The chemical structure of the O-polysaccharide isolated from the lipopolysaccharide of Pectobacterium brasiliense IFB5527, a phytopathogenic bacterium of high economic importance.

Pectobacterium brasiliense is a widespread plant pathogenic bacterium classified to the Pectobacteriaceae family, which causes significant economic losses because of the developed soft rot and blackleg symptoms on potatoes and a wide spectrum of crops, vegetables, and ornamentals. One of the key virulence factors is a lipopolysaccharide due to its involvement in efficient colonisation of plant tissues and overcoming the host defence mechanisms. Thus, we structurally characterised the O-polysaccharide from the LPS of P. brasiliense strain IFB5527 (HAFL05) using chemical methods followed by GLC and GLC-MS as well as 1D and 2D NMR spectroscopy. The analyses revealed that the polysaccharide repeating unit consists of Fuc, Glc, GlcN and an unusual N-formylated 6-deoxy amino sugar, Qui3NFo, and has the structure shown below.

The structure of O-polysaccharide isolated from the type strain of Pectobacterium versatile CFBP6051T containing an erwiniose - higher branched monosaccharide.

Utilizing sugar, methylation, and absolute configurations analyses as well as NMR spectroscopy, the chemical repeating unit of the O-specific polysaccharide of Pectobacteriumversatile CFBP6051T was identified as: The polymer contains residues of an unusual, higher-branched monosaccharide, named erwiniose (3,6,8-trideoxy-4-C-(R-1-hydroxyethyl)-d-gulo-octose). Comparison of the P. versatile CFBP6051T O-polysaccharide with those isolated from strains of other Pectobacterium species indicated high differentiation in their structures within this genus.

Capillary zone electrophoresis of bacterial extracellular vesicles: A proof of concept.

The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs.

Detection of Endogenous Phthalates in Bacterial Pathogens of Plants and Animals.

The endogenous esters of orthophthalic acid, dibutyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP), have been first detected in bacterial pathogens of plants (Clavibacter michiganensis ssp. sepedonicus, Pectobacterium carotovorum ssp. carotovorum, Rhizobium rhizogenes, Rhizobium radiobacter) and bacterial pathogens of animal (Escherichia coli).

Pectobacterium zantedeschiae sp. nov. a new species of a soft rot pathogen isolated from Calla lily (Zantedeschia spp.).

Four Gram-negative, rod-shaped pectinolytic bacterial strains designated as 2M, 9M, DPMP599 and DPMP600 were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Strains 2M and 9M were isolated from Calla lily bulbs cultivated in Central Poland. DPMP599 and DPMP600 strains were isolated from Calla lily leaves from plants grown in Serbia. Phylogenetic analyses based on nine housekeeping genes (gapA, gyrA, icdA, pgi, proA, recA, recN, rpoA, and rpoS), as well as phylogeny based on the 381 most conserved universal proteins confirmed that Pectobacterium zantedeschiae strains were distantly related to the other Pectobacterium, and indicated Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium parmentieri and Pectobacterium wasabiae as the closest relatives. Moreover, the analysis revealed that Pectobacterium zantedeschiae strains are not akin to Pectobacterium aroidearum strains, which were likewise isolated from Calla lily. The genome sequencing of the strains 2M, 9M and DPMP600 and their comparison with whole genome sequences of other Pectobacterium type strains confirmed their distinctiveness and separate species status within the genus based on parameters of in silico DNA-DNA hybridization and average nucleotide identity (ANI) values. The MALDI-TOF MS proteomic profile supported the proposition of delineation of the P. zantedeschiae and additionally confirmed the individuality of the studied strains. Based on of all of these data, it is proposed that the strains 2M, 9M, DPMP599, and DPMP600 isolated from Calla lily, previously assigned as P. atrosepticum should be reclassified as Pectobacterium zantedeschiae sp. nov. with the strain 9MT (PCM2893=DSM105717=IFB9009) as the type strain.

Pectobacterium atrosepticum exopolysaccharides: identification, molecular structure, formation under stress and in planta conditions.

