Biochemical properties of CumA multicopper oxidase from plant pathogen, Pseudomonas syringae

2021 ◽  
Author(s):  
Konan Ishida ◽  
Yuya Tsukamoto ◽  
Masaki Horitani ◽  
Tomohisa Ogawa ◽  
Yoshikazu Tanaka
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Cell ◽  
Plant Pathogens ◽  
Plant Cell Wall ◽  
Copper Ions ◽  
Multicopper Oxidase ◽  
Type I ◽  
Multicopper Oxidases ◽  
Oxidation Activity ◽  
Wide Range

Abstract Multicopper oxidases have a wide range of substrate specificity to be involved in various physiological reactions. Pseudomonas syringae, a plant pathogenic bacterium, has a multicopper oxidase, CumA. Multicopper oxidases have ability to degrade plant cell wall component, lignin. Once P. syringae enter apoplast and colonize, they start to disrupt plant immunity. Therefore, deeper understanding of multicopper oxidases from plant pathogens, help to invent measures to prevent invasion into plant cell, which bring agricultural benefits. Several biochemical studies have reported lower activity of CumA compared with other multicopper oxidase called CotA. However, the mechanisms underlying the difference in activity have not yet been revealed. In order to acquire insight into them, we conducted a biophysical characterization of PsCumA. Our results show that PsCumA has weak type I copper EPR signal, which is essential for oxidation activity. We propose that difference in the coordination of copper ions may decrease reaction frequency.

scholarly journals Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Erika Berenice Martínez-Ruiz ◽  
Myriel Cooper ◽  
Jimena Barrero-Canosa ◽  
Mindia A. S. Haryono ◽  
Irina Bessarab ◽  
...  
Keyword(s):  
Calvin Cycle ◽  
Multicopper Oxidase ◽  
Genomic Islands ◽  
Biotechnological Applications ◽  
High Similarity ◽  
Multicopper Oxidases ◽  
Metabolic Potential ◽  
Natural Habitats ◽  
Manganese Oxidation ◽  
Wide Range

Abstract Background Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. Results Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. Conclusions The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.

scholarly journals Immunocytochemical Localization of HrpA and HrpZ Supports a Role for the Hrp Pilus in the Transfer of Effector Proteins from Pseudomonas syringae pv. tomato Across the Host Plant Cell Wall

2001 ◽  
Vol 14 (3) ◽  
pp. 394-404 ◽  
Author(s):  
Ian R. Brown ◽  
John W. Mansfield ◽  
Suvi Taira ◽  
Elina Roine ◽  
Martin Romantschuk
Keyword(s):  
Cell Wall ◽  
Pseudomonas Syringae ◽  
Plant Cell ◽  
Type Iii Secretion ◽  
Plant Cell Wall ◽  
Immunogold Labeling ◽  
Effector Proteins ◽  
Basal Bodies ◽  
Type Iii

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 μm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

scholarly journals Adaptor Scaffoldins: An Original Strategy for Extended Designer Cellulosomes, Inspired from Nature

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Johanna Stern ◽  
Sarah Moraïs ◽  
Raphael Lamed ◽  
Edward A. Bayer
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Soluble Sugars ◽  
Single Type ◽  
Wall Material ◽  
Type I ◽  
Type Ii ◽  
Recombinant Enzymes ◽  
Designer Cellulosome

