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[Adsorbed receptors for Erwinia carotovor subsp. carotovora macromolecular bacteriocins].

Study of nature of receptors for macromolecular bacteriocins Erwinia carotovora subsp. carotovora has shown that lipopolysaccharide (LPS) of cell membrane is an attaching structure for them. It has been established that enzymic treatment of LPS preparation with its further deproteinization by phenol is necessary for isolation of biologically active lipopolysaccharide. The process of absorption by LPS has been studied quantitatively and it has been shown that it is a low-efficient receptor as compared with LPS included in the native cell membranes. An approach has been proposed for the first time to the estimation of monosaccharide composition of LPS-receptor based on relations between bacteriocin sensitivity and content of monosaccharides. Study of six strains of E. carotovora subsp. carotovora from different sources has shown that the structure of LPS-receptors includes mannose, fucose, xylose, ramnose and two lipophylic monosaccharides of unknown nature. A conclusion has been made that S-LPS (0-chain) is that part which contains the sites of attachment of macromolecular carotovoricins.

Adsorption↗

[Characteristics of bacterial lipopolysaccharides depending on extraction method].

Carbohydrate-containing biopolymers have been isolated from Erwinia carotovora subsp. atroseptica 549 by two methods--the aqueous-phenol and with using physiological solution--with addition and without addition of ethylenediaminetetraacetate (EDTA). The biopolymers yield from the cells of bacteria are shown to depend on the extraction method. Lipopolysaccharide-protein complex have been isolated by the sparing method. The purest lipopolysaccharide have been isolated by the aqueousphenol method. The preliminary treatment of cells by EDTA increased the biopolymers output. Carbohydrate containing biopolymers isolated from the cells of bacteria by different methods possess the similar qualitative composition of monosaccharides but they differ in the quantitative content of monosaccharides, spectrum of fatty acids of lipid components as well as in the protein content.

Biopolymers↗

[Isolation and preliminary characterization of cryptic plasmids from Erwinia carotovora].

Of the fifty-two Erwinia carotovora strains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovora strains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovora strains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, 47.7-kbp pCA 6-2. Three E. carotovora subsp. carotovora strains and one E. carotovora subsp. atroseptica strain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.

DNA, Bacterial↗

[Temperate bacteriophage ZF40 of Erwinia carotovora: phage particle structure and DNA restriction analysis].

Structural organization of the temperate bacteriophage ZF40 of Erwinia carotovora was studied. Phage ZF40 proved to be a typical member of the Myoviridae family (morphotype A1). Phage particles consist of an isometric head 58.3 nm in diameter and a contractile 86.3-nm-long tail with a complex basal plate and short tail fibers (31.5 nm). Phage tail sheath, a truncated cone in shape, is characterized by specific packaging of structural subunits. The ZF40 phage genome is 45.8 kb in size, as determined by restriction analysis, and contains DNA cohesive ends. The ZF40 phage of Erwinia carotovora is assumed to be a new species of bacteriophages specific for enterobacteria.

DNA, Viral↗

[Study of Erwinia carotovora phage resistance with the use of temperate bacteriophage ZF40].

The causes of the unique phage resistance of the pectinolytic phytopathogenic strains of Erwinia carotovora were studied with the use of temperate bacteriophage ZF40. It was shown that, in these bacteria, the bacteriophage-cell interaction can be substantially blocked at the adsorption level. An adequate indicator for studying the temperate bacteriophages of erwinias was developed on the basis of mutants resistant to macromolecular bacteriocins. Various restriction-modification systems, which influence cell resistance to bacteriophages, were revealed for the first time in E. carotovora. The phage resistance was shown to be determined by the wide occurrence of homoimmune temperate viruses in pectinolytic erwinias.

Bacteriocins↗

[Study of population dissociation of collection strains of Erwinia carotovora].

