Crystal structure of pectocin M1 reveals diverse conformations and interactions during its initial step via the ferredoxin uptake system.

Pectocin M1 (PM1), the bacteriocin from phytopathogenic Pectobacterium carotovorum which causes soft rot disease, has a unique ferredoxin domain that allows it to use FusA of the plant ferredoxin uptake system. To probe the structure-based mechanism of PM1 uptake, we determined the X-ray structure of full-length PM1, containing an N-terminal ferredoxin and C-terminal catalytic domain connected by helical linker, at 2.04 Å resolution. Based on published FusA structure and NMR data for PM1 ferredoxin domain titrated with FusA, we modeled docking of the ferredoxin domain with FusA. Combining the docking models with the X-ray structures of PM1 and FusA enables us to propose the mechanism by which PM1 undergoes dynamic domain rearrangement to translocate across the target cell outer membrane.

Immune Receptor Signaling and the Mushroom Body Mediate Post-ingestion Pathogen Avoidance.

In spite of the positive effects of bacteria on health, certain species are harmful, and therefore, animals must weigh nutritional benefits against negative post-ingestion consequences and adapt their behavior accordingly. Here, we use Drosophila to unravel how the immune system communicates with the brain, enabling avoidance of harmful foods. Using two different known fly pathogens, mildly pathogenic Erwinia carotovora (Ecc15) and highly virulent Pseudomonas entomophila (Pe), we analyzed preference behavior in naive flies and after ingestion of either of these pathogens. Although survival assays confirmed the harmful effect of pathogen ingestion, naive flies preferred the odor of either pathogen to air and also to harmless mutant bacteria, suggesting that flies are not innately repelled by these microbes. By contrast, feeding assays showed that, when given a choice between pathogenic and harmless bacteria, flies-after an initial period of indifference-shifted to a preference for the harmless strain, a behavior that lasted for several hours. Flies lacking synaptic output of the mushroom body (MB), the fly's brain center for associative memory formation, lost the ability to distinguish between pathogenic and harmless bacteria, suggesting this to be an adaptive behavior. Interestingly, this behavior relied on the immune receptors PGRP-LC and -LE and their presence in octopaminergic neurons. We postulate a model wherein pathogen ingestion triggers PGRP signaling in octopaminergic neurons, which in turn relay the information about the harmful food source directly or indirectly to the MB, where an appropriate behavioral output is generated.

QCQuan: A Web Tool for the Automated Assessment of Protein Expression and Data Quality of Labeled Mass Spectrometry Experiments.

In the context of omics disciplines and especially proteomics and biomarker discovery, the analysis of a clinical sample using label-based tandem mass spectrometry (MS) can be affected by sample preparation effects or by the measurement process itself, resulting in an incorrect outcome. Detection and correction of these mistakes using state-of-the-art methods based on mixed models can use large amounts of (computing) time. MS-based proteomics laboratories are high-throughput and need to avoid a bottleneck in their quantitative pipeline by quickly discriminating between high- and low-quality data. To this end we developed an easy-to-use web-tool called QCQuan (available at qcquan.net ) which is built around the CONSTANd normalization algorithm. It automatically provides the user with exploratory and quality control information as well as a differential expression analysis based on conservative, simple statistics. In this document we describe in detail the scientifically relevant steps that constitute the workflow and assess its qualitative and quantitative performance on three reference data sets. We find that QCQuan provides clear and accurate indications about the scientific value of both a high- and a low-quality data set. Moreover, it performed quantitatively better on a third data set than a comparable workflow assembled using established, reliable software.

A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase.

Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U gprot-1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0 ) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1 ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.

[Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase]. / Podavlenie aktivnosti telomerazy leikoznykh kletok mutantymi formami L-asparaginazy Rhodospirillum rubrum.

The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

[PEGylated recombinant L-asparaginase from Erwinia carotovora: Production, properties, and potential applications].

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.

Globin domain interactions control heme pocket conformation and oligomerization of globin coupled sensors.

