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1.
Pectobacterium carotovorum subsp. carotovorum, a member of the Enterobacteriaceae family, is an important plant-pathogenic bacterium causing significant economic losses worldwide. P. carotovorum subsp. carotovorum bacteriophage My1 was isolated from a soil sample. Its genome was completely sequenced and analyzed for the development of an effective biological control agent. Sequence and morphological analyses revealed that phage My1 is a T5-like bacteriophage and belongs to the family Siphoviridae. To date, there is no report of a Pectobacterium-targeting siphovirus genome sequence. Here, we announce the complete genome sequence of phage My1 and report the results of our analysis.  相似文献   

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Two different bacteriocins, carotovoricin and carocin S1, had been found in Pectobacterium carotovorum subsp. carotovorum, which causes soft-rot disease in diverse plants. Previously, we reported that the particular strain Pcc21, producing only one high-molecular-weight bacteriocin, carried a new antibacterial activity against the indicator strain Pcc3. Here, we report that this new antibacterial activity is due to a new bacteriocin produced by strain Pcc21 and named carocin D. Carocin D is encoded by the caroDK gene located in the genomic DNA together with the caroDI gene, which seems to encode an immunity protein. N-terminal amino acid sequences of purified carocin D were determined by Edman degradation. In comparison with the primary translation product of caroDK, it was found that 8 amino acids are missing at the N terminus. This finding proved that carocin D is synthesized as a precursor peptide and that 8 amino acids are removed from its N terminus during maturation. Carocin D has two putative translocation domains; the N-terminal and C-terminal domains are homologous to those of Escherichia coli colicin E3 and Pseudomonas aeruginosa S-type pyocin, respectively. When caroDK and caroDI genes were transformed into carocin D-sensitive bacteria such as Pcc3, the bacteria became resistant to this bacteriocin. Carocin D has one putative DNase domain at the extreme C terminus and showed DNase activity in vitro. This bacteriocin had slight tolerance to heat but not to proteases. The caroDK gene was present in only 5 of 54 strains of P. carotovorum subsp. carotovorum. These results indicate that carocin D is a third bacteriocin found in P. carotovorum subsp. carotovorum, and this bacteriocin can be readily expressed in carocin D-sensitive nonpathogenic bacteria, which may have high potential as a biological control agent in the field.Pectobacterium carotovorum subsp. carotovorum is a Gram-negative phytopathogen responsible for soft rot, blackleg, or stem rot in various commercially important plants, including Chinese cabbage and potato. Bacterial soft rot is found throughout Korea and causes serious yield loss in the field, in transit, and in storage. Pathogenesis in P. carotovorum subsp. carotovorum is dependent on production of plant cell wall-degrading enzymes that are actively secreted by the bacterium. Various aspects of epidemiology of the disease caused by this phytopathogen are relatively well understood, but no efficient method is available to control the disease (21).Some bacteria, including plant pathogens, produce one or more antibacterial peptides called bacteriocins. Bacteriocins were originally defined as ribosomally synthesized proteinaceous compounds that killed strains of the same or closely related species (20). They are potent, often highly specific toxins that are usually produced under stressful conditions, causing the rapid elimination of neighboring cells that are not immune or resistant to their effects (14). Elucidation of the ecological significance of inhibitory substances such as bacteriocins produced by plant pathogens is important for understanding factors that affect population dynamics on plant surfaces. Thus, the exploitation of narrow-spectrum bacteriocins is an attractive strategy for targeted attack against bacterial diseases in plant disease control (10).Among bacteriocins produced by Gram-negative bacteria, colicins and S-type pyocins have been intensively studied. Colicins and S-type pyocins are produced by Escherichia coli and Pseudomonas aeruginosa, respectively. They consist of two proteins, one responsible for antimicrobial activity (the killing protein) and the other for immunity (the immunity protein). The killing proteins are organized in functional domains, with receptor-binding, translocation, and DNase (RNase) activity (9, 15). Their gene promoters, located upstream of the structural genes, include conserved DNA regions, the so-called SOS box of colicins, and the P box of S-type pyocins, and they are inducible by DNA-damaging agents such as mitomycin C (MMC) (1, 19). Colicins and S-type pyocins need to interact with specific membrane receptors on target cells for their activities, and these specific interactions determine the spectrum of target cells, which is generally very narrow. The host strain is protected from its own bacteriocin through interaction with the immunity protein that is coproduced with the bacteriocin. It has been proposed that bacteriocins may play a key role in bacterial population dynamics (16).Two bacteriocins have been reported in P. carotovorum subsp. carotovorum. One is carotovoricin, a high-molecular-weight bacteriocin, which contains a lysis cassette and a gene cluster for a structural protein and is located in the chromosomal DNA (13, 22). Sequence comparisons showed high homology between carotovoricin and bacteriophage proteins (22). Electron microscopy showed that carotovoricin has an antenna-like structure, with a base plate and tail fibers. Another bacteriocin is a low-molecular-weight bacteriocin, carocin S1, which consists of a killing protein and an immunity protein. Production of carocin S1 is induced by glucose and lactose (4). The carocin S1 gene is homologous to the pyocin S3 and pyocin AP41 genes of P. aeruginosa (4).Because soft-rot disease in Chinese cabbage is destructive and no efficient control method is known, development of new control methods against the pathogen P. carotovorum subsp. carotovorum is desirable, and any method should be safe for humans and environmentally friendly. The use of bacteriocins may be one of the most feasible methods that satisfies both criteria. Although the rapid occurrence of resistant mutants may limit the efficacy of bacteriocin as a control method, use of combinations of several different bacteriocins will help to overcome this.In this study, a new low-molecular-weight bacteriocin, carocin D, and its immunity gene were identified and characterized. This new bacteriocin has a rare feature in that it has two translocation domains. Additionally, the domain structure of carocin D suggests that it may have arisen from a chimera of two different bacteriocins: one from colicin E of E. coli and the other from pyocin of P. aeruginosa.  相似文献   

