Luminescence-based detection of Erwinia carotovora associated with rotting potato tubers

A luminescence-based marker system has been used to follow rotting of potato tubers by Erwinia carotovora. The early stages of infection could be detected by visual observation of areas of luminescence developing from the point of inoculation. During later stages luminescence was limited to peripheral regions and to central regions where the structure of the potato was destroyed. Luminometry demonstrated reduction in aerobic activity of infecting cells due to oxygen limitation. The system enables rapid and sensitive detection of active marked populations and distinction between aerobic and fermentative activity.     

Extracellular polysaccharide of Erwinia chrysanthemi A350 and ribotyping of Erwinia chrysanthemi spp

Erwinia chrysanthemi spp. are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by the E. chrysanthemi strain A350, which is a lacZ- mutant of the wild type strain 3937, pathogenic to Saintpaulia, has been determined using a combination of chemical and physical techniques including methylation analysis, low-pressure gel-filtration and anion-exchange chromatography, high-pH anion-exchange chromatography, partial acid hydrolysis, mass spectrometry and 1- and 2D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain A350 contains D-GalA, together with L-Rhap and D-Galp in a 1:4:1 ratio. Evidence is presented for the following hexasaccharide repeat unit: [structure: see text] All the Erwinia chrysanthemi spp. studied to date have been analyzed by ribotyping and collated into families, which are consistent with the related structures of their EPS.     

Physiological ageing of potato tubers: A Review

Numerous theories have been proposed to describe the complex process of ageing in biological systems. Two general groups of ageing theories currently exist: 1) stochastic where the accumulation of random molecular damage leads to loss of information vital to the cell; and, 2) systemic where an organised, genetically based sequence of metabolic activities leads to programmed ageing. Whether these are acceptable models of ageing in potato tubers is unknown although the tuber could provide a useful experimental system for studying ageing. An initial requirement for advancing the concept of ageing in potato tubers must centre on the development of a suitable ageing index. A review of the literature suggests that a modified approach to ‘sprouting capacity’ and ‘incubation period’ may allow tuber ageing to be described in mathematical terms that would, in turn, facilitate the development of a physiological ageing index as well as temperature sensitive predictive models. Although a number of biochemical studies of ageing have been pursued, the development of adequate biomarkers has yet to achieve a coordinated level of development as found in a range of organisms. For example, ageing in other biological systems may be viewed as an outcome of an accumulation of random molecular damage and may be primarily caused by a changing balance between reactive oxygen species and diminishing levels of protective agents such as superoxide dismutase, alpha‐tocopherol or vitamin C. The exploration of these and similar problems in the context of appropriate modelling approaches should allow a better understanding of physiological ageing in potato tubers.     

Comparison of methods for estimating Erwinia carotovora numbers on potato tubers

Two methods for estimation of the numbers of Erwinia carotovora on potato tubers were compared using 17 naturally infected stocks. The 'wash' method estimates the surface count, whereas the 'peel' method is based on a subcuticular count. A correlation of 0.79 was obtained. Comparisons between methods were also made using a bactericide, Myacide As, and on stocks during chitting. Erwinia carotovora counts were reduced in treated tubers with the 'wash' method, but not with the 'peel' method.     

Time-resolved fluoroimmunoassay as a method for detection of Erwinia chrysanthemi in potato peel extracts

J.M. VAN DER WOLF. 1993. Time-resolved fluoroimmunoassay (TR-FIA) was compared with double antibody sandwich (DAS)-ELISA for detection of Erwinia chrysanthemi antigens in potato peel extracts. Pure cultures were used to optimize TR-FIA with respect to the microplate washing procedure and dilution buffer compositions.
The detection threshold level for spiked potato peel extracts with the optimized TR-FIA format was 10⁵ cells ml^-1 as for the detection level of DAS-ELISA. The signal to background ratios at concentrations above 10⁵ cells ml^-1 were higher with TR-FIA than with DAS-ELISA. The dynamic range of TR-FIA was also superior to that of DAS-ELISA.
It can be concluded that TR-FIA is an attractive alternative to DAS-ELISA as a detection method for Erw. chrysanthemi, especially when quantification is required.     

Osmoregulated periplasmic glucans of Erwinia chrysanthemi

We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of Erwinia chrysanthemi. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000-0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and (1)H and (13)C nuclear magnetic resonance analyses. OPGs produced by E. chrysanthemi are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of beta-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by beta-1,6 linkages in a random way. This glucan backbone may be substituted by O-acetyl and O-succinyl ester-linked residues.     

recA-genes of Erwinia chrysanthemi ENA49

The recA gene of Erwinia chrysanthemi ENA49 has been cloned in vivo in Escherichia coli K12, recA13 cells using the plasmid pULB113. On the basis or DNA repair and recombination deficiencies complementation, of restoration of the inducible "SOS"-response functions the functional identity of the cloned gene with the recA gene was concluded. The recA gene was localized in the 18th min region of the chromosomal genetical map of Erwinia chrysanthemi ENA49 between the genes proA and pheA.     