Globin coupled sensors (GCS) are O2-sensing proteins used by bacteria to monitor the surrounding gaseous environment. To investigate the biphasic O2 dissociation kinetics observed for full-length GCS proteins, isolated globin domains from Pectobacterium carotovorum ssp. carotovorum (PccGlobin), and Bordetella pertussis (BpeGlobin), have been characterized. PccGlobin is found to be dimeric, while BpeGlobin is monomeric, indicating key differences in the globin domain dimer interface. Through characterization of wild type globin domains and globin variants with mutations at the dimer interface and within the distal pocket, dimerization of the globin domain is demonstrated to correlate with biphasic dissociation kinetics. Furthermore, a distal pocket tyrosine is identified as the primary hydrogen bond donor, while a secondary hydrogen bond donor within the distal heme pocket is involved in conformation(s) that lead to the second O2 dissociation rate. These findings highlight the role of the globin dimer interface in controlling properties of both the heme pocket and full-length GCS proteins.

Structural determinants in bacterial 2-keto-3-deoxy-D-gluconate dehydrogenase KduD for dual-coenzyme specificity.

Short-chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2-keto-3-deoxy-d-gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG-producing SDR enzymes (NADPH-dependent A1-R and NADH-dependent A1-R') involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes-KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)-prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5-diketo-3-deoxy-d-gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X-ray crystallography. Both PcaKduD and SpyDhuD consist of a three-layered structure, α/ß/α, with a coenzyme-binding site in the Rossmann fold; similar to enzymes A1-R and A1-R', both arrange the two short and long loops close to the coenzyme-binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1-R' but not A1-R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2' coenzyme region were determined to be structurally crucial for dual-coenzyme specificity in PcaKduD by structural comparison of the NADH- and NADPH-specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual-coenzyme specificity. Proteins 2016; 84:934-947. © 2016 Wiley Periodicals, Inc.

[Recombinant cephalosporin-acid synthesase: optimisation of expression in E.coli cells, immobilisation and application for biocatalytic cefazolin synthesis].

Cephalosporin acid synthetase (CASA) is responsible for specific to synthesis of cephalosporin-acids, its expression in Escherichia coli cells is accompanied by accumulation of unprocessed insoluble precursor. In order to optimize conditions of recombinant CASA production we have studied the effects of several parameters of strain cultivation, including growth media composition, temperature, and inoculation dose. Also plasmids for production of CASA variants with the signal sequence of Erwinia carotovora L-asparaginase (ansCASA) and "leaderless" CASA were created in search of more efficient expression constructs. Removal of the N-terminal secretion signal sequence reduced the production of functionally active CASA more than 10-fold and inhibited strain growth. Insertion of the L-asparaginase signal sequence increased the specific enzyme activity in the resultant recombinant strain. The ansCASA producing strain was used to develop the method of immobilization of the recombinant enzyme on an epoxy-activated macroporous acrylic support. The resultant biocatalyst performed effective synthesis of cefazolin from 3-[(5-methyl-1,3,4-thiadiazol-2-il)-thiomethyl]-7- aminocephalosporanic acid (MMTD-7-ACA) and methyl ester of 1(H)-tetrazolilacetic acid (МETzAA), under mild conditions a transformation level of MMTD-7-ACA to cefazolin of 95% is reached.

[PEG-chitosan branched copolymers to improve the biocatalytic properties of Erwinia carotovora recombinant L-asparaginase].