4.
Seven Gram-negative, rod-shaped pectinolytic bacteria strains designated as IFB5227, IFB5228, IFB5229, IFB5230, IFB5231, IFB5232, IFB5636, isolated from potato tubers cultivated in Peru at high altitude (2400–3800 m) were subjected to polyphasic analyses that revealed their distinctiveness from the other Pectobacterium species. Phylogenetic analyses based on five housekeeping genes (gyrA, recA, recN, rpoA and rpoS) clearly showed strains separateness, simultaneously indicating Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium parmentieri and Pectobacterium betavasculorum as the closest relatives. In silico DNA–DNA hybridization of strain IFB5232T with other Pectobacterium type strains revealed significant drop in DDH value below 70%, which is a prerequisite to distinguish Pectobacterium peruviense. The ANI values supported the proposition of delineation of the P. peruviense. Genetic REP-PCR fingerprint and detailed MALDI-TOF MS proteomic profile sealed the individuality of the studied strains. However, phenotypic assays do not indicate immense differences.Provided results of analyses performed for seven Peruvian strains are the basis for novel species distinction and reclassification of the strains IFB5227-5232 and IFB5636, previously classified as Pectobacterium carotovorum subsp. carotovorum. Here, we propose to establish the IFB5232 isolate as a type strain (=PCM2893T = LMG30269T = SCRI179T) with the name Pectobacterium peruviense sp. nov.  相似文献   

5.
《Process Biochemistry》2010,45(2):223-229
Bacterial l-asparaginase has been widely used as therapeutic agent in the treatment of various lymphoblastic leukemia diseases. Studies on localization and production of novel glutaminase-free l-asparaginase were performed using Pectobacterium carotovorum MTCC 1428. The localization of l-asparaginase was carried out using cell fractionation techniques. The activity of l-asparaginase was found to be 85 and 77% in the cytoplasm of P. carotovorum MTCC 1428 grown on medium containing l-asparagine and combination of l-asparagine and glucose respectively. Among the tested carbon sources, l-asparagine or the combination of l-asparagine and glucose was found to be the most suitable carbon sources to maximize the production of l-asparaginase. The maximum production of l-asparaginase was observed to be 14.56 U/ml (26.92 U/mg of protein) at 4 and 2 g/l of l-asparagine and glucose respectively. Yeast extract, l-asparagine and peptone have shown significant effect on the production of l-asparaginase. P. carotovorum MTCC 1428 has assimilated l-asparagine as an essential carbon source for maximizing the production of l-asparaginase.  相似文献   

