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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 -
Microbiology Resource Announcements Sep 2021Erwinia chrysanthemi S3-1 is a bacterial soft rot pathogen of the white-flowered calla lily. The complete genome sequence of the strain was determined and used to...
Erwinia chrysanthemi S3-1 is a bacterial soft rot pathogen of the white-flowered calla lily. The complete genome sequence of the strain was determined and used to reclassify the strain as Dickeya dadantii subsp. . The sequence will be useful to study plant host-driven speciation in strains of D. dadantii.
PubMed: 34528816
DOI: 10.1128/MRA.00620-21 -
Phytopathology Dec 2023Fire blight, caused by , is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause...
Fire blight, caused by , is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in . We showed that adding TS108 to an in vitro culture of repressed the expression of several T3SS regulon genes, including the master regulator gene . Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of , which encodes a key activator of . Additionally, TS108 has no impact on the expression of T3SS in or , suggesting that its inhibition of the T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.
Topics: Type III Secretion Systems; Erwinia amylovora; Plant Diseases; Anti-Bacterial Agents; Malus; Bacterial Proteins
PubMed: 37344783
DOI: 10.1094/PHYTO-04-23-0111-SA -
Frontiers in Plant Science 2020Plant defense stimulators, used in crop protection, are an attractive option to reduce the use of conventional crop protection products and optimize biocontrol...
Plant defense stimulators, used in crop protection, are an attractive option to reduce the use of conventional crop protection products and optimize biocontrol strategies. These products are able to activate plant defenses and thus limit infection by pathogens. However, the effectiveness of these plant defense stimulators remains erratic and is potentially dependent on many agronomic and environmental parameters still unknown or poorly controlled. The developmental stage of the plant as well as its fertilization, and essentially nitrogen nutrition, play major roles in defense establishment in the presence of pathogens or plant defense stimulators. The major nitrogen source used by plants is nitrate. In this study, we investigated the impact of plant developmental stage and nitrate nutrition on its capacity to mount immune reactions in response to two plant defense stimulators triggering two major defense pathways, the salicylic acid and the jasmonic acid pathways. We show that optimal nitrate nutrition is needed for effective defense activation and protection against the pathogenic bacteria and pv. . Using an defense signaling mutant, we showed that nitrate dependent protection against requires a functional gene. Our results indicate that the efficacy of plant defense stimulators is strongly affected by nitrate nutrition and the developmental stage. The nitrate dependent efficacy of plant defense stimulators is not only due to a metabolic effect but also invloves NPR1 mediated defense signaling. Plant defense stimulators may have opposite effects on plant resistance to a pathogen. Together, our results indicate that agronomic use of plant defense stimulators must be optimized according to nitrate fertilization and developmental stage.
PubMed: 32528493
DOI: 10.3389/fpls.2020.00583 -
Frontiers in Microbiology 2023For a coherent response to environmental changes, bacterial evolution has formed a complex transcriptional regulatory system comprising classical DNA binding proteins...
For a coherent response to environmental changes, bacterial evolution has formed a complex transcriptional regulatory system comprising classical DNA binding proteins sigma factors and modulation of DNA topology. In this study, we investigate replication-induced gene copy numbers - a regulatory concept that is unlike the others not based on modulation of promoter activity but on replication dynamics. We show that a large fraction of genes are predominantly affected by transient copy numbers and identify cellular functions and central pathways governed by this mechanism in . Furthermore, we show quantitatively that the previously observed spatio-temporal expression pattern between different growth phases mainly emerges from transient chromosomal copy numbers. We extend the analysis to the plant pathogen and the biotechnologically relevant organism . The analysis reveals a connection between growth phase dependent gene expression and evolutionary gene migration in these species. A further extension to the bacterial kingdom indicates that chromosome evolution is governed by growth rate related transient copy numbers.
PubMed: 37152747
DOI: 10.3389/fmicb.2023.1119878 -
Frontiers in Microbiology 2024The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The species complex is a...
The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The species complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels. The strain 1D1609 genome encodes one main T6SS gene cluster and four genes (i.e., ), each encoding a spike protein as an effector carrier. A previous study reported that associated gene 2, named encodes a His-Me finger nuclease toxin (also named HNH/ENDO VII nuclease), contributing to DNase-mediated antibacterial activity. However, the functions and roles of other putative effectors remain unknown. In this study, we identified associated gene 2 () that encodes another His-Me finger nuclease but with a distinct Serine Histidine Histidine (SHH) motif that differs from the AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in using DNAse activity assay and fluorescence microscopy. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues the phenotypes of growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retains the cell elongation phenotype, while V2a induces cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activity and . Notably, V2c also mediated the DNA degradation and cell elongation of the target cell in the context of interbacterial competition. Importantly, V2a and V2c exhibit different capacities against different bacterial species and function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, .
PubMed: 38426053
DOI: 10.3389/fmicb.2024.1351590 -
International Journal of Molecular... Nov 2019The prominent antibacterial and quorum sensing (QS) inhibition activity of aromatic plants can be used as a novel intervention strategy for attenuating bacterial...
The prominent antibacterial and quorum sensing (QS) inhibition activity of aromatic plants can be used as a novel intervention strategy for attenuating bacterial pathogenicity. In the present work, a total of 29 chemical components were identified in the essential oil (EO) of leaves by gas chromatography-mass spectrometry (GC-MS). The principal component was methyleugenol, followed by methyl trans-cinnamate, with relative contents of 90.46% and 4.25%, respectively. Meanwhile, the antibacterial activity and the QS inhibitory activity of EO were first evaluated here. Antibacterial activity assay and MIC detection against seven pathogens ( Onc5, ATCC25933, spp., ATCC25922, MG1, PAO1 and ATCC31532) demonstrated that ATCC25933 and MG1 had the higher sensitivity to EO, while PAO1 displayed the strongest resistance to EO. An anti-QS (anti-quorum sensing) assay revealed that at sub-minimal inhibitory concentrations (sub-MICs), EO strongly interfered with the phenotype, including violacein production, biofilm biomass, and swarming motility, as well as -hexanoyl-L-homoserine lactone (C6-HSL) production (i.e., a signaling molecule in ATCC31532) of . Detection of C6-HSL indicated that EO was capable of not only inhibiting C6-HSL production in , but also degrading the C6-HSL. Importantly, changes of exogenous C6-HSL production in CV026 revealed a possible interaction between EO and a regulatory protein (cviR). Additionally, quantitative real-time polymerase chain reaction (RT-qPCR) analysis demonstrated that the expression of QS-related genes (, , , , , , , and ) was significantly suppressed. Conclusively, these results indicated that EO can act as a potential antibacterial agent and QS inhibitor (QSI) against pathogens, preventing and controlling bacterial contamination.
Topics: Anti-Bacterial Agents; Bacteria; Biofilms; Gas Chromatography-Mass Spectrometry; Melaleuca; Microbial Sensitivity Tests; Oils, Volatile; Phenotype; Plant Extracts; Plant Leaves; Quorum Sensing
PubMed: 31739398
DOI: 10.3390/ijms20225696