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Pharmaceutics Mar 2022L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different... (Review)
Review
L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different biochemical, physicochemical and pharmacological properties are found in many organisms, including bacteria, fungi, algae, plants and mammals. To date, asparaginases from and (formerly known as ) are widely used in hematology for the treatment of lymphoblastic leukemias. However, their medical use is limited by side effects associated with the ability of these enzymes to hydrolyze L-glutamine, as well as the development of immune reactions. To solve these issues, gene-editing methods to introduce amino-acid substitutions of the enzyme are implemented. In this review, we focused on molecular analysis of the mechanism of enzyme action and to optimize the antitumor activity.
PubMed: 35335974
DOI: 10.3390/pharmaceutics14030599 -
Frontiers in Microbiology 2022, a plant soft-rot pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors, infecting a wide variety of monocotyledonous and...
, a plant soft-rot pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors, infecting a wide variety of monocotyledonous and dicotyledonous plants and causing serious losses to the production of economic crops. In order to alleviate the problem of pesticide resistance during bacterial disease treatment, compounds targeting at T3SS have been screened using a bioreporter. After screening by Multifunctional Microplate Reader and determining by flow cytometer, five compounds including salicylic acid (SA), p-hydroxybenzoic acid (PHBA), cinnamyl alcohol (CA), p-coumaric acid (PCA), and hydrocinnamic acid (HA) significantly inhibiting promoter activity without affecting bacterial growth have been screened out. All the five compounds reduced hypersensitive response (HR) on non-host tobacco leaves and downregulated the expression of T3SS, especially the master regulator encoding gene . Inhibition efficacy of the five compounds against soft rot were also evaluated and results confirmed that the above compounds significantly lessened the soft-rot symptoms caused by 3937 on potato, CL3 on taro, EC1 on rice, and MS2 on banana seedlings. Findings in this study provide potential biocontrol agents for prevention of soft-rot disease caused by spp.
PubMed: 35273588
DOI: 10.3389/fmicb.2022.839025 -
Frontiers in Microbiology 2022species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (). Using random barcode transposon-site...
species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (). Using random barcode transposon-site sequencing (RB-TnSeq), we generated genome-wide mutant fitness profiles of 3937, ME23, and 67-19 isolates collected after passage through several and conditions. Though all three strains are pathogenic on potato, 3937 is a well-characterized model while strains ME23 and 67-19 are recent isolates. Strain ME23 specifically was identified as a representative strain from a 2014 outbreak on potato. This study generated comparable gene fitness measurements across ecologically relevant conditions for both model and non-model strains. Tubers from the potato cultivars "Atlantic," "Dark Red Norland," and "Upstate Abundance" provided highly similar conditions for bacterial growth. Using the homolog detection software PyParanoid, we matched fitness values for orthologous genes in the three bacterial strains. Direct comparison of fitness among the strains highlighted shared and variable traits important for growth. Bacterial growth in minimal medium required many metabolic traits that were also essential for competitive growth , such as amino acid, carbohydrate, and nucleotide biosynthesis. Growth in tubers specifically required the pectin degradation gene . Disruption in three putative DNA-binding proteins had strain-specific effects on competitive fitness in tubers. Though the Soft Rot can cause disease with little host specificity, it remains to be seen the extent to which strain-level variation impacts virulence.
PubMed: 35145503
DOI: 10.3389/fmicb.2022.778927 -
The Journal of Biological Chemistry Jan 2022The catabolism of pectin from plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel...
The catabolism of pectin from plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce massive pectinolytic activity during the maceration phase. Previous studies identified the role of a positive feedback loop specific to the pectin-degradation pathway, whereas the precise signals controlling the dynamics of pectate lyase expression were unclear. Here, we show that the latter is controlled by a metabolic switch involving both glucose and pectin. We measured the HPLC concentration profiles of the key metabolites related to these two sources of carbon, cAMP and 2-keto-3-deoxygluconate, and developed a dynamic and quantitative model of the process integrating the associated regulators, cAMP receptor protein and KdgR. The model describes the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights that their activity is controlled by a mechanism of carbon catabolite repression, which directly controls the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitatively different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, justifying their evolutionary conservation as separate genes in this species.
Topics: Bacterial Proteins; Catabolite Repression; Dickeya; Digestion; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Pectins; Polysaccharide-Lyases
PubMed: 34826421
DOI: 10.1016/j.jbc.2021.101446 -
Scientific Reports Oct 2021RecA plays a central role in DNA repair and is a main actor involved in recombination and activation of the SOS response. It is also used in the context of... (Comparative Study)
Comparative Study
RecA plays a central role in DNA repair and is a main actor involved in recombination and activation of the SOS response. It is also used in the context of biotechnological applications in recombinase polymerase isothermal amplification (RPA). In this work, we studied the biological properties of seven RecA variants, in particular their recombinogenic activity and their ability to induce the SOS response, to better understand the structure-function relationship of RecA and the effect of combined mutations. We also investigated the biochemical properties of RecA variants that may be useful for the development of biotechnological applications. We showed that Dickeya dadantii RecA (DdRecA) had an optimum strand exchange activity at 30 °C and in the presence of a dNTP mixture that inhibited Escherichia coli RecA (EcRecA). The differences between the CTD and C-tail of the EcRecA and DdRecA domains could explain the altered behaviour of DdRecA. D. radiodurans RecA (DrRecA) was unable to perform recombination and activation of the SOS response in an E. coli context, probably due to its inability to interact with E. coli recombination accessory proteins and SOS LexA repressor. DrRecA strand exchange activity was totally inhibited in the presence of chloride ions but worked well in acetate buffer. The overproduction of Pseudomonas aeruginosa RecA (PaRecA) in an E. coli context was responsible for a higher SOS response and defects in cellular growth. PaRecA was less inhibited by the dNTP mixture than EcRecA. Finally, the study of three variants, namely, EcPa, EcRecAV1 and EcRecAV2, that contained a combination of mutations that, taken independently, are described as improving recombination, led us to raise new hypotheses on the structure-function relationship and on the monomer-monomer interactions that perturb the activity of the protein as a whole.