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Twenty-one salts were tested for their effects on the growth of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum. In liquid medium, 11 salts (0.2 M) exhibited strong inhibition of bacterial growth. The inhibitory action of salts relates to the water-ionizing capacity and the lipophilicity of their constituent ions.Different biochemical mechanisms have been put forth to explain the antimicrobial activity of organic and inorganic salts, including inhibition of several steps of the energy metabolism (benzoate, bicarbonate, propionate, sorbate, and sulfite salts) (2, 3, 11, 16, 17, 19, 25) and complexation to DNA and RNA (aluminum and sulfites) (12, 13, 15, 20, 27, 28). However, little is known about the physicochemical basis for the general antimicrobial action of salts. The objective of this work was to gain an understanding of the relationship between the inhibitory action of salts on bacterial growth and their physicochemical properties by using the bacteria Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) and Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica). These bacteria are responsible for soft rot, a disease of economic importance affecting numerous stored vegetable crops (14, 22).Pectobacterium carotovorum subsp. carotovorum (strain Ecc 1367) and P. atrosepticum (strain Eca 709), provided by the Laboratoire de Diagnostic en Phytoprotection (MAPAQ, Québec, Canada), were grown in 250-ml flasks containing 50 ml of 20% tryptic soy broth (Difco Laboratories, Becton Dickinson, Sparks, MD) amended with salts (200 mM) or unamended (control), by incubation at 24°C with agitation (150 rpm; Lab-Line Instruments Inc., Melrose Park, IL) for 24 h. The pHs of the media were not adjusted but varied with the type of salts, unless stated otherwise. Flasks were inoculated with 100 μl of each bacterial suspension (1 × 107 CFU/ml). Bacterial growth was determined by turbidimetry at 600 nm with a UV/visible spectrophotometer (Ultrospec 2000; Pharmacia Biotech Ltd, Cambridge, United Kingdom), using appropriate blanks. Results were expressed as the percentage of growth inhibition compared with the growth of the control. A completely randomized experimental design with three replicates was used, the experimental unit being a flask. Analysis of variance was carried out with the GLM (general linear model) procedure of SAS (SAS Institute, Cary, NC) software. When they were significant (P < 0.05), treatment means were compared using Fisher''s protected least-significant-difference test.Among the 21 salts tested, sodium carbonate, sodium metabisulfite, trisodium phosphate, aluminum lactate, aluminum chloride, sodium bicarbonate, sodium propionate, ammonium acetate, aluminum dihydroxy acetate, potassium sorbate, and sodium benzoate exhibited strong inhibition (≥97%) of the growth of both P. carotovorum subsp. carotovorum and P. atrosepticum (Table (Table1).1). Calcium chloride, sodium formate, sodium acetate, ammonium hydrogen phosphate, and sodium hydrogen phosphate exhibited a moderately inhibitory effect; sodium lactate and tartrate had no effect. On the other hand, ammonium chloride, potassium chloride, and sodium chloride stimulated the growth of P. atrosepticum.

TABLE 1.