ABSTRACTDesigner cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The largest designer cellulosome reported to date is a chimeric scaffoldin that contains 6 cohesins. This scaffoldin represented a technical limit of sorts, since adding another cohesin proved problematic, owing to resultant low expression levels, instability (cleavage) of the scaffoldin polypeptide, and limited numbers of available cohesin-dockerin specificities—the hallmark of designer cellulosomes. Nevertheless, increasing the number of enzymes integrated into designer cellulosomes is critical, in order to further enhance degradation of plant cell wall material. Adaptor scaffoldins comprise an intermediate type of scaffoldin that can both incorporate various enzymes and attach to an additional scaffoldin. Using this strategy, we constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional scaffoldin, and a carbohydrate-binding module for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by the incorporation of an appropriate type II cohesin. The resultant extended designer cellulosome comprised 8 recombinant enzymes—4 xylanases and 4 cellulases—thereby representing a potent enzymatic cocktail for solubilization of natural lignocellulosic substrates. The contribution of the adaptor scaffoldin clearly demonstrated that proximity between the two scaffoldins and their composite set of enzymes is crucial for optimized degradation. After 72 h of incubation, the performance of the extended designer cellulosome was determined to be approximately 70% compared to that of native cellulosomes.IMPORTANCEPlant cell wall residues represent a major source of renewable biomass for the production of biofuels such as ethanol via breakdown to soluble sugars. The natural microbial degradation process, however, is inefficient for achieving cost-effective processes in the conversion of plant-derived biomass to biofuels, either from dedicated crops or human-generated cellulosic wastes. The accumulation of the latter is considered a major environmental pollutant. The development of designer cellulosome nanodevices for enhanced plant cell wall degradation thus has major impacts in the fields of environmental pollution, bioenergy production, and biotechnology in general. The findings reported in this article comprise a true breakthrough in our capacity to produce extended designer cellulosomes via synthetic biology means, thus enabling the assembly of higher-order complexes that can supersede the number of enzymes included in a single multienzyme complex.

scholarly journals A pattern-triggered immunity-related phenolic, acetosyringone, boosts rapid inhibition of a diverse set of plant pathogenic bacteria

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ágnes Szatmári ◽  
Ágnes M. Móricz ◽  
Ildikó Schwarczinger ◽  
Judit Kolozsváriné Nagy ◽  
Ágnes Alberti ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Membrane ◽  
Pseudomonas Syringae ◽  
Bacterial Cell ◽  
Plant Pathogens ◽  
Bacterial Attachment ◽  
Bacterial Metabolism ◽  
Free Form ◽  
Wide Range ◽  
Bacterial Cell Membrane

Abstract Background Acetosyringone (3,5-dimethoxy-4-hydroxyacetophenone, AS) is a syringyl-type phenolic compound rarely found in plants in free form. It has been shown earlier to inhibit the growth of Pseudomonas bacteria in the presence of hydrogen peroxide and peroxidase (AS mix). Results We detected elevated levels of free AS in Nicotiana tabacum and N. benthamiana plants after inducing pattern-triggered immunity (PTI) by injecting bacterial elicitor flg22, or pathogenicity-mutant Pseudomonas syringae pv. syringae 61 hrcC- bacteria; but not after inoculations with compatible or incompatible pathogens at the time of PTI onset. In this study, we demonstrate that the antibacterial effect of the AS mix is general, as growth of several Gram-negative and -positive phytopathogenic bacteria was characteristically inhibited. The inhibition of bacterial metabolism by the AS mix was rapid, shown by the immediate drop of luminescence intensity of P. syringae pv. tomato DC3000 lx strain after addition of AS mix. The mechanism of the bacteriostatic effect was investigated using fluorescent reporter dye assays. SYTOX Green experiments supported others’ previous findings that the AS mix does not result in membrane permeabilization. Moreover, we observed that the mode of action could be depolarization of the bacterial cell membrane, as shown by assays carried out with the voltage sensitive dye DIBAC4(3). Conclusions Level of free acetosyringone is elevated during plant PTI responses in tobacco leaves (N. tabacum and N. benthamiana). When combined with hydrogen peroxide and peroxidase (AS mix), components of the mix act synergistically to inhibit bacterial metabolism and proliferation rapidly in a wide range of plant pathogens. This effect is related to depolarization rather than to permeabilization of the bacterial cell membrane. Similar AS mixture to the in vivo model might form locally at sites of invading bacterial attachment to the plant cells and the presence of acetosyringone might have an important role in the inhibition of bacterial proliferation during PTI.

scholarly journals Leafhopper Viral Pathogens: Ultrastructure of salivary gland infection of Taastrup-like virus inPsammotettix alienusDahlbom; and a novel Rhabdovirus inHomalodisca vitripennis(Germar) Hemiptera: Cicadellidae

2018 ◽  
Author(s):  
Thorben Lundsgaard ◽  
Wayne B. Hunter ◽  
Scott Adkins
Keyword(s):  
Next Generation Sequencing ◽  
Plant Pathogens ◽  
Insect Pests ◽  
Economic Losses ◽  
Type I ◽  
Next Generation ◽  
Viral Pathogen ◽  
Thin Sections ◽  
Wide Range ◽  
Generation Sequencing