A new approach to the regulation of catalytic properties of medically relevant enzymes has been proposed using the novel recombinant preparation of L-asparaginase from Erwinia carotovora (EwA), a promising antitumor agent. New branched co-polymers of different composition based on chitosan modified with polyethylene glycol (PEG) molecules, designated as PEG-chitosan, have been synthesized. PEG-chitosan copolymers were further conjugated with EwA. In order to optimize the catalytic properties of asparaginase two types of conjugates differing in their architecture have been synthesized: (1) crown-type conjugates were synthesized by reductive amination reaction between the reducing end of the PEG-chitosan copolymer and enzyme amino groups; (2) multipoint-conjugates were synthesized using the reaction of multipoint amide bond formation between PEG-chitosan amino groups and carboxyl groups of the enzyme in the presence of the Woodward's reagent. The structure and composition of these conjugates were determined by IR spectroscopy. The content of the copolymers in the conjugates was controlled by the characteristic absorption band of C-O-C bonds in the PEG structure at the frequency of 1089 cm-1. The study of catalytic characteristics of EwA preparations by conductometry showed that at physiological pH values the enzyme conjugates with PEG-chitosan with optimized structure and the optimal composition demonstrated 5-8-fold higher catalytic efficiency (kcat/Km) than the native enzyme. To certain extent, this can be attributed to favorable shift of pH-optima in result of positively charged amino-groups introduction in the vicinity of the active site. The proposed approach, chito-pegylation, is effective for regulating the catalytic and pharmacokinetic properties of asparaginase, and is promising for the development of prolonged action dosage forms for other enzyme therapeutics.

[Cross-immunogenicity of various bacterial L-asparaginases].

AIM: Evaluate immune response in mice against various L-asparaginases and determine their cross-immunogenicity. MATERIALS AND METHODS: The studies were carried out in C57Bl(6j) line mice. Immunogenicity of L-asparaginases was studied: Escherichia coli type II (recombinant) (Medak, Germany) (EcA); Erwinia carotovora type II (ErA); Yersinia pseudotuberculosis type II (YpA); Rhodospirillum rubrum type I (RrA); Wollinella succinogenes type II (WsA). Immune response against the administered antigens was determined in EIA. RESULTS: Y. pseudotuberculosis L-asparaginase was the most immunogenic, E. coli--the least immunogenic. E. carotovora, R. rubrum, W. succinogenes asparaginases displayed intermediate immunogenicity. The results of cross-immunogenicity evaluation have established, that blood sera of mice, that had received YpA, showed cross-immunogenicity against all the other L-asparaginase preparations except E. carotovora. During immunization with E. coli L-asparaginase the developed antibodies also bound preparation from E. carotovora. Sera from mice immunized with W. succinogenes, E. carotovora and R. rubrum L-asparaginases had cross-reaction only with EcA and did not react with other preparations. CONCLUSION: Cross-immunogenicity of the studied L-asparaginases was determined. A sequence of administration of the studied preparation is proposed that allows to minimize L-asparaginase neutralization by cross-reacting antibodies.

[Catalytic properties of enzymes from Erwinia carotovora involved in transamination of phenylpyruvate].

Km for L-phenylalanine, L-glutamic acid, L-aspartic acid, and the corresponding keto acids were calculated, as well as Vmax, was measured for the following pairs of substrates: L-phenylalanine-2-ketoglutarate, L-phenylalanine-oxaloacetate, L-glutamic acid-phenylpyruvate, and L-aspartic acid-phenylpyruvate for aminotransferases PATI, PAT2, and PAT3 from Erwinia carotovora catalyzing transamination of phenylpyruvate. The ping-pong bi-bi mechanism was shown for the studied aminotransferases. The substrate inhibition (Ks) of PAT3 with 2-ketoglutarate and oxaloacetate was 10.23 +/- 3.20 and 3.73 +/- 1.99 mM, respectively.

Field enhanced bacterial sample stacking in isotachophoresis using wide-bore capillaries.

The isotachophoretic analysis of different bacterial strains was studied using capillaries with different internal diameters from 50 to 250 µm. Several injection modes were investigated and compared in order to improve the limit of detection of bacteria by capillary isotachophoresis. A system suitability test obtained from the separation voltage was developed to ensure reliable results. As expected, the use of wider bore capillaries improved the analytical sensitivity of the isotachophoretic method when compared to the 50 µm capillary. With the optimized conditions, the isotachophoretic method presented in this work allows the quantification of Erwinia carotovora (Gram negative bacteria) with a limit of detection as low as ~3000 cells mL(-1). The proposed methodology does not require any additive in the electrolyte such a fluorescent or chromophoric dye to reach these limits of detection.

[Species composition of agents of the horsetail common (Equisetum arvense L.) bacteriosises].