Effect of salts on the growth of P. atrosepticum and P. carotovorum subsp. carotovorum
Salt (0.2 M)apHbOsmotic pressure (atm)cGrowth inhibition (%)d
P. atrosepticumP. carotovorum subsp. carotovorum
Aluminum dihydroxy acetate [Al(OH)2C2H3O2]4.99.79100 a100 a
Aluminum chloride (AlCl3·6H2O)2.519.57100 a100 a
Aluminum lactate [Al(C3H5O3)3]3.419.57100 a100 a
Ammonium acetate (NH4C2H3O2)7.29.79100 a100 a
Ammonium chloride (NH4Cl)7.09.79−18 dND
Ammonium hydrogen phosphate [(NH4)2HPO4]8.314.6843 b23 c
Calcium chloride (CaCl2·2H2O)5.814.6885 a70 b
Potassium chloride (KCl)7.39.79−27 dND
Potassium sorbate (KC6H7O2)7.79.79100 a97 a
Sodium acetate (NaC2H3O2·3H2O)7.49.7963 bND
Sodium benzoate (NaC7H5O2)7.49.79100 a100 a
Sodium bicarbonate (NaHCO3)8.19.79100 a100 a
Sodium carbonate (Na2CO3)10.614.68100 a100 a
Sodium chloride (NaCl)7.29.79−29 dND
Sodium formate (NaCHO2)7.39.7924 cND
Sodium lactate (C3H5O3Na)7.39.793 cND
Sodium metabisulfite (Na2S2O5)4.519.57100 a100 a
Sodium hydrogen phosphate (Na2HPO4)8.714.6869 b61 b
Sodium propionate (NaC3H5O2)7.49.79100 a99 a
Sodium tartrate (Na2C4H4O6·2H2O)7.314.682 cND
Trisodium phosphate (Na3PO4·12H2O)11.919.57100 a100 a
Open in a separate windowaSalts were purchased from Sigma Chemical Co. (St. Louis, MO), except for ammonium acetate (BDH Inc., Toronto, Canada), sodium chloride (BDH), sodium bicarbonate (BDH), and aluminum lactate (Aldrich Chemical, Milwaukee, WI).bpH of the medium amended with each salt.cOsmotic pressure of the salt solution was calculated using van’t Hoff''s equation, Π = iRTc, where R is the gas constant, T is the absolute temperature (K), c is the concentration of the salt (mol/liter), and i is the number of ions into which the salt dissociates in solution.dPercentage of growth inhibition compared to growth of the control. Each value represents the mean of three replicates. Values in the same column followed by the same letter are not significantly different according to Fisher''s protected least-significant-difference test (P > 0.05). ND, not determined. Negative values signify bacterial growth stimulation.Several factors in the salt solutions can contribute to bacterial growth inhibition. Elevated osmolarity due to salt addition may trigger the osmoregulatory process, causing an increased maintenance metabolism and leading to reduction in bacterial growth. Thus, we calculated the osmotic pressure (Π) of salt solutions using van''t Hoff''s equation (26). As shown in Table Table1,1, salts with comparable osmolarities displayed complete or no bacterial growth inhibition, indicating that osmotic stress or reduction in water activity alone may not have brought about the inhibition of the bacterial growth. Therefore, other factors may play a role.The acidity or alkalinity of the medium resulting from the addition of some of the salts can have profoundly adverse effects on bacterial growth. Extreme pH conditions can lead to denaturation of proteins like enzymes present on the cell surface, depolarization of transport for essential ions and nutrients, modification of cytoplasmic pH, and DNA damage (12, 18). Table Table11 shows that the addition of aluminum lactate, aluminum chloride, and sodium metabisulfite, whose ΔpHs (ΔpH = |7.5 [the optimal pH for growth] − the pH of the salt-amended medium|) are ≥3, strongly acidified the medium, whereas the addition of sodium carbonate and trisodium phosphate strongly increased the pH (ΔpH ≥ 3.1). Except for ammonium acetate, sodium acetate, sodium bicarbonate, and the preservative salts (potassium sorbate, sodium benzoate, and sodium propionate), whose ΔpHs are <1, all the other salts generally display inhibitory effects when ΔpH values are ≥1 (Fig. (Fig.1).1). Based on this result, the effect of the highly acidic or alkaline salts (which strongly affected the pH of the medium) on the growth of P. atrosepticum was evaluated at pH 7.5. Sodium carbonate and sodium metabisulfite completely inhibited bacterial growth at pH 7.5, as they did at pHs 10.6 and 4.5, respectively; trisodium phosphate (pH 11.9) exhibited a slightly lower inhibitory effect (growth inhibition of 83.2%) at pH 7.5. These observations suggest that growth inhibition by sodium carbonate, sodium metabisulfite, and trisodium phosphate cannot be attributed solely to extreme pH and passive proton transfer (extreme pH) across the bacterial membrane. Since aluminum salts precipitate at pH 7.5 (due to formation of hydrated aluminum hydroxide), it was not possible to test their inhibitory effect at pH 7.5.Open in a separate windowFIG. 