Comparative genomics, pangenomics, and phenomic studies of Pectobacterium betavasculorum strains isolated from sugar beet, potato, sunflower, and artichoke: insights into pathogenicity, virulence determinants, and adaptation to the host plant.

Introduction: Bacteria of genus Pectobacterium, encompassing economically significant pathogens affecting various plants, includes the species P. betavasculorum, initially associated with beetroot infection. However, its host range is much broader. It causes diseases of sunflower, potato, tomato, carrots, sweet potato, radish, squash, cucumber, and chrysanthemum. To explain this phenomenon, a comprehensive pathogenomic and phenomic characterisation of P. betavasculorum species was performed. Methods: Genomes of P. betavasculorum strains isolated from potato, sunflower, and artichoke were sequenced and compared with those from sugar beet isolates. Metabolic profiling and pathogenomic analyses were conducted to assess virulence determinants and adaptation potential. Pathogenicity assays were performed on potato tubers and chicory leaves to confirm in silico predictions of disease symptoms. Phenotypic assays were also conducted to assess the strains ability to synthesise homoserine lactones and siderophores. Results: The genome size ranged from 4.675 to 4.931 kbp, and GC % was between 51.0% and 51.2%. The pangenome of P. betavasculorum is open and comprises, on average, 4,220 gene families. Of these, 83% of genes are the core genome, and 2% of the entire pangenome are unique genes. Strains isolated from sugar beet have a smaller pangenome size and a higher number of unique genes than those from other plants. Interestingly, genomes of strains from artichoke and sunflower share 391 common CDS that are not present in the genomes of other strains from sugar beet or potato. Those strains have only one unique gene. All strains could use numerous sugars as building materials and energy sources and possessed a high repertoire of virulence determinants in the genomes. P. betavasculorum strains were able to cause disease symptoms on potato tubers and chicory leaves. They were also able to synthesise homoserine lactones and siderophores. Discussion: The findings underscore the adaptability of P. betavasculorum to diverse hosts and environments. Strains adapted to plants with high sugar content in tissues have a different composition of fatty acids in membranes and a different mechanism of replenishing nitrogen in case of deficiency of this compound than strains derived from other plant species. Extensive phenomics and genomic analyses performed in this study have shown that P. betavasculorum species is an agronomically relevant pathogen.

Comprehensive phenomic and genomic studies of the species, Pectobacterium cacticida and proposal for reclassification as Alcorniella cacticida comb. nov.

Introduction: Pectobacterium cacticida was identified as the causative agent of soft rot disease in cacti. Due to a high potential of spread in the face of global warming, the species poses a significant threat to horticultural and crop industry. The aim of this study was to revise the genomic, physiology and virulence characteristics of P. cacticida and update its phylogenetic position within the Pectobacterium genus. Methods: Whole genome sequences of five P. cacticida strains were obtained and subjected to comprehensive genomic and phylogenomic data analyses. We assessed the presence of virulence determinants and genes associated with host and environmental adaptation. Lipidomic analysis, as well as biochemical and phenotypic assays were performed to correlate genomic findings. Results: Phylogenomic analysis revealed that P. cacticida forms a distinct lineage within the Pectobacterium genus. Genomic evaluation uncovered 516 unique proteins, most of which were involved in cellular metabolism. They included genes of carbohydrate metabolism and transport and ABC transporters. The main differing characteristics from other Pectobacterium species were the lack of a myo-inositol degradation pathway and the presence of the malonate decarboxylase gene. All tested strains were pathogenic towards Opuntia spp., chicory, Chinese cabbage, and potato, but exhibited only mild pathogenicity towards carrot. Discussion: This study sheds light into the genomic characteristics of P. cacticida and highlights the pathogenic potential of the species. Unique genes found in P. cacticida genomes possibly enhance the species' survival and virulence. Based on phylogenomic analyses, we propose the reclassification of P. cacticida to a new genus, Alcorniella comb. nov.

Gas Chromatography-Mass Spectrometry Profiling of Volatile Metabolites Produced by Some Bacillus spp. and Evaluation of Their Antibacterial and Antibiotic Activities.

