Phytopathogenic Bacterium Pectobacterium carotovorum Cryptic Plasmids Distribution.

Information on the extrachromosomal elements occurrence in phytopathogenic bacterium Pectobacterium carotovorum is insuffciently presented in modern scientifc literature. Data on the pectobacteria plasmid content are random. Aim: The aim was to study the Pectobacterium carotovorum plasmids spectra, cryptic plasmids distribution and general characteristics. Materials and Methods: Plasmid spectra of 54 strains of different origins were studied. Standard hot alkaline Kado and Liu method was used to isolate plasmids DNA [Kado C.J., Liu S.-T. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 1981; 145 (3): 1365­1373]. Results: It was found that 16 strains contained plasmids of various sizes. Isolated plasmids belonged to four discrete size classes: 2.5 ­ 6.8, 9.8 ­ 16.7, 47.7 ­ 64.5 and 129 kb. Approximately 50 % of the isolated P. carotovorum plasmids belonged to the second discrete size class with a size of 8.7 ­ 10.4 kb. Four large 129 kb P. carotovorum plasmids had unique primary DNA sequence according to results of restriction analysis. Conclusions: Pectobacteria plasmids isolation results correlate with data obtained earlier that 30 % of strains contained plasmids [Tovkach FI. [Isolation and preliminary characterization of cryptic plasmids from Erwinia carotovora]. Microbiology. 2001; 70 (6): 804­810. Russian]. Strains` plasmid maintenance was associated with environmental and ecological niches where bacteria persisted. These extrachromosomal DNAs may present silent "selfsh", and probably prophage