In the present study, we identified exopolysaccharides of the harmful phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 and characterized the molecular structure of these polymers. The synthesis of the target polysaccharides was shown to be induced under starvation conditions. Moreover, intensive accumulation of exopolysaccharides occurred during the colonization by bacteria of the xylem vessels of infected plants, where microorganisms formed specific 3D "multicellular" structures-bacterial emboli. Thus, the identified polymers are likely to be involved in the adaptation and virulence of bacteria of Pectobacterium genus.

Structure of the bacterial plant-ferredoxin receptor FusA.

Iron is a limiting nutrient in bacterial infection putting it at the centre of an evolutionary arms race between host and pathogen. Gram-negative bacteria utilize TonB-dependent outer membrane receptors to obtain iron during infection. These receptors acquire iron either in concert with soluble iron-scavenging siderophores or through direct interaction and extraction from host proteins. Characterization of these receptors provides invaluable insight into pathogenesis. However, only a subset of virulence-related TonB-dependent receptors have been currently described. Here we report the discovery of FusA, a new class of TonB-dependent receptor, which is utilized by phytopathogenic Pectobacterium spp. to obtain iron from plant ferredoxin. Through the crystal structure of FusA we show that binding of ferredoxin occurs through specialized extracellular loops that form extensive interactions with ferredoxin. The function of FusA and the presence of homologues in clinically important pathogens suggests that small iron-containing proteins represent an iron source for bacterial pathogens.

The structure of O-polysaccharides isolated from plant pathogenic bacteria Pectobacterium wasabiae IFB5408 and IFB5427.

O-Polysaccharides were isolated from the lipopolysaccharides of two strains of plant pathogenic bacteria Pectobacterium wasabiae isolated in Poland in 2013 (IFB5408 and IFB5427). The purified polysaccharides were analyzed using 1D and 2D NMR spectroscopy ((1)H, DQF-COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY, and HMBC) and the chemical methods. Sugar and methylation analyses of native polysaccharides, absolute configuration assignment of constituent monosaccharides together with NMR spectroscopy data revealed that the chemical structures of both O-polysaccharides are the same.

Characterization of Dickeya and Pectobacterium species by capillary electrophoretic techniques and MALDI-TOF MS.

Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.

Penicillin V acylase from Pectobacterium atrosepticum exhibits high specific activity and unique kinetics.

Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria.

Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake.

The colicin-like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram-negative bacteria by parasitizing nutrient uptake systems. We have structurally characterized the colicin M-like bacteriocin, pectocin M2, which is active against strains of Pectobacterium spp. This unusual bacteriocin lacks the intrinsically unstructured translocation domain that usually mediates translocation of these bacteriocins across the outer membrane, containing only a single globular ferredoxin domain connected to its cytotoxic domain by a flexible α-helix, which allows it to adopt two distinct conformations in solution. The ferredoxin domain of pectocin M2 is homologous to plant ferredoxins and allows pectocin M2 to parasitize a system utilized by Pectobacterium to obtain iron during infection of plants. Furthermore, we identify a novel ferredoxin-containing bacteriocin pectocin P, which possesses a cytotoxic domain homologous to lysozyme, illustrating that the ferredoxin domain acts as a generic delivery module for cytotoxic domains in Pectobacterium.

Nicotiflorin, rutin and chlorogenic acid: phenylpropanoids involved differently in quantitative resistance of potato tubers to biotrophic and necrotrophic pathogens.