AbstractViruses that are pathogenic to insect pests can be exploited as biological control agents. Viruses that are pathogenic to beneficial insects and other arthropods, as in honey bees, silk worms, and shrimp, cause millions of dollars of losses to those industries. Current advances in next generation sequencing technologies along with molecular and cellular biology have produced a wealth of information about insect viruses and their potential applications. Leafhoppers cause economic losses as vectors of plant pathogens which significantly reduce the worlds’ food crops. Each year more viruses are discovered primarily through the use of next generation sequencing of the leafhopper hosts. The diversity of viruses from leafhoppers demonstrates a wide range of taxonomic members that includes genomes of DNA or RNA from families like: Reoviridae, Iridoviridae, Dicistroviridae, Iflaviridae, and others yet to be classified. Discussed is a recent viral pathogen isolated from the leafhopperPsammotettix alienus, name Taastrup Virus. Taastrup virus (TV) is a novel virus with a RNA genome, a Filovirus-like morphology, being tentatively placed within theMononegavirales. AdultPsammotettix alienusinfected with TV, showed the highest concentration of virions in salivary glands, consisting of a principal gland (type I-VI-cells) and an accessory gland. Examination of thin sections revealed enveloped particles, about 1300 nm long and 62 nm in diameter, located singly or in paracrystalline arrays in canaliculi of type III- and IV-cells. In gland cells with TV particles in canaliculi, granular masses up to 15 μm in diameter were present in the cytoplasm. These masses are believed to be viroplasms, the sites for viral replication. TV particles were observed at the connection between a canaliculus and the salivary duct system. A TV-like virus with strongly similar morphology was discovered in the ornamental plant,Liriope, near Fort Pierce, Florida, USA. When the virus was inoculated to a leafhopper cell culture, HvWH, made from the glassy-winged sharpshooter,Homalodisca vitripennis(Germar), the cells rapidly degraded with 100% mortality in 48 hours. These two instances are the only reported cases of this newly discovered viral pathogen of leafhoppers.

scholarly journals Arabidopsis thaliana Cells: A Model to Evaluate the Virulence of Pectobacterium carotovorum

2010 ◽  
Vol 23 (2) ◽  
pp. 139-143 ◽  
Author(s):  
Meriam Terta ◽  
Mohamed Kettani-Halabi ◽  
Khadija Ibenyassine ◽  
Daniel Tran ◽  
Patrice Meimoun ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Death ◽  
Plant Cell ◽  
Plant Pathogens ◽  
Plant Cell Wall ◽  
Pectate Lyase ◽  
Soft Rot ◽  
Pectobacterium Carotovorum ◽  
Cell Wall Degrading Enzymes ◽  
Suspension Cells

Pectobacterium carotovorum are economically important plant pathogens that cause plant soft rot. These enterobacteria display high diversity world-wide. Their pathogenesis depends on production and secretion of virulence factors such as plant cell wall–degrading enzymes, type III effectors, a necrosis-inducing protein, and a secreted virulence factor from Xanthomonas spp., which are tightly regulated by quorum sensing. Pectobacterium carotovorum also present pathogen-associated molecular patterns that could participate in their pathogenicity. In this study, by using suspension cells of Arabidopsis thaliana, we correlate plant cell death and pectate lyase activities during coinfection with different P. carotovorum strains. When comparing soft rot symptoms induced on potato slices with pectate lyase activities and plant cell death observed during coculture with Arabidopsis thaliana cells, the order of strain virulence was found to be the same. Therefore, Arabidopsis thaliana cells could be an alternative tool to evaluate rapidly and efficiently the virulence of different P. carotovorum strains.