Bacillus species produce different classes of antimicrobial and antioxidant substances: peptides or proteins with different structural compositions and molecular masses and a broad range of volatile organic compounds (VOCs), some of which may serve as biomarkers for microorganism identification. The aim of this study is the identification of biologically active compounds synthesized by five Bacillus species using gas chromatography coupled to mass spectrometry (GC-MS). The current study profoundly enhances the knowledge of antibacterial and antioxidant metabolites ensuring the unambiguous identification of VOCs produced by some Bacillus species, which were isolated from vegetable samples of potato, carrot, and tomato. Phylogenetic and biochemical studies were used to identify the bacterial isolates after culturing. Phylogenetic analysis proved that five bacterial isolates BSS12, BSS13, BSS16, BSS21, and BSS25 showed 99% nucleotide sequence similarities with Bacillus safensis AS-08, Bacillus cereus WAB2133, Bacillus acidiproducens NiuFun, Bacillus toyonesis FORT 102, and Bacillus thuringiensis F3, respectively. The crude extract was prepared from bacterial isolates to assess the antibiotic resistance potency and the antimicrobial potential against various targeted multidrug-resistant strains, including yeast strains such as Candida albicans, Candida krusei, and bacterial strains of Enterococcus hirae, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus group B, Streptococcus mutans, Shigella sonnei, Salmonella enteritidis, Serratia marcescens, Pseudomonas aeruginosa, and Proteus vulgaris. GC-MS analysis of bacterial strains found that VOCs from Bacillus species come in a variety of chemical forms, such as ketones, alcohols, terpenoids, alkenes, etc. Overall, 69 volatile organic compounds were identified from five Bacillus species, and all five were found to share different chemical classes of volatile organic components, which have a variety of pharmacological applications. However, eight antibacterial compounds with different concentrations were commonly found in all five species: acetoin, acetic acid, butanoic acid, 2-methyl-, oxime-, methoxy-phenyl, phenol, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, nonanoic acid, and hexadecanoic acid, methyl. The present study has demonstrated that bacterial isolates BSS25, BSS21, and BSS16 display potent inhibitory effects against Candida albicans, while BSS25, BSS21, and BSS13 exhibit the ability to restrain the growth and activity of Candida krusei. Notably, BSS25 and BSS21 are the only isolates that demonstrate substantial inhibitory activity against Klebsiella aerogenes. This disparity in inhibitory effects could be attributed to the higher concentrations of acetoin in BSS25 and BSS21, whereas BSS16 and BSS13 have relatively elevated levels of butanoic acid, 2-methyl-. Certainly, the presence of acetoin and butanoic acid, 2-methyl-, contributes to the enhanced antibacterial potential of these bacterial strains, in conjunction with other organic volatile compounds and peptides, among other factors. The biology and physiology of Bacillus can be better understood using these results, which can also be used to create novel biotechnological procedures and applications. Moreover, because of its exceptional ability to synthesize and produce a variety of different antibacterial compounds, Bacillus species can serve as natural and universal carriers for antibiotic compounds in the form of probiotic cultures and strains to fight different pathogens, including mycobacteria.

The International Trade of Ware Vegetables and Orna-Mental Plants-An Underestimated Risk of Accelerated Spreading of Phytopathogenic Bacteria in the Era of Globalisation and Ongoing Climatic Changes.

Bacteria of the genus Pectobacterium are globally occurring pathogens that infect a broad spectrum of plants. The plant cell wall degrading enzymes allow them to cause diseases like soft rot and blackleg. Worldwide trade and exchange of plant material together with the accompanying microorganisms contributed to the rapid spread and consequently the acquisition of new traits by bacteria. The 161 pectinolytic strains were isolated from symptomless vegetables and ornamental plants acquired from Polish and foreign local food markets. All strains except four Dickeya isolates were identified as belonging to the Pectobacterium genus by PCR with species-specific primers and recA gene sequencing. The newly isolated bacteria were assigned to eight species, P. versatile (50 strains), P. carotovorum (33), P. brasiliense (27), P. atrosepticum (19), P. parmentieri (12), P. polaris (11), P. parvum (3) and P. odoriferum (2). ERIC PCR and phenotypic characteristics revealed high heterogeneity among P. carotovorum, P. brasiliense and P. versatile isolates. Moreover, a subset of the newly isolated strains was characterised by high tolerance to changing environmental conditions such as salinity, pH and water availability. These bacteria can effectively macerate the tissues of various plants, including potato, chicory and orchid. Our results indicate that Pectobacterium strains isolated from internationally traded, symptomless vegetables and ornamental plants have high potential for adaptation to adverse environmental conditions and to infect various host plants. These features may contribute to the success of the genus Pectobacterium in spreading between different climatic zones and facilitate the colonisation of different ecological niches.

Quality Control of Bacterial Extracellular Vesicles with Total Protein Content Assay, Nanoparticles Tracking Analysis, and Capillary Electrophoresis.

Extracellular vesicles (EVs) were isolated from Pectobacterium zantedeschiae culturing media using direct ultracentrifugation (UC), iodixanol cushion ultracentrifugation (ICUC), and iodixanol density gradient ultracentrifugation (IDGUC) techniques. The isolates were characterized with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A satisfactory correlation (R2 > 0.94) between quantitative results obtained with BCA, NTA and CE was achieved only for isolates obtained with the IDGUC. The correlation between protein content and CE was proved to be related to the isolates' purity. The NTA was found unable to provide reliable information on EVs quantity in samples isolated with UC and ICUC, due to the co-isolated particulate impurities. Moreover, the work reports polysaccharides, used as culturing media components, as a potential source of bias of quantitation with total protein content assay and NTA. The study demonstrates the advantageous selectivity of CE in quality control of EVs and its ability to differentiate subpopulations of EVs of Pectobacterium.

Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System.

Bacteria of genus Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produces large MVs (100-300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that the MVs of ß-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in Pectobacterium strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an effective secretion system.

Investigation of selected parameters of capillary zone electrophoresis method for analysis of isolates of outer membrane vesicles.