1.Relationship between ΔpH (|7.5 [the optimal pH for growth] − the pH of the salt-amended medium|) and growth inhibition of Pectobacterium atrosepticum. 1, Sodium chloride; 2, potassium chloride; 3, ammonium chloride; 4, sodium tartrate; 5, sodium lactate; 6, sodium formate; 7, ammonium hydrogen phosphate; 8, sodium acetate; 9, sodium hydrogen phosphate; 10, calcium chloride; 11, ammonium acetate; 12, sodium benzoate; 13, sodium propionate; 14, potassium sorbate; 15, sodium bicarbonate; 16, aluminum dihydroxy acetate; 17, sodium metabisulfite; 18, sodium carbonate; 19, aluminum lactate; 20, aluminum chloride; 21, trisodium phosphate.The dissociation of salts in aqueous medium generates ionic species which can participate in proton exchange reactions with water molecules. The capacity of an ion to dissociate water is an intrinsic characteristic, determined by its pK value (pKa for acidic species or pKb for basic ones) (4, 21, 24). For an ionic strength of >0.1 M, pKa and pKb values of the ions are more accurate when they are defined as apparent constants (pK′a or pK′b) in terms of the activities of hydronium and hydroxyl ions, ionic species concentrations and activity coefficients (6). Thus, for the acidic ions, we have the equation ), and for the basic anions, pK′b = pKb + log(γHB/γB), where pK′a and pK′b are the apparent acidity constant and basicity constant, respectively; is the activity coefficient of the conjugate base (B); and γHB is that of the acidic (HB) species. The activity coefficient (γ) of the species i can be expressed as a function of ionic strength (μ), using the Güntelberg approximation of the Debye-Hückel equation (21), as follows: −log γi=[(0.51Zi2 μ1/2)/(1 + μ1/2)], where Zi is the charge on the species i, and μ is the ionic strength. Thus, log(/γHB) = [(0.51μ1/2)/(1 + μ1/2)] (), and log(γHB/) = −[(0.51μ1/2)/(1 + μ1/2)] ().Polytropic acid-potentiating ions (bicarbonate, carbonate, monohydrogen phosphate, phosphate, sulfite, and tartrate) in an aqueous solution can exist as (n + 1) possible species for which the parent acid is HnA. These species may coexist in equilibrium under certain pH conditions. For these ions, pK′a or pK′b were expressed as the means of the coexisting species at a specified pH. Calculated values for pK′a of acidic anions and cations and calculated values for pK′b of basic anions are presented in Table Table2.2. Figure Figure2A2A shows a sigmoidal relationship between the inhibitory effect of salts on bacterial growth and the pK′b value of the basic ions (with a common cation, sodium or potassium, in the salt) and the pK′a value of the acidic ions (with a common anion, chloride, in the salt). The plot exhibits a sharp linear relationship in the pK′ range of 8.0 to 12.0. Below the pK′ value of 8.0, inhibition is maximal, whereas above the pK′ value of 11.0, ions appear to stimulate growth (growth was maximal above the pK′ value of 12). This result demonstrates that the capacity of the constitutive ions of the salts to either donate or subtract protons to water molecules, either in the growth environment (as reflected in the modification of the medium pH) or in the developing cells, generally plays a role in their inhibitory action. The consequent transmembrane pH gradient generated leads to a passive H+ transport across the microbial membrane and to acidification (in the case of ions with low pK′a) or alkalinization (in the case of ions with low pK′b) of the cytoplasm, once the capacity for proton-coupled active transport is outstripped. In both cases, proton exchange with outer membrane proteins will destabilize these proteins, their interaction with membrane lipids, and ultimately, their function in solute transport, leading to growth inhibition. The modification of cytoplasmic pH can also alter nucleic acid structures and functions and contribute to growth inhibition (18).Open in a separate windowFIG. 2.(A) Relationship between the growth inhibition of Pectobacterium atrosepticum and the apparent basicity constant (pK′b,•) of basic anions with common Na+ (or K+) cations in the salt, the apparent acidity constant (pK′a,○) of acidic bisulfite anion (HSO3), and the cations with common Cl ions in the salt. (B) Relationship between the growth inhibition of Pectobacterium atrosepticum and the addition parameter (pK′ + pPo/w) combining the partition coefficient (Po/w) and pK′b (•) of basic anions (common cation, Na+ or K+, in the salt) or pK′a (○) of cations (common anion, Cl, in the salt) and the acidic bisulfite anion (HSO3).

