibited by EGCG-TuMV VNPs, contrary to free EGCG, which induced higher amounts of biofilm mass in a concentration-dependent manner. Taken together, our results open highly promising perspectives for the antimicrobial exploitation of EGCG-TuMV VNPs.
PubMed: 35025325
DOI: 10.1021/acsabm.9b01161 -
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 -
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 -
Biochemistry Oct 2017Agrobacterium fabrum induces tumor growth in susceptible plant species. The upregulation of virulence genes that occurs when the bacterium senses plant-derived compounds...
Agrobacterium fabrum induces tumor growth in susceptible plant species. The upregulation of virulence genes that occurs when the bacterium senses plant-derived compounds is enhanced by acidic pH and limiting inorganic phosphate. Nutrient starvation may also trigger the stringent response, and purine salvage is among the pathways expected to be favored under such conditions. We show here that phosphate limitation induces the stringent response, as evidenced by production of (p)ppGpp, and that the xdhCSML operon encoding the purine salvage enzyme xanthine dehydrogenase is upregulated ∼15-fold. The xdhCSML operon is under control of the TetR family transcription factor XdhR; direct binding of ppGpp to XdhR attenuates DNA binding, and the enhanced xdhCSML expression correlates with increased cellular levels of (p)ppGpp. Xanthine dehydrogenase may also divert purines away from salvage pathways to form urate, the ligand for the transcription factor PecS, which in the plant pathogen Dickeya dadantii is a key regulator of virulence gene expression. However, urate levels remain low under conditions that produce increased levels of xdhCSML expression, and neither acidic pH nor limiting phosphate results in induction of genes under control of PecS. Instead, expression of such genes is induced only by externally supplemented urate. Taken together, our data indicate that purine salvage is favored during the stringent response induced by phosphate starvation, suggesting that control of this pathway may constitute a novel approach to modulating virulence. Because bacterial purine catabolism appears to be unaffected, as evidenced by the absence of urate accumulation, we further propose that the PecS regulon is induced by only host-derived urate.
Topics: Agrobacterium; Bacterial Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Phosphates; Purines; Uric Acid; Virulence Factors; Xanthine Dehydrogenase
PubMed: 29027458
DOI: 10.1021/acs.biochem.7b00844