Unusual dissociants of Erwinia carotovora subsp. atroseptica (EAT) population that emerge with high frequency (18-94%) during long-term storage of cultures were found. The dissociants (Poe-phenotype) differ from parental forms by slow growth and long development of the colour of colonies on EMB agar though they are not Lac-mutants. More than 60% of Poe-mutants of strain EAT 39A are characterized by 8- and 150-fold decrease of stability to erythromycin and oleandomicin, correspondingly. They are also sensitive to colicin-like carotovoricin, which is induced by mytomicin C, to carotovoricin of initial strain EAT 39A and to autocin, which they produce themselves during induction. A multiple phenotype of these dissociants (dissociants of the second type) is labeled: PoeOmSErSBnSAu. Its appearance as well as the appearance of pure Poe-variants is not associated with usual mutations and a loss of residential plasmids by cells. Population dissociants of the both types--Poe and PoeApSOmS--were also obtained from E. carotovora subsp. carotovora (J2) during growth at supraoptimal temperature. Poe-variants are more often observed in representatives of subspecies atroseptica than carotovora and can be useful when studying pathogeneity of these practically important erwinias.

Pectobacterium carotovorum↗

[Defective lysogeny in Erwinia carotovora].

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128-192 nm in length and 13-21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.

Defective Viruses↗

[Method for studying horizontal transfer of plasmids in Erwinia carotovora].

A simple method to determine frequency characteristics of the horizontal transfer of antibiotic resistance by transconjugative plasmids of various incompatibility groups into Erwinia carotovora strains has been developed. The proposed method for selection of E. carotovora transconjugates provides for the use of selective medium with pectin and corresponding antibiotic, which permits carrying out reliable counterselection of the donor (Escherichia, Salmonella). The proposed method helped to study horizontal transfer of plasmids RP4 (Inc P), R 391 (Inc J) and pKM101 (Inc N) to some E. carotovora strains and to demonstrate their inheritance as extrachromosomal DNA in Erwinia cells.

Anti-Bacterial Agents↗

[In vitro self assembly of supermolecular structures after spontaneous disassembly of carotovoricins].

The self-assembly of supramolecular structures (empty sheaths and polysheaths of the macromolecular Erwinia carotovora bacteriocins) was studied by electron microscopy in the course of 1- to 2-year incubation of phage particles at 4 degrees C. This study showed that the empty sheaths and polysheaths of the bacteriocins of eight E. carotovora strains spontaneously assemble at the self-assembly centers (or crystallization centers), which have a diameter of 26-65 nm and contain a dense proteinaceous material. The self-assembly center consists of two components, a primer and the structural protein of contracted sheaths. Empty sheaths assembled in the crystallization centers are polar structures synthesized through the stepwise head-to-tail polymerization of monomeric units. The supramolecular structures of two E. carotovora 62A bacteriocins are assembled in a different way. At the early stages of their self-assembly, a reticular structure is formed, which then transforms into very long polysheaths composed of monomers. Along with polysheaths, rounded or lamplike structures 33-117 nm in size composed of the subunits of contracted sheath are produced. Carotovoricins may serve as suitable objects for the study of the self-assembly of elementary biological structures.

Bacteriocins↗

[Type III secretion system in Se9R can recognize and secret harpin in Erwinia amylovora].

Bacterial pathogens of both animals and plants use type III secretion machines to secret virulence proteins. The signal that leads to tye type III secretion is encoded as stem loop structure in the messenger RNA. Harpin, produced by Erwinia anylovora is secreted via type III secretion. Erwinia carotovora Se9R and Erwinia amylovora belong to the same genus and they use type III secretion machines to secret virulence proteins. We used PCR to amplify hrpN from pCPP430 which harbors hrpN gene cluster, and cloned it to pGEM-T vector to get pWGF1, which was then chemically transformed into DH5 alpha (pCPP430hrpN-) and electrotransformed into Se9R. DH5 alpha (pCPP430hrpN-/pWGF1) induced hypersensitive response on tomato leaf and Se9R(pWGF1) showed significantly reduced virulence than Se9R on Chinese cabbage leaf. Western-blotting analysis of the CFEP of Se9R(pWGF1) showed production of harpin protein. These results suggest the type III secretion machine in Se9R can recognize secretion singal in the gene of harpin in Erwinia amylovora and secret it as a biologically native form.