TABLE 2.

Calculated apparent values for acidity, pK′a, and basicity, pK′ba
SaltBasic anion
Cation and acidic anion
pHIonic species or species in equilibriumpK′bpHIonic species or species in equilibriumpK′a
Sodium acetate7.4Acetate9.5
Sodium benzoate7.4Benzoate10.0
Sodium bicarbonate8.1H2CO3/HCO3b7.7
Sodium carbonate10.6HCO3/CO32−6.1
Sodium formate7.3Formate10.4
Sodium hydrogen phosphate8.7H2PO4/HPO42−9.8
Sodium lactate7.3Lactate11.1
Trisodium phosphate11.9HPO42−/PO43−5.3
Sodium propionate7.4Propionate9.3
Potassium sorbate7.7Sorbate9.4
Sodium tartrate7.3Tartrate2−10.6
Sodium chloride7.2Cl17.2
Sodium metabisulfite4.5SO2·H2O/HSO34.0
Aluminum chloride2.5Al3+6.2
Calcium chloride5.8Ca2+13.4
Potassium chloride7.3K+16.2
Sodium chloride7.2Na+15.0
Ammonium chloride7.0NH4+9.5
Open in a separate windowaCalculation of pK′ was performed according to Edsall and Wyman (6). pH values were measured at 0.2 M.bIncludes CO2·H2O and H2CO3.However, the water-ionizing capacity of the constituent ions of the salts and the consequent modification of the pH of the medium are not the sole factors accounting for growth inhibition, as suggested by the exceptional inhibitory actions of benzoate, propionate, and sorbate (Fig. (Fig.11 and and2A).2A). These ions provide a higher inhibition than is expected from their pK′ values (pK′b values of 10.0, 9.3, and 9.4, respectively), while the pH of their solution is optimal for bacterial growth (pHs of 7.4, 7.4, and 7.7, respectively). This suggests that they possess additional characteristics mediating their action, in addition to their water-ionization property. In fact, these preservative agents have been shown to be active either as undissociated acids (like other weak acids) or as anions (7, 8), due to their possibly hydrophobic nature which would allow them to interact with lipid constituents of the cell envelope of gram-negative bacteria such as Pectobacterium spp., and to modify their functionality (5), resulting in growth inhibition. They can also cross the cell envelope due to their lipophilicity, and their acidification inside the cell can cause additional adverse effects.Thus, we determined the octanol/water partition coefficient (Po/w), an indicator of the lipophilic character of a compound, for the effective salts with common sodium (or potassium) or chloride ions. The Po/w coefficients of the salts were determined in duplicate by using the general solvent-solvent separation procedure (9). Equal volumes (50 ml) of 1-octanol (Sigma Chemical Co., St. Louis, MO) and bidistilled water were poured into a separating flask and thoroughly shaken for 5 min. Four grams of each salt was then added, and the flask content was thoroughly mixed three times for 5 min each time, with a rest period of 5 min after each agitation. After complete separation (20 to 24 h at room temperature), the two phases were recovered separately in different flasks, and the concentration of the accompanying ion of the salt was measured in each phase by atomic absorption (model 3300 unit; Perkin-Elmer, Ueberlinger, Germany). The Po/w coefficient was calculated as the ratio of the concentration of ion in 1-octanol to the concentration of ion in the aqueous phase. Sodium benzoate was found to be the most lipophilic (Po/w = 1.41 × 10−2), followed by potassium sorbate (Po/w = 7.6 × 10−3) and sodium metabisulfite (Po/w = 2.0 × 10−4). Most other salts, sodium chloride (reference salt), sodium bicarbonate and carbonate, sodium propionate, sodium acetate, calcium chloride, and aluminum chloride mainly remained in the aqueous phase (Po/w = 2.0 × 10−5 to 5.0 × 10−5). This lipophilic characteristic of benzoate and sorbate ions would result from a reduced charge density in their molecules (due to the conjugated double bonds in their molecules). An addition parameter, pK′ + pPo/w, which combines the two properties of salts ions, i.e., the water-ionizing capacity (pK′) and the lipophilicity (pPo/w = −log Po/w), appears to provide a more general basis for the inhibitory effect of salts (Fig. (Fig.2B).2B). This suggests that while the dissociation constant of ions plays a major role in growth inhibition, as seen in Fig. Fig.2A,2A, the lipophilic character of the preservative-salt ions confers to them an added ability to penetrate the cell envelope and to inhibit bacterial growth (5, 10). The exclusion of ammonium (lower inhibition than expected from its pK′a value) and calcium (higher inhibition than expected from its pK′a value) ions from the sigmoidal pattern portrayed in Fig. Fig.2B2B would have resulted from their interactions with water and other molecules (NH4+) (1) or from cell membrane destabilization (Ca2+) (23).In conclusion, the study has shown that several salts (0.2 M concentration), including aluminum dihydroxy acetate, aluminum chloride, aluminum lactate, ammonium acetate, potassium sorbate, sodium benzoate, sodium metabisulfite, sodium bicarbonate, sodium carbonate, sodium propionate, and trisodium phosphate, strongly inhibited the growth of P. carotovorum subsp. carotovorum and P. atrosepticum. In addition, the study has established for the first time a basic sigmoidal relationship between the antimicrobial activity of the salts and the physicochemical characteristics of their constituent ions, namely their water-ionizing capacity and their lipophilicity. The constituent ions of the highly inhibiting salts generally displayed a high capacity to ionize water molecules (low pK′a or pK′b values) (Al3+, CO32−, PO43−, HCO3, and HSO3) or a high lipophilicity (benzoate and sorbate), and these two parameters in combination with known biochemical activities of salts ions would affect bacterial growth.  相似文献   

8.
Enzymatic degradation of N-acyl homoserine lactone (NAHL) was found to interfere with the quorum sensing (QS) system and related functions in several soil bacteria. In this research, the NAHL lactonase gene aiiA was amplified using aiiA-7F/aiiA7R PCR primers from the quorum sensing inhibitor rhizobacterium Bacillus sp. strain DMS133, and cloned. The plasmid pME7075, carrying the DMS133 aiiA gene under the constitutive lac promoter, was introduced into the plant pathogen Pectobacterium carotovorum EMPCC, creating strain EMPCC/aiiA. Heterologous expression of the DMS133 aiiA gene in EMPCC severely reduced the accumulation of the NAHL throughout growth, and completely prevented pigmentation of the CV026 bioreporter strain. Virulence analysis revealed that the P. carotovorum strain EMPCC/aiiA expressing AiiA lactonase had drastically reduced tissue maceration activity compared with the wild type EMPCC strain. These results provide evidence that AiiA plays an important role in the quorum quenching ability of Bacillus sp. DMS133 whose AHL degradation capacity was investigated previously. In addition, the communication signal-inactivation approach represents a promising strategy for the prevention of diseases in which virulence is regulated by QS signal molecules.  相似文献   