Elucidation of the crystal structure ofCoriolopsis caperatalaccase: restoration of the structure and activity of the native enzyme from the T2-depleted form by copper ions

2015 ◽  
Vol 71 (4) ◽  
pp. 854-861 ◽  
Author(s):  
Olga A. Glazunova ◽  
Konstantin M. Polyakov ◽  
Tatyana V. Fedorova ◽  
Pavel V. Dorovatovskii ◽  
Olga V. Koroleva
Keyword(s):  
Copper Ions ◽  
Large Family ◽  
Native Enzyme ◽  
Multicopper Oxidases ◽  
Wide Range ◽  
Inorganic Substrates ◽  
Structure And Activity ◽  
Copper Centre ◽  
High Redox Potential

Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. A new laccase was isolated from the basidiomyceteCoriolopsis caperatastrain 0677 and its amino-acid sequence was determined. According to its physicochemical properties and spectroscopic features, the laccase fromC. caperatais a high redox-potential blue laccase. Attempts to crystallize the native enzyme were unsuccessful. The copper type 2-depleted (T2D) laccase was prepared and crystallized. The structure of T2D laccase fromC. caperatawas solved at 1.6 Å resolution, and attempts to reconstruct the T2 copper centre were performed using Cu+and Cu2+ions. The structure of T2D+Cu+laccase was solved at 1.89 Å resolution. It was shown that the T2D+Cu+laccase structure contained four copper ions in the active site. Reconstruction could not be achieved when the T2D laccase crystals were treated with CuSO4.

scholarly journals Isolation and utilization of cellulosic elements from the plant cell wall

Botany ◽  
2020 ◽  
Vol 98 (1) ◽  
pp. 77-80
Author(s):  
Derek G. Gray
Keyword(s):  
Plant Cell ◽  
Liquid Crystalline ◽  
Plant Cell Wall ◽  
Cellulose Nanofibrils ◽  
Polarized Light ◽  
Renewable Materials ◽  
Tropical Plants ◽  
Aqueous Gels ◽  
Wide Range ◽  
Commercial Applications

Two very different types of cellulosic materials that may be isolated from plant-based sources are reviewed herein. We have shown that long helical cellulosic coils may be isolated by gentle chemical treatment of leaf petioles. The coils stem from protoxylem elements and are readily distinguished from other cellulosic components by polarized light microscopy, forming single- or multistranded left-handed helices. More widely known are the nanometer thickness cellulosic elements that may also be isolated from plant cell walls. These nanocelluloses can be subdivided into two main families. Cellulose nanocrystals (CNCs) are less than 250 nm long and are usually made by sulfuric acid hydrolysis. CNCs form stable liquid crystalline suspensions in water that dry to give iridescent coloured films, analogous to those found in some tropical plants. Cellulose nanofibrils (CNFs) are much longer and may have carboxyl groups on the surface. Both CNCs and CNFs are intrinsically hydrophilic and form useful aqueous gels and viscosity modifiers. By altering the surface chemistry, the nanocelluloses can also be incorporated as strengthening agents in composite materials. A wide range of commercial applications for these renewable materials has been proposed.

Structure and molecular evolution of multicopper blue proteins

2010 ◽  
Vol 1 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Hirofumi Komori ◽  
Yoshiki Higuchi
Keyword(s):  
Molecular Evolution ◽  
Nitrite Reductase ◽  
Quaternary Structure ◽  
Multicopper Oxidase ◽  
Protein Family ◽  
Type I ◽  
Multicopper Oxidases ◽  
Different Types ◽  
Diverse Groups ◽  
Insight Into

AbstractThe multicopper blue protein family, which contains cupredoxin-like domains as a structural unit, is one of the most diverse groups of proteins. This protein family is divided into two functionally different types of enzymes: multicopper oxidase and nitrite reductase. Multicopper oxidase catalyzes the oxidation of the substrate and then reduces dioxygen. The structures of many multicopper oxidases are already known, and until recently they were classified into two main groups: the three- and six-domain types. Both function as monomers and have three spectroscopically different copper sites: Types I (blue), II, and III (tri-nuclear). Nitrite reductase is a closely related protein that contains Types I and II (mono-nuclear) coppers but reduces nitrite instead of dioxygen. Nitrite reductase, which consists of two domains, forms a homotrimer. Multicopper oxidase and nitrite reductase share similar structural architectures and also contain Type I copper. Therefore, it is proposed that they have a common ancestor protein. Recently, some two-domain type multicopper oxidases have been found and their crystal structures have been determined. They have a trimeric quaternary structure and contain an active site at the molecular interface such as nitrite reductase. These results support previous hypotheses and provide an insight into the molecular evolution of multicopper blue proteins.

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