Bacterial Outer Membrane Proteins↗

[The study of Erwinia carotovora bacteriocins in the nalidixic acid resistant Erwinia carotovora].

A novel approach is proposed for the study of the macromolecular bacteriocins of Erwinia carotovora (MCTVs). The approach lies in that the bacteriocinogeny of pectolytic erwinia is studied using a lawn of a bacterial mutant resistant to nalidixic acid, an inducer of MCTVs. The high efficiency of this approach was demonstrated by studying carotovoricins in 104 different E. carotovora strains, 88% of which bear MCTVs, distinguished by the morphology of zones of induced lysis on a lawn of susceptible cells, the lysis pattern, and some other characteristics. Preliminary studies by this approach showed that there is no correlation between the occurrence of MCTVs in particular E. carotovora strains and the habitat of the host plants from which these strains were isolated. There are grounds to believe that the approach proposed can also be used for investigating bacterial lysogeny.

Bacteriocins↗

[The bactericidal activity of lectins from nitrogen-fixing bacilli].

Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 micrograms/ml). Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp. citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A. brasilense 245. The bacillar lectins incubated with Rhizobium and Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function. We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.

Azospirillum brasilense↗

Purifications and properties of orotidine-phosphorolyzing enzyme and purine nucleoside phosphorylase from Erwinia carotovora AJ 2992.

An orotidine-phosphorolyzing enzyme and a purine nucleoside phosphorylase (PNPase) of Erwinia carotovora AJ 2992, which is a potent producer of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), an antiviral agent, from orotidine and 1,2,4-triazole-3-carboxamide (TCA), were purified 23-fold and 103-fold, respectively. At each purification step, the orotidine-phosphorolyzing enzyme was always co-purified with an uridine phosphorylase (UPase) and its activity could not be separated from that of the UPase after it showed as a single band on SDS-polyacrylamide gel electrophoresis. These results suggest that this enzyme may be identical with UPase. The purified enzyme had a molecular weight of 68,000 +/- 2,000, and seemed to be a dimer. The optimal temperatures and pH values were 60 degrees C and 6.0 for orotidine phosphorolysis, and 70 degrees C and 7.0 for uridine phosphorolysis. The Michaelis constants for uridine and orotidine were 0.75 mM and 10.87 mM, respectively, at 40 degrees C. The PNPase of E. carotovora AJ 2992 had a molecular weight of 58,000 +/- 2,000 and seemed to be a dimer. The Michaelis constants for inosine and guanosine were 1.92 mM and 1.85 mM, respectively, at 40 degrees C. The PNPase was completely inactivated by p-chloromercuribenzoate.

Amino Acid Sequence↗

[Mu-induced deletions and mutations of Erwinia carotovora chromosomes, including resident prophages E105 and 59].

The plasmid RP4::Mu cts62 in stably inherited by Erwinia carotovora 268 strain. Under the conditions of thermoinduction bacteriophage Mu is segregated and completely eliminated more intensively than in Escherichia coli cells. At thermoinduction the transposition of bacteriophage Mu cts62 into different chromosomal sites takes place, causing the induction of chlorate resistant and auxotrophic mutants with the frequency of 10(-4). Two clones deficient in production of 2 of the 4 resident prophages of Erwinia carotovora 268 strain were found among Mu-induced mutants. The deleted prophages are E105 and 59. DNA-DNA hybridization has revealed the complete and partial deletions of bacteriophage E105 with the level of L-asparaginase production in the cells remaining intact. The damage of the prophage 59 is probably caused by point mutations or short deletions.

Bacteriophages↗