9.
Plant stomata function in disease resistance by restricting bacteria entry inside leaves. During plant-bacteria interactions, stomatal closure is initiated by the recognition of Microbe-Associated Molecular Patterns (MAMPs). Recently, we have shown that the Lectin Receptor Kinase V.5 (LecRK-V.5) negatively regulates bacterium- and MAMP-induced stomatal closure upstream of Reactive Oxygen Species (ROS) production mediated by abscisic acid signaling. Closed stomata in lecrk-V.5 mutants are correlated with constitutive high level of ROS in guard cells. Consequently, lecrk-V.5 mutants are more resistant to hemi-biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000). In this report, we further investigate the role of LecRK-V.5 in resistance against necrotrophic bacteria Pectobacterium carotovorum ssp. carotovorum (Pcc). Upon surface-inoculation lecrk-V.5 mutants exhibited enhanced resistance against Pcc whereas a wild-type level of resistance was observed using infiltration-inoculation, an inoculation method that bypasses the epidermal barrier. Enhanced resistance of dip-inoculated lecrk-V.5 mutants against necrotrophic bacteria, that induce different defense responses than hemi-biotrophic bacteria, further suggests a possible role for LecRK-V.5 in stomatal immunity.  相似文献   

10.
The aim of this study was characterized Pectobacterium carotovorum subsp. carotovorum (Pcc) the causal pathogen of watermelon soft rot disease in Iran. Of fifty bacterial isolates with white grey and convex colonies on nutrient agar obtained from symptomatic watermelon, ten isolates were selected for further tests. Pathogenicity tests results showed that all test isolates developed typical water‐soak symptoms after 2 days and signs of soft rot began 4 days after inoculation on watermelon fruits. Based on the phenotypic properties, the isolates were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rDNA sequences of isolates were 99% similar to the corresponding 16S rDNA sequence of the reference Pcc isolate. BOX and ERIC‐PCR analysis indicated that genetic diversity was present among the isolated Pcc isolates did not relate to the geographic location isolated from. To the best of our knowledge, this is the first study of biochemical and genotypic characterization of Pcc isolates the causal agents of soft rot disease on watermelon, in Iran.  相似文献   

11.
【目的】为了研究鞭毛钩基因flgK在胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovorum subsp.carotovorum,P.c.c)的功能。【方法】本研究采用两亲同源交换法构建了基因缺失突变体ΔflgKpcc并构建了互补菌株ΔflgKpcc-KH,测定突变体及其互补菌株的菌体形态、运动性、致病因子、致病性等表型。【结果】与野生菌株PccS1相比,ΔflgKpcc鞭毛缺失,菌体易沉降,在0.3%半固体培养基上运动能力明显降低,生长速率无明显变化,但是纤维素酶和蛋白酶的活性、生物膜形成能力明显下降,对感病寄主的致病力显著减弱。基因互补可以使上述突变表型恢复。【结论】实验表明,鞭毛基因flgK突变导致了菌体的运动性降低、病原菌毒性相关的酶活力下降,从而导致致病力下降。  相似文献   

12.
To identify bacteria causing soft rot and blackleg in potato in Finland, pectinolytic enterobacteria were isolated from diseased potato stems and tubers. In addition to isolates identified as Pectobacterium atrosepticum and Dickeya sp., many of the isolated strains were identified as Pectobacterium carotovorum subsp. carotovorum. Phylogenetic analysis and biochemical tests indicated that one of the isolates from potato stems resembled Pectobacterium wasabiae. Furthermore, two blackleg‐causing P. carotovorum strains recently isolated in Europe clustered with P. wasabiae, suggesting that at least some of these isolates were originally misidentified. All the other Finnish P. carotovorum isolates resembled the subsp. carotovorum type strain in biochemical tests but could be clustered into two distinct groups in the phylogenetic analysis. One of the groups mainly contained strains isolated from diseased tubers, whereas the other mainly included isolates from potato stems. In contrast to the tuber isolates, the stem isolates lacked genes in Type III secretion genes, were not able to elicit a hypersensitive response in tobacco leaves and produced only small amounts of autoinducers in the stationary phase in vitro. P. wasabiae isolate was able to cause similar amount of blackleg‐like symptoms as P. atrosepticum in a field experiment with vacuum‐infiltrated tubers, whereas both P. atrosepticum and P. carotovorum isolates reduced emergence and delayed growth more than P. wasabiae. Our findings confirm the presence of P. wasabiae in Finland and show that the Finnish P. carotovorum subsp. carotovorum isolates can be divided into two groups with specific characteristics and possibly also different ecologies.  相似文献   