The capillary zone electrophoresis (CZE) has recently been proposed by our group as a novel technique for outer membrane vesicles (OMVs) characterization (J. Chromatography 1621 (2020) 461047). In present work the impact of selected parameters of CZE method on OMVs isolates analysis was assessed. It was shown that the extension of sample injection plug length significantly improves the detectability of macromolecular aggregates in CZE. Moreover, a negligible adsorption of OMVs to both uncoated and polymer-modified (poly(DMA-GMA-MAPS)) capillary walls was proven. Finally, the relaxation effect as well as deformation/polarization of vesicles were demonstrated to affect OMVs electrophoretic mobility. The significance of these findings was discussed.

Haplotypes of butyrylcholinesterase K-variant and their influence on the enzyme activity.

Butyrylcholinesterase (BChE) is a serine hydrolase widely distributed throughout the body. It provides protection against administrated or inhaled poisons by hydrolyzing or sequestering the toxic compounds. The most frequent genetic variant of BCHE gene - K variant (p.A539T) is located in the C-terminal tetramerization domain, outside of the catalytic center. Many studies tried to reveal the nature of the lower activity of BChE K-variant but results and conclusions were often contradictory. The aim of this study is to estimate K allele frequency and its coexisting alterations in BCHE gene in a population of 162 individuals, as well as, assess influence on the enzyme activity in serum. We present three haplotypes of BChE-K variant, two of them coexist in strong linkage disequilibrium with alterations in 5'UTR (rs1126680), intron 2 (rs55781031) or in exon 2 (rs1799807). We demonstrate a negative role of these alterations on enzyme activity. By oneself BCHE-K (with no other alterations in BCHE gene) haplotype exhibits wild type enzyme activity. Based on our previous and presented results we conclude that SNPs localized outside the coding sequence, in 5'UTR or/and in intron 2 of BCHE gene, but not solely in K-variant alteration (p.A539T) itself, are responsible for reduced enzyme activity.

Pectobacterium polonicum sp. nov. isolated from vegetable fields.

Gram-stain-negative, rod-shaped pectinolytic bacteria strains designated as DPMP315T, DPMP316, DPMP317 and DPMP318 isolated from groundwater sampled from a vegetable field in the North of Poland, were subjected to the polyphasic analyses. Multilocus sequence analyses based on five housekeeping genes (gyrA, recA, recN, rpoA and rpoS) revealed their distinctiveness from the other species of the genus, simultaneously indicating that the newly described species, Pectobacterium punjabense, as well as Pectobacterium parmentieri and P. wasabiae, to be the closest relatives. In silico DNA-DNA hybridization (<43.1 %) and average nucleotide identity (<92.5 %) values of strain DPMP315T with other type strains of species of the genus Pectobacterium supported the delineation of the novel strain as representing a novel species. The phenotypic comparisons, fatty acid methyl esters compositions, genetic rep PCR fingerprint and detailed whole-cell MALDI-TOF mass spectrometry proteomic profiles permitted the differentiation of Polish strains from the type strains of all other known species of the genus Pectobacterium. The results of polyphasic analyses performed for four Polish strains are the basis for the distinction of the novel species. Here, we propose to establish DPMP315T as a type strain (=PCM3006T=LMG 31077T) with the name Pectobacterium polonicum sp. nov.

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.

Activity and polymorphisms of butyrylcholinesterase in a Polish population.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents. In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 3'UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (<2000 U/L and >5745 U/L) or with DN (Dibucaine Number) or FN (Fluoride-Number) values outside the reference range (DN < 78 and FN < lower than wild type). In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A > G or c.1253G > T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 5'UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity ≥6000 U/L were wild type. To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes.

Bacterial membranes are the target for antimicrobial polysiloxane-methacrylate copolymer.

Antibacterial polysiloxane polymers with pending tert-butylamine groups are a novel class of compounds that are compatible with silicone elastomers, but their mechanism of action is not well understood. The research into their action mechanism was conducted on a polysiloxane copolymer grafted with tert-butylaminoethyl methacrylate and covalently attached fluorescein. Fluorometric measurements results suggest that the polymer forms a stable link with bacteria. The results of ß-galactosidase enzyme assay with the use of ortho-nitrophenyl-ß-galactoside as a substrate show that the polymer has a damaging effect on bacterial membranes. The scanning and transmission electron micrographs of Escherichia coli cells incubated with the polymer prove further that the polymer's site of action is bacterial cell membranes. In order to investigate the polymer interaction with bacterial membranes the fluorescein labelled polymer was incubated with bacterial cells and membranes isolation and identification method was next applied. The E. coli membrane fractions were identified by light scattering, protein content, oxidase NADH activity and N-phenylnaphtylamine fluorescence measurements, as well as electron microscopy. Oxidase NADH and N-phenylnaphtylamine were the inner membrane markers. The bacterial membranes were then tested for the presence of the polymer. The experiments gave evidence that the copolymer binds to the inner bacterial membrane. Further studies, where the copolymer was incubated with isolated mixed (inner and outer) membrane fractions, proved that the copolymer exerts more destructive effect on E. coli outer membrane. The damaging effect on the membranes is concentration dependent.

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.