Bacterial diseases of weeds horsetail common (Equisetum arvense L.) were revealed in the crops of wheat and soya in the fields of Kyiv and Vinnitsia Regions of Ukraine. The distinctive symptoms of bacterial affections on the root neck, on stalks of vegetative and spore shoots, on twigs were brown, dark brown or almost black necrotic spots of oblong form. The necroses increased in size, embraced the stalks. The stalks broke, the plants dried up. Patterns of affected plants, isolated and identified phytopathogenic bacteria Pseudomonas syringae, Pectobacterium carotovorum subsp. carotovorum, Pantoea agglomerans and Curtobacterium sp. were analyzed These bacteria caused pathological process on the horsetail common, wheat and soy under the conditions of artificial inoculation. The composition of bacteria species was different in different years depending on temperature conditions of vegetative period.

Enhanced accumulation of secondary metabolites in hairy root cultures of Scutellaria lateriflora following elicitation.

Hairy root cultures of Scutellaria lateriflora were established using Agrobacterium rhizogenes A4 and produced acteoside (18.5 mg g(-1) dry wt), baicalin (14.5 mg g(-1) dry wt) and wogonoside (12 mg g(-1) dry wt). Yeast extract (50 µg ml(-1)) increased acteoside production 1.4-fold and flavone production 1.7-fold after 7 and 14 days of elicitation. Addition of Pectobacterium carotovorum lysate in the stationary phase of the hairy root culture stimulated only the accumulation of wogonin to 30 mg g(-1) dry wt. The production of wogonin in hairy roots could be associated with its role as a phytolaexin.

Study of antibacterial activity by capillary electrophoresis using multiple UV detection points.

A new methodology for an antibacterial assay based on capillary electrophoresis with multiple UV detection points has been proposed. The possible antibacterial activity of cationic molecules on bacteria (Gram-positive and Gram-negative) is studied by detecting the bacteria before, during, and after their meeting with the cationic antibacterial compound. For that, a UV area imaging detector having two loops and three detection windows was used with a 95 cm ×100 µm i.d. capillary. In the antibacterial assay, the bacteria (negatively charged) and the cationic molecules were injected separately from each end of the capillary. The bacteria were mobilized by anionic ITP mode while cationic molecules migrate in the opposite direction under conditions close to CZE. The cationic molecules were injected into the capillary as a broad band (injected volume about 16% of the volume of the capillary) to prevent dilution of the sample during the electrophoretic process. Bacteriolytic activity, as well as strong interactions between the small antibacterial molecules and the bacteria, can be investigated within a few minutes. The assay was used to study the antibacterial activity of dendrigraft poly-L-lysines on Micrococcus luteus and Erwinia carotovora. Because dendrigraft poly-L-lysines are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications.

Biophysical investigations into the interactions of endotoxins with bile acids.

The interaction of selected endotoxin preparations (lipid A from Erwinia carotovora and LPS Re and Ra from Salmonella enterica sv. Minnesota strains R595 and R60, respectively) with selected bile acids was investigated biophysically. Endotoxin aggregates were analyzed for their gel-to-liquid crystalline phase behavior, the type of their aggregates, the conformation of particular functional groups, and their Zeta potential in the absence and presence of the bile acids by applying Fourier-transform infrared spectroscopy, differential scanning calorimetry, measurements of the electrophoretic mobility, and synchrotron radiation X-ray scattering. In addition, the ability of the endotoxins to induce cytokines in human mononuclear cells was tested in the absence and presence of varying concentrations of bile acids. The data show that the endotoxin:bile acid interaction is not governed by Coulomb forces, rather a hydrophobic interaction takes place. This leads to an enhanced formation of the inherent cubic aggregate structures of the endotoxins, concomitant with a slight disaggregation, as evidenced by freeze-fracture electron microscopy. Parallel to this, the addition of bile acids increased the bioactivity of lipid A and, to a lower degree, also that of the tested rough mutant LPS at lower concentrations of the endotoxin preparation, a finding similar as reported for the interaction of other agents such as hemoglobin. These data imply that there are general mechanisms that govern the expression of biological activities of endotoxins.