13.
Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.  相似文献   

14.
Microbial expansins act on plant cell walls similarly to plant expansins, albeit their loosening activity levels are tenfold lesser compared to plant expansins. We report the characterization of an expansin-like gene from the plant pathogen Pectobacterium carotovorum, named exl1. PcExl1 is an acidic protein that binds cellulose (Avicel), and weakens filter paper. The acidic nature of PcExl1 confers different binding properties when compared to Bacillus subtilis BsEXLX1, which is a basic protein. PcExl1 binding to wheat cell wall increased when acidic components were depleted, reaching a similar level to the binding to Avicel, indicating that cellulose is the target of PcExl1.  相似文献   

15.
JH Lee  H Shin  S Ji  S Malhotra  M Kumar  S Ryu  S Heu 《Journal of virology》2012,86(16):8899-8900
Pectobacterium carotovorum subsp. carotovorum is a phytopathogen causing soft rot disease on diverse plant species. To control this plant pathogen, P. carotovorum subsp. carotovorum-targeting bacteriophage PP1 was isolated and its genome was completely sequenced to develop a novel biocontrol agent. Interestingly, the 44,400-bp genome sequence does not encode any gene involved in the formation of lysogen, suggesting that this phage may be very useful as a biocontrol agent because it does not make lysogen after host infection. This is the first report on the complete genome sequence of the P. carotovorum subsp. carotovorum-targeting bacteriophage, and it will enhance our understanding of the interaction between phytopathogens and their targeting bacteriophages.  相似文献   

16.
胡萝卜软腐果胶杆菌是世界十大植物病原菌之一,主要侵染十字花科的经济作物和观赏花卉。文中从胡萝卜软腐果胶杆菌的基因组中克隆1个抗菌基因cpxP(GeneID:29704421),将其构建在原核表达质粒pET-15b上,并转化至大肠杆菌Escherichia coli BL21 (DE3)进行表达,经纯化后进行稳定性和抑菌实验。结果显示,IPTG的诱导终浓度为1mmol/L,实现了蛋白的高效外源表达,纯化后电泳无杂蛋白残留,且该蛋白具有良好的热稳定性和pH稳定性。CpxP蛋白抑菌试验结果显示其对胡萝卜切片的抑菌率可达到44.89%,对马铃薯切片的抑菌率可达到59.41%。为进一步解释其抑菌机理,研究该蛋白的空间结构可为软腐病的防治和新型蛋白农药靶点研究提供新思路。  相似文献   

17.
Aims: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. Methods and Results: Several calibration steps were required to distinctly gate the GFP‐labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. Conclusions: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. Significance and Impact of the Study: The combination of time‐ and cost‐saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.  相似文献   

18.
19.
Abstract

In this study, an antagonistic yeast isolate, Wickerhamiella versatilis was considered as a promising biocontrol agent against Pectobacterium carotovorum subsp. Carotovorum (Pcc) the causal agent of soft rot disease of potato. Antagonistic yeast inhibited the growth of Pcc in vitro, and reducing the soft rot severity of infected potato tubers (cv. Diamant) under greenhouse conditions. Consequently, cellulase and pectinase hydrolytic activities in infected potato tubers with yeast?+?Pcc were decreased compared with infected tubers with Pcc. The histological characterization of treated potato tubers with antagonistic yeast W. versatilis using scanning electron microscope showed the accumulation of extracellular substances that may induce plant resistant and protects potato tubers from hydrolysis and damages. This study may introduce the possibility of using the antagonistic yeast isolate, as a biocontrol agent against soft rot of potato tubers.  相似文献   

20.
Bacteria and plant derived volatile organic compounds have been reported as the chemical triggers that elicit induced resistance in plants. Previously, volatile organic compounds (VOCs), including acetoin and 2,3-butanediol, were found to be emitted from plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis GB03, which had been shown to elicit ISR and plant growth promotion. More recently, we reported data that stronger induced resistance could be elicited against Pseudomonas syringae pv maculicola ES4326 in plants exposed to C13 VOC from another PGPR Paenibacillus polymyxa E681 compared with that of strain GB03. Here, we assessed whether another long hydrocarbon C16 hexadecane (HD) conferred protection to Arabidopsis from infection of a biotrophic pathogen, P. syringae pv maculicola and a necrotrophic pathogen, Pectobacterium carotovorum subsp carotovorum. Collectively, long-chain VOCs can be linked to a plant resistance activator for protecting plants against both biotrophic and necrotrophic pathogens at the same time.  相似文献   

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