Lipid-degrading enzymes from potato tubers

Three varieties of potato were used to investigate the activity of lipolytic acyl hydrolase, (LAH) and lipoxygenase, (LOX), for a short period after harvest. Both enzymes displayed very low levels of activity during the first few days, followed by an increase in later storage, with the hydrolase activity of Désirée tubers remaining low. An inverse relationship was found between the total LOX activity and the percentage of activity obtained in a particulate form. Only when the total LOX content was below 0.7 units (μmol/g/min fr. wt), was it possible to obtain a highly active particulate fraction. LAH particulate activity was dependent upon both enzymes remaining low. Protoplasts were isolated by the use of cell-degrading enzymes. When the total LOX activity in the tubers was low, 50% of this activity could be obtained in intact protoplasts. Once the LOX concentration in the tubers had risen, fewer intact protoplasts were isolated. No particulate activity of either enzyme was found when these protoplasts were lysed. The two lipid-degrading enzymes were not located in the amyloplasts.     

Beta-aspartylpeptides as substrates of L-asparaginases from Escherichia coli and Erwinia chrysanthemi

L-Asparaginase is known to catalyze the hydrolysis of L-asparagine to L-aspartic and ammonia, but little is known about its action on peptides. When we incubated L-asparaginases purified either from Escherichia coli or Erwinia chrysanthemi - commonly used as chemotherapeutic agents because of their antitumour activity - with eight small beta-aspartylpeptides such as beta-aspartylserineamide, beta-aspartylalanineamide, beta-aspartylglycineamide and beta-aspartylglycine, we found that both L-asparaginases could catalyze the hydrolysis of five of them yielding L-aspartic acid and amino acids or peptides. Our data show that L-asparaginases can hydrolyze beta-aspartylpeptides and suggest that L-asparaginase therapy may affect the metabolism of beta-aspartylpeptides present in human body.     

Hydrodynamic properties of oxidized extracellular polysaccharides from Erwinia chrysanthemi spp

The molecular weights of the native polysaccharides of Erwinia chrysanthemi strains range from 1.8 to 7.1 x 10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions. The effect on the hydrodynamic properties of the polysaccharides by adding carboxyl groups to increase the charge density is studied, with particular reference to their molecular weight (MW), viscosity and conformation. In general, it is found that periodate oxidation of the extracellular polysaccharides of E. chrysanthemi strains, Ech9Sm6 and Ech6S+, introduces little change in the hydrodynamic properties of the resulting polyaldehydes. However, bromine oxidation at neutral pH of the polyaldehydes results in polycarboxylate biopolymers that show significant reduction in MW and viscosity, but they are still characteristic polyanions.     

Cold-lability of phosphofructokinase from potato tubers

The aim of this work was to study the cold-lability of phosphofructokinase from tubers of Solanum tuberosum cv Record, a variety that exhibits low temperature sweetening. The enzyme was purified by affinity chromatography and samples were examined by differential scanning calorimetry. Power-time curves were recorded for cooling and warming between 293 and 265 K. This revealed an exothermic dissociation, centred on 286 K, as the temperature was lowered. The latter temperature is close to that at which the tubers start to sweeten. It is suggested that hydrophobic interactions that contribute to the stability of the active configuration of the oligomeric enzyme are weakened at low temperatures, and that this causes spontaneous dissociation and consequent loss of activity of the enzyme. The results are discussed in relation to low temperature sweetening of potatoes.     

Pyruvated galactose and oligosaccharides from Erwinia chrysanthemi Ech6 extracellular polysaccharide

The acidic extracellular polysaccharide of Ech6 was depolymerized by fuming HCl. The pyruvated sugars were isolated and characterized by methods that included a combination of low-pressure gel-filtration and high-pH anion-exchange chromatographies, methylation linkage analyses, mass (GC-MS and MALDI-TOF MS) and 1H NMR (1D and 2D) spectroscopies. The following pyruvated sugars were obtained: 4,6-O-(1-carboxyethylidene)-D-Galp; 4,6-O-(1-carboxyethylidene)- alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-D-Galp; 4,6-O-(1-carboxyethylidene)-alpha-D-Galp-(1-->4)-alpha-D-GlcAp- (1-->3)-alpha-D-Galp-(1-->3)-L-Fucp; 4,6-O-(1-carboxyethylidene)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3) -alpha-D-Galp-(1-->3)-L-[beta-D-Glcp-(1-->4)]-Fucp. These oligosaccharides present potential haptenes for the development of specific antibodies and confirm the partial structure proposed previously for the extracellular polysaccharide from Erwinia chrysanthemi Ech6 [Yang, B. Y.; Gray, J. S. S.; Montgomery, R. Int. J. Biol. Macromol., 1994, 16, 306-312].     

