Extracellular polysaccharide of Erwinia chrysanthemi CU643.

Erwinia chrysanthemi are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by E. chrysanthemi strain CU643, pathogenic to Philodendron, has been determined using a combination of chemical and physical techniques including methylation analysis, high- and low-pressure gel-filtration and anion-exchange chromatography, high-pH anion-exchange chromatography, partial acid hydrolysis, mass spectrometry, and 1- and 2-D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain CU643 is a linear polysaccharide containing L-Rhap, D-Galp, and D-GlcAp in the ratio 4:1:1. Evidence is presented for the following hexasaccharide repeat unit: -->3)-beta-D-Galp-(1-->2)-alpha-L-Rhap-(1-->4)-beta-D-GlcAp- (1-->2)-alpha-L- Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->(1 ).     

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.     

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].     

Extracellular polysaccharides of modified strains of Erwinia spp.

The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain A2148 has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl-linkage analysis, and 1D 1H NMR spectroscopy. The polysaccharide is structurally similar, if not identical, to the EPS produced by E. chrysanthemi strain A350. A streptomycin-resistant strain of E. chrysanthemi Ech6 (Ech6S(+)) has been generated and has an elevated production of EPS, as does a streptomycin-resistant strain (Ech9Sm6) of E. chrysanthemi Ech9. These modified E. chrysanthemi spp. have been ribotyped and found to be closely related to their parent strains.     

beta-Elimination of glucosyluronic residues during methylation of an acidic polysaccharide from Erwinia chrysanthemi CU 643

The Erwinia chrysanthemi CU643 EPS has a linear hexasaccharide repeating unit in which a 4-linked uronic acid residue is present. The EPS was methylated by either the NaOH-Me2SO-MeI or Li-dimsyl procedure. MALDI-TOF MS analysis of the methylated products indicates that the beta-eliminative degradation occurs during the methylation, as characterized by serial fragments of the hexasaccharide repeating units. The degradation was clearly defined from the methylation of a glucosyluronic-containing pyruvated pentasaccharide.     

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.     

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.     

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.     

Lactose and melibiose metabolism in Erwinia chrysanthemi.

A Lac+ mutant of Erwinia chrysanthemi was isolated from the Lac- wild type on lactose agar. beta-Galactosidase was expressed independently of lactose transport in both the mutant and the wild type, and neither strain expressed thiogalactoside transacetylase. Lactose transport and alpha-galactosidase, constitutive in the Lac+ strain, were coordinately induced in the Lac- strain by melibiose and raffinose but not by isopropyl-beta-D-thiogalactopyranoside or thiomethyl-beta-D-galactopyranoside. Melibiose was a strong inhibitor of both the melibiose- and the raffinose-induced lactose permeases, whereas raffinose was a strong inhibitor of only the raffinose-induced lactose permease.     

Spread of Erwinia chrysanthemi in Kalanchoë blossfeldiana

Meristems were excised from young and 8-month-old Kalanchoë blossfeldiana naturally-infected with Erwinia chrysanthemi (Echr). From heavily infected plants the bacteria were identified in meristems from the very top of the plant as well as from meristems situated lower on the plant. In other naturally-infected plants Echr was only found at the stem base, depending on how advanced (old) the infection was. Repetition of identification on meristem samples stored in the refrigerator for1 week revealed more positive samples than in the first test. Infection trials showed a quick spread of bacteria from stem base to apex via xylem vessels in some of the inoculated plants. Spread of Echr via capillary mats from inoculated to non-inoculated Kalanchoë plants were found after 5.5 months growth on the capillary mat. The time lapse before transmission may vary with the seasons of the year.     

Expanded linkage map of Erwinia chrysanthemi strain 3937

In this paper we describe the chromosomal location of various loci in Erwinia chrysanthemi strain 3937. Auxotrophic markers were obtained by chemical mutagenesis, antibiotic resistances were isolated spontaneously and mutations in sugar utilization were obtained by means of Mu insertions. These markers were located on the genetic linkage map of strain 3937 by using a conjugative system mediated by RP4::mini-Mu plasmids which permitted transfer of genetic material from any point of origin. The location of these markers was compared to that of previously located mutations. Many genes involved in pectinolysis were also located on the E. chrysanthemi 3937 map. These results permitted us to present a new genetic map containing 61 markers distributed over 34 widely scattered loci on the chromosome. Some pairs of markers giving high cotransfer frequencies were tested for cotransduction mediated by the generalized transducing phage phi-EC2; nine cotransducing pairs were found. It appears that the chromosomal locations of many of these loci are quite different to those of the well-known enterobacterium Escherichia coli but seem similar to those described for other E. chrysanthemi strains.     

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi

The "family 9 polysaccharide lyase" pectate lyase L (Pel9A) from Erwinia chrysanthemi comprises a 10-coil parallel beta-helix domain with distinct structural features including an asparagine ladder and aromatic stack at novel positions within the superhelical structure. Pel9A has a single high affinity calcium-binding site strikingly similar to the "primary" calcium-binding site described previously for the family Pel1A pectate lyases, and there is strong evidence for a common second calcium ion that binds between enzyme and substrate in the "Michaelis" complex. Although the primary calcium ion binds substrate in subsite -1, it is the second calcium ion, whose binding site is formed by the coming together of enzyme and substrate, that facilitates abstraction of the C5 proton from the sacharride in subsite +1. The role of the second calcium is to withdraw electrons from the C6 carboxylate of the substrate, thereby acidifying the C5 proton facilitating its abstraction and resulting in an E1cb-like anti-beta-elimination mechanism. The active site geometries and mechanism of Pel1A and Pel9A are closely similar, but the catalytic base is a lysine in the Pel9A enzymes as opposed to an arginine in the Pel1A enzymes.     

Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes

Erwinia chrysanthemi 3937 secretes four major pectate lyase isoenzymes (PL, EC 4.2.2.2) and one endocellulase (Cx, EC 3.2.1.4). A genomic library of this strain was constructed in the Lambda L47-1 vector, and screened for the presence of PL and Cx on pectate and caboxymethylcellulose agar. Among the seven Cx-positive phage clones, three were shown to encode an enzyme of the same mol. wt. as the one found in the culture supernatant of strain 3937. The 34 PL-positive phage clones were analyzed by electrofocusing and could, according to the PL they produced, be arranged in five classes. Phages from three classes produced three different single PL, named PLb, c and d. No common fragment was evidenced between the inserts of the phages of these three classes. This demonstrated that, in strain 3937, PLb, C, and d were encoded by three different genes called pelB, C, and D. Furthermore, our results suggest the existence of two additional genes encoding PLa and e. In addition, a pectin methylesterase gene was found closely linked to pelD.     

Fermentation of d-Xylose and l-Arabinose to Ethanol by Erwinia chrysanthemi

Erwinia spp. are gram-negative facultative anaerobes within the family Enterobacteriacae which possess several desirable traits for the conversion of pentose sugars to ethanol, such as the ability to ferment a broad range of carbohydrates and the ease with which they can be genetically modified. Twenty-eight strains of Erwinia carotovora and E. chrysanthemi were screened for the ability to ferment d-xylose to ethanol. E. chrysanthemi B374 was chosen for further study on the basis of its superior (4%) ethanol tolerance. We have characterized the fermentation of d-xylose and l-arabinose by the wild type and mutants which bear plasmids containing the pyruvate decarboxylase gene from Zymomonas mobilis. Expression of the gene markedly increased the yields of ethanol (from 0.7 up to 1.45 mol/mol of xylose) and decreased the yields of formate, acetate, and lactate. However, the cells with pyruvate decarboxylase grew only one-fourth as fast as the wild type and tolerated only 2% ethanol. Alcohol tolerance was stimulated by the addition of yeast extract to the growth medium. Xylose catabolism was characterized by a high saturation constant K(s) (4.5 mM).     

Mapping and regulation of the cel genes in Erwinia chrysanthemi

Summary Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the fusions, (iii) recombinational exchange between the plasmidic mutated and the wild-type chromosomal alleles. These mutations allowed mapping of celZ between ura and pan and celY between xyl and met on the linkage map of E. chrysanthemi. The -galactosidase activity of these strains indicated that celZ is expressed in the late exponential and stationary growth phases, while celY expression is almost undetectable.     

Mutations of ousA alter the virulence of Erwinia chrysanthemi

A negative correlation was observed between the aggressiveness of several Erwinia chrysanthemi strains on potato tuber and their osmotic tolerance. The disruption of the ousA gene encoding the major osmoprotectant uptake system highly enhanced bacterial virulence on potato tubers. The ousA disruption also increased the maceration efficiency on potato tubers under anaerobic conditions. In the absence of oxygen, pectate lyase (Pel) production was significantly higher in the tissue macerated with the ousA- strain than with the wild type. Oxygen content is significantly different between infected and healthy tissues; therefore, ousA may be a contributory factor in the infection progression within the host. In minimal medium, ousA disruption enhanced Pel production and pelE expression only under micro-aerobiosis conditions. The effect on Pel was reversed by reintroduction of the ousA gene. The osmoprotectectants glycine betaine, proline betaine, and pipecolic acid are known to be taken up via OusA and to have an inhibitory effect on Pel production. However, their effects on Pel activity were not (glycine betaine) or only weakly (proline and pipecolic acid) affected by ousA disruption. Furthermore, no correlation was observed between their effects on Pel activities and their osmoprotection efficacies. The results demonstrate a relationship between E. chrysanthemi pathogenicity factors and the activity of ousA under low oxygen status. The evidence indicates that ousA and osmoprotectant effects on Pel are not linked to osmoregulation and that complex regulations exist between Pel production, ousA, and osmoprotection via compounds liberated during the plant infection.     

Fusion of Erwinia chrysanthemi spheroplasts in an electric fields

A new approach has been elaborated for electrofusion of Erwinia chrysanthemi spheroplasts. The new approach consists of superimposition of high voltage impulses on the pellet of tightly contacting cells in the course of centrifugation. The mixture of spheroplasts of two genetically marked strains was placed into the special centrifuge chambers and spinned for 15 min at 2500 g to get a compressed pellet between chamber electrodes. Three successive pulses of 6.6 kv/cm amplitude and 30 microseconds duration were applied to spheroplast pellet during centrifugation. Fusion products were viable and after plating on the surface of hypertonic medium regenerated to the rod forms. As a result, the hybrid clones carrying the markers of both parents were isolated.