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Biomolecular NMR Assignments Oct 2022The ability to interact and adapt to the surrounding environment is vital for bacteria that colonise various niches and organisms. One strategy developed by...
The ability to interact and adapt to the surrounding environment is vital for bacteria that colonise various niches and organisms. One strategy developed by Gram-negative bacteria is to secrete exoprotein substrates via the type II secretion system (T2SS). The T2SS is a proteinaceous complex spanning the bacterial envelope that translocates folded proteins such as toxins and enzymes from the periplasm to the extracellular milieu. In the T2SS, a cytoplasmic ATPase elongates in the periplasm the pseudopilus, a non-covalent polymer composed of protein subunits named pseudopilins, and anchored in the inner membrane by a transmembrane helix. The pseudopilus polymerisation is coupled to the secretion of substrates. The T2SS of Dickeya dadantii secretes more than 15 substrates, essentially plant cell wall degrading enzymes. In D. dadantii, the major pseudopilin or the major subunit of the pseudopilus is called OutG. To better understand the mechanism of secretion of these numerous substrates via the pseudopilus, we have been studying the structure of OutG by NMR. Here, as the first part of this study, we report the H, N and C backbone and sidechain chemical shift assignment of the periplasmic domain of OutG and its NMR derived secondary structure.
Topics: Adenosine Triphosphatases; Bacterial Proteins; Dickeya; Nuclear Magnetic Resonance, Biomolecular; Periplasm; Polymers; Protein Binding; Protein Subunits; Type II Secretion Systems
PubMed: 35482172
DOI: 10.1007/s12104-022-10085-4 -
Molecular Plant Pathology Aug 2022Bacteria use signal transduction systems to sense and respond to their external environment. The two-component system CpxA/CpxR senses misfolded envelope protein stress...
Bacteria use signal transduction systems to sense and respond to their external environment. The two-component system CpxA/CpxR senses misfolded envelope protein stress and responds by up-regulating envelope protein factors and down-regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS-inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c-di-GMP regulators were also up-regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c-di-GMP phenotypes in biofilm formation and swimming. Increased production of cellular c-di-GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c-di-GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.
Topics: Bacterial Proteins; Cyclic GMP; Dickeya; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Virulence
PubMed: 35460168
DOI: 10.1111/mpp.13219 -
PloS One 2022Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for Dickeya virulence....
Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for Dickeya virulence. Previous studies showed that the D. dadantii T2SS secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins possess a signal peptide and are first addressed to the periplasm before their recruitment by Out. T2SS-specific secretion signals remain unknown which prevents in silico identification of T2SS substrates. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced and encoding proteins with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and that svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.
Topics: Bacterial Proteins; Dickeya; Enterobacteriaceae; Gammaproteobacteria; Plant Diseases; Type II Secretion Systems; Virulence Factors
PubMed: 35417462
DOI: 10.1371/journal.pone.0265075 -
Microbiology Spectrum Apr 2022Dickeya dadantii is a phytopathogenic bacterium that causes diseases on a wide range of host plants. The pathogen secretes pectate lyases (Pel) through the type II...
Dickeya dadantii is a phytopathogenic bacterium that causes diseases on a wide range of host plants. The pathogen secretes pectate lyases (Pel) through the type II secretion system (T2SS) that degrades the cell wall in host plants. The virulence of is controlled by the second messenger cyclic diguanylate monophosphate (c-di-GMP), and the homeostasis of c-di-GMP is maintained by a number of diguanylate cyclases and phosphodiesterases. Deletion of a phosphodiesterase repressed transcription, and such repression can be suppressed by an additional deletion in . VfmE is an AraC type of transcriptional regulator in the Vfm quorum-sensing system. Our results suggest that VfmE is a c-di-GMP effector that functions as an activator of at low c-di-GMP concentrations and a repressor of at high c-di-GMP concentrations through regulation of the transcriptional activator SlyA. Multiple sequence alignment with known c-di-GMP effectors identified an RWIWR motif in VfmE that we demonstrate is required for the c-di-GMP binding. Mutation of R93D in the RxxxR motif eliminates the c-di-GMP-related phenotypes in Pel activity. Our results show that VfmE is not only a quorum-sensing regulator but also a c-di-GMP effector, suggesting that integrates the c-di-GMP signaling network with the Vfm quorum-sensing pathway during environmental adaptation. How bacteria integrate environmental cues from multiple sources to appropriately regulate adaptive phenotypes is a central question in microbiology. In Dickeya dadantii, the quorum-sensing regulator VfmE controls the key virulence factor pectate lyase (Pel). Here, we demonstrate that VfmE also binds to c-di-GMP, resulting in VfmE functioning as an activator of at low c-di-GMP concentrations and repressor of at high c-di-GMP concentrations. The RWIWR motif in VfmE is required for c-di-GMP binding, and mutation of the motif in the mutant R93D eliminates the c-di-GMP-related phenotypes in Pel activity. We propose that VfmE is an important mediator to integrate quorum-sensing signals with c-di-GMP to collectively regulate pathogenesis.
Topics: Bacterial Proteins; Cyclic GMP; Dickeya; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Polysaccharide-Lyases
PubMed: 35352959
DOI: 10.1128/spectrum.01805-21 -
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