Secretion processing and activation of Erwinia chrysanthemi proteases

E chrysanthemi, a phytopathogenic enterobacterium, secretes several enzymes into the medium such as pectinases cellulases and proteases. It also produces 3 distinct and antigenically related extracellular proteases. The proteases secretion pathway seems to be distinct from that of the other extracellular enzymes since pleiotropic mutants impaired in cellulase and pectinase secretion are unimpaired in protease secretion. E chrysanthemi proteases B and C secretion occurs without an N-terminal signal peptide and is dependent upon specific secretion functions which are encoded by genes adjacent to the protease structural genes. This secretion pathway might be analogous to the alpha-hemolysin secretion pathway in E coli. Protection against intracellular proteolytic activity is achieved by 2 distinct mechanisms: the proteases are synthesized as inactive precursors with an N-terminal extension of 15 aminoacids (protease B) and 17 aminoacids (protease C) absent in the mature active extracellular enzymes; an intracellular specific protease inhibitor is produced by some E chrysanthemi strains.     

Aromatic alcohol dehydrogenase from potato tubers

The purification of NADP specific aromatic alcohol dehydrogenase is reported. The properties of the enzyme suggest that it should be classified as E.C. 1.1.1.2. Kinetic constants for a number of substrates are reported. The relative rates of reaction of a variety of substituted benzaldehydes have been found to correlate with Hammett's sigma values yielding a biphasic relationship. The physiological significance of the enzyme is briefly discussed.     

Protein trypsin inhibitor from potato tubers

A protein of 22 kDa designated as PKTI-22 was isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and purified to homogeneity using CM-Sepharose CL-6B ion-exchange chromatography. The protein efficiently suppressed the activity of trypsin, affected chymotrypsin less, and did not affect subtilisin Carlsberg. The N-terminal sequence of PKTI-22 (20 amino acid residues) was found to be highly homologous with the amino acid sequences of the potato Kunitz-type proteinase inhibitors of group B (PKPI-B) that were aligned from the corresponding gene sequences and was identical to the sequence (from the 2nd to the 20th residue) of the recombinant protein PKPI-B10. These data together with the observed similarity of the properties of two proteins indicate that the PKTI-22 protein is encoded by the PKPI-B10 gene.     

Post-infectional metabolites from infected potato tubers

The C-1′ epimers of the sesquiterpenoids 2-(1′,2′-dihydroxy-1′-methylethyl)-6,10-dimethylspiro[4,5]dec-6,9-dien-8-one and 2-(1′,2′-dihydroxy-1′-methylethyl)-6,10-dimethyl-9-hydroxyspiro[4,5]dec-6-en-8-one were isolated from potato tubers infected with Phoma foveata and Fusarium spp., in addition to 4,4a,5,6,7-hexahydro-3-hydroxy-6-(1′,2′-dihydroxy-1′-methylethyl)-4-methyl-2(3H)-naphthalenone, N-trans-p-coumaroyl tyramine and N-trans-feruloyl tyramine. Three of the compounds are novel.     

Aliphatic alcohol dehydrogenase from potato tubers

Potato tubers are shown to contain at least 3 alcohol dehydrogenases, one active with NAD and aliphatic alcohols, one active with NADP and terpene alcohols and one active with NADP and aromatic alcohols. The purification of the aliphatic alcohol dehydrogenase is described and its activity with a wide range of substrates is reported. On the basis of substrate specificity, the enzyme is shown to resemble yeast alcohol dehydrogenase rather than liver alcohol dehydrogenase. The enzyme shows high activity with and high affinity for ethanol, activity and affinity decline as the chain length is increased from ethanol to butanol, but a further increase in chain length leads to increased affinity for the alcohol. The physiological significance of the results is briefly discussed.     

Erwinia and yellow-pigmentedEnterobacter isolates from human sources

Sixty-five strains of gram-negative, yellow-pigmented bacilli, including four chromogenicEnterobacter strains and 55 anaerogenic and six aerogenicErwinia strains, were isolated from human sources. The genusErwinia contained two groups; an anaerogenic group which produced aggregates of bacteria (symplasmata) in the syneresis water of slant cultures and biconvex bodies in colonies on agar medium, and an aerogenic group which lacked these characteristics.Erwinia was differentiated fromEnterobacter, since the latter possessed dihydrolase and decarboxylase activity and demonstrated resistance to cephalothin.