Physiological and molecular mechanisms underlying quantitative resistance of plants to pathogens are still poorly understood, but could depend upon differences in the intensity or timing of general defense responses. This may be the case for the biosynthesis of phenolics which are known to increase after elicitation by pathogens. We thus tested the hypothesis that differences in quantitative resistance were related to differential induction of phenolics by pathogen-derived elicitors. Five potato cultivars (Solanum tuberosum, L.) spanning a range of quantitative resistance were treated with a concentrated culture filtrate (CCF) of Phytophthora infestans or purified lipopolysaccharides (LPS) from Pectobacterium atrosepticum. The kinetic of phenolics accumulation was followed and a set of typical phenolics was identified: chlorogenic acid, phenolamides and flavonols including rutin (quercetin-3-O-rutinoside) and nicotiflorin (kaempferol-3-O-rutinoside). Our results showed that CCF but not LPS induced differential accumulation of major phenolics among cultivars. Total phenolics were related with resistance to P. atrosepticum but not to P. infestans. However, nicotiflorin was inversely related with resistance to both pathogens. Rutin, but not nicotiflorin, inhibited pathogen growth in vitro at physiological concentrations. These data therefore suggest that (i) several phenolics are candidate markers for quantitative resistance in potato, (ii) some of these are pathogen specific although they are produced by a general defense pathway, (iii) resistance marker molecules do not necessarily have antimicrobial activity, and (iv) the final content of these target molecules-either constitutive or induced-is a better predictor of resistance than their inducibility by pathogen elicitors.

The role of secretion systems and small molecules in soft-rot Enterobacteriaceae pathogenicity.

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.

Chemical structure of the O-polysaccharide isolated from Pectobacterium atrosepticum SCRI 1039.

The lipopolysaccharide (LPS) of the bacterium Pectobacterium atrosepticum SCRI 1039 was hydrolyzed and the products were separated. A study of the obtained O-polysaccharide by means of chemical methods, GLC, GLC-MS, and NMR spectroscopy allowed us to identify a branched polymer with a pentasaccharide repeating unit of the structure shown below, in which the fucose residue was partially O-acetylated at C-2, C-3 or C-4.

A processed noncoding RNA regulates an altruistic bacterial antiviral system.

The ≥ 10³° bacteriophages on Earth relentlessly drive adaptive coevolution, forcing the generation of protective mechanisms in their bacterial hosts. One such bacterial phage-resistance system, ToxIN, consists of a protein toxin (ToxN) that is inhibited in vivo by a specific RNA antitoxin (ToxI); however, the mechanisms for this toxicity and inhibition have not been defined. Here we present the crystal structure of the ToxN-ToxI complex from Pectobacterium atrosepticum, determined to 2.75-Å resolution. ToxI is a 36-nucleotide noncoding RNA pseudoknot, and three ToxI monomers bind to three ToxN monomers to generate a trimeric ToxN-ToxI complex. Assembly of this complex is mediated entirely through extensive RNA-protein interactions. Furthermore, a 2'-3' cyclic phosphate at the 3' end of ToxI, and catalytic residues, identify ToxN as an endoRNase that processes ToxI from a repetitive precursor but is regulated by its own catalytic product.

Cloning, expression, and characterization of a new phytase from the phytopathogenic bacterium Pectobacterium wasabiae DSMZ 18074.

The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072+/-47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50 degrees C, respectively. The Km value was 0.17 mM, with a Vmax of 1,714 micromol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium.

Host-extract induced changes in the secretome of the plant pathogenic bacterium Pectobacterium atrosepticum.

Pectobacterium atrosepticum is a Gram-negative plant pathogenic bacterium that causes rotting in potato stems and tubers. The secreted proteins of this pathogen were analyzed with proteomics from culture supernatant of cells grown in minimal medium supplemented with host extracts. More than 40 proteins were identified, among them known virulence determinants, such as pectic enzymes, metalloprotease, and virulence protein Svx, along with flagella proteins, GroEL and cyclophilin-type chaperones and several hypothetical proteins or proteins with unknown function. Some of the identified proteins may be involved in utilization of nutrients or transport of minerals. Northern and real-time RT-PCR analyses suggested that most of the proteins upregulated by plant extract were transcriptionally regulated. Among the identified proteins were VgrG and four homologs of hemolysin-coregulated proteins (Hcps). A mutant strain lacking one of the hcp genes was not affected in virulence, while a bacterial strain overexpressing the same gene was shown to have increased virulence, which suggests that these proteins may be new virulence determinants of P. atrosepticum. Comparison of the secretomes of wild type cells and hrcC mutant defective in Type III secretion suggested that the production of the identified proteins was independent of functional Type III secretion system.