Looking inside the box: using Raman microspectroscopy to deconstruct microbial biomass stoichiometry one cell at a time.

Stoichiometry of microbial biomass is a key determinant of nutrient recycling in a wide variety of ecosystems. However, little is known about the underlying causes of variance in microbial biomass stoichiometry. This is primarily because of technological constraints limiting the analysis of macromolecular composition to large quantities of microbial biomass. Here, we use Raman microspectroscopy (MS), to analyze the macromolecular composition of single cells of two species of bacteria grown on minimal media over a wide range of resource stoichiometry. We show that macromolecular composition, determined from a subset of identified peaks within the Raman spectra, was consistent with macromolecular composition determined using traditional analytical methods. In addition, macromolecular composition determined by Raman MS correlated with total biomass stoichiometry, indicating that analysis with Raman MS included a large proportion of a cell's total macromolecular composition. Growth phase (logarithmic or stationary), resource stoichiometry and species identity each influenced each organism's macromolecular composition and thus biomass stoichiometry. Interestingly, the least variable peaks in the Raman spectra were those responsible for differentiation between species, suggesting a phylogenetically specific cellular architecture. As Raman MS has been previously shown to be applicable to cells sampled directly from complex environments, our results suggest Raman MS is an extremely useful application for evaluating the biomass stoichiometry of environmental microorganisms. This includes the ability to partition microbial biomass into its constituent macromolecules and increase our understanding of how microorganisms in the environment respond to resource heterogeneity.

Discovery of a quorum sensing modulator pharmacophore by 3D small-molecule microarray screening.

The screening of large arrays of drug-like small-molecules was traditionally a time consuming and resource intensive task. New methodology developed within our laboratories provides an attractive low cost, 3D microarray-assisted screening platform that could be used to rapidly assay thousands of compounds. As a proof-of-principle the platform was exploited to screen a number of quorum sensing analogs. Quorum sensing is used by bacterium to initiate and spread infection; in this context its modulation may have significant clinical value. 3D microarray slides were probed with fluorescently labeled ligand-binding domains of the LuxR homolog CarR from Erwinia carotovora subsp. carotovora. The 3D microarray platform was used to discover the biologically active chloro-pyridine pharmacophore, which was validated using a fluorometric ligand binding assay and ITC. Analogs containing the chloro-pyridine pharmacophore were found to be potent inhibitors of N-acyl-homoserine-lactone (AHL) mediated quorum sensing phenotypes in Serratia (IC(50) = ∼5 µM) and Pseudomonas aeruginosa (IC(50) = 10-20 µM).

Novel low-temperature-active phytase from Erwinia carotovora var.carotovota ACCC 10276.

A phytase with high activity at low temperatures has great potential for feed applications, especially in aquaculture. Therefore, this study used a degenerate PCR and TAIL PCR to clone a phytase gene from Erwinia carotovora var. carotovota, the cause of soft rot of vegetables in the ground or during cold storage. The full-length 2.5-kb fragment included an open reading frame of 1,302 bp and encoded a putative phytase of 45.3 kDa with a 50% amino acid identity to the Klebsiella pneumoniae phytase. The phytase contained the active site RHGXRXP and HD sequence motifs that are typical of histidine acid phosphatases. The enzyme was expressed in Escherichia coli, purified, and displayed the following characteristics: a high catalytic activity at low temperatures (retaining over 24% activity at 5 degrees C) and remarkably thermal lability (losing >96% activity after incubation at 60 degrees C for 2 min). The optimal phytase activity occurred at pH 5.5 and approximately 40 degrees C, and the enzyme activity rapidly decreased above 40 degrees C. When compared with mesophilic counterparts, the phytase not only exhibited a high activity at a low temperature, but also had a low K(m) and high k(cat). These temperature characteristics and kinetic parameters are consistent with low-temperature-active enzymes. To our knowledge, this would appear to be the first report of a low-temperature-active phytase and its heterogeneous expression.