Topics: DNA-Binding Proteins; Deinococcus; Dickeya; Escherichia coli; Escherichia coli Proteins; Pseudomonas aeruginosa; Rec A Recombinases; Species Specificity
PubMed: 34702889
DOI: 10.1038/s41598-021-00589-9 -
Microorganisms Sep 2021(Pbr) 1692 is an aggressive phytopathogen affecting a broad host range of crops and ornamental plants, including potatoes. Previous research on animal pathogens, and a...
(Pbr) 1692 is an aggressive phytopathogen affecting a broad host range of crops and ornamental plants, including potatoes. Previous research on animal pathogens, and a few plant pathogens, revealed that Outer Membrane Vesicles (OMVs) are part of Gram-negative bacteria's (GNB) adaptive toolkit. For this reason, OMV production and subsequent release from bacteria is a conserved process. Therefore, we hypothesized that OMVs might transport proteins that play a critical role in causing soft rot disease and in the survival and fitness of Pbr1692. Here, we show that the potato pathogen, Pbr1692, releases OMVs of various morphologies in Luria Bertani media at 31 °C. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) confirmed the production of OMVs by Pbr1692 cells. Transmission Electron Microscopy showed that these exist as chain-, single-, and double-membrane morphologies. Mass spectrometry followed by Gene Ontology, Clusters of Orthologous Groups, Virulence Factor, CAZymes, Antibiotic Resistance Ontology, and Bastion6 T6SE annotations identified 129 OMV-associated proteins with diverse annotated roles, including antibiotic stress response, virulence, and competition. Pbr1692 OMVs contributed to virulence in potato tubers and elicited a hypersensitive response in leaves. Furthermore, Pbr1692 OMVs demonstrated antibacterial activity against .
PubMed: 34576813
DOI: 10.3390/microorganisms9091918 -
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 -
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 -
Frontiers in Microbiology 2021is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the...
is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the transcription of its genes encoding the main virulence functions, little information is available on the post-transcriptional regulation of these functions. We investigated the involvement of the RNA chaperones Hfq and ProQ in the production of the main virulence functions. Phenotypic assays on the and mutants showed that inactivation of resulted in a growth defect, a modified capacity for biofilm formation and strongly reduced motility, and in the production of degradative extracellular enzymes (proteases, cellulase, and pectate lyases). Accordingly, the mutant failed to cause soft rot on chicory leaves. The mutant had reduced resistance to osmotic stress, reduced extracellular pectate lyase activity compared to the wild-type strain, and reduced virulence on chicory leaves. Most of the phenotypes of the and mutants were related to the low amounts of mRNA of the corresponding virulence factors. Complementation of the double mutant by each individual protein and cross-complementation of each chaperone suggested that they might exert their effects via partially overlapping but different sets of targets. Overall, it clearly appeared that the two Hfq and ProQ RNA chaperones are important regulators of pathogenicity in This underscores that virulence genes are regulated post-transcriptionally by non-coding RNAs.
PubMed: 34248909
DOI: 10.3389/fmicb.2021.687484 -
International Journal of Molecular... Jun 2021Coumarins belong to a group of secondary metabolites well known for their high biological activities including antibacterial and antifungal properties. Recently, an...
Coumarins belong to a group of secondary metabolites well known for their high biological activities including antibacterial and antifungal properties. Recently, an important role of coumarins in plant resistance to pathogens and their release into the rhizosphere upon pathogen infection was discovered. It is also well documented that coumarins play a crucial role in the growth under Fe-limited conditions. However, the mechanisms underlying interplay between plant resistance, accumulation of coumarins and Fe status, remain largely unknown. In this work, we investigated the effect of both mentioned factors on the disease severity using the model system of Arabidopsis/ spp. molecular interactions. We evaluated the disease symptoms in Arabidopsis plants, wild-type Col-0 and its mutants defective in coumarin accumulation, grown in hydroponic cultures with contrasting Fe regimes and in soil mixes. Under all tested conditions, Arabidopsis plants inoculated with IFB0099 strain developed more severe disease symptoms compared to lines inoculated with 3937. We also showed that the expression of genes encoding plant stress markers were strongly affected by IFB0099 infection. Interestingly, the response of plants to 3937 infection was genotype-dependent in Fe-deficient hydroponic solution.
Topics: Arabidopsis; Coumarins; Dickeya; Disease Resistance; Disease Susceptibility; Hydroponics; Iron; Plant Diseases; Plant Leaves; Plants; Stress, Physiological
PubMed: 34208600
DOI: 10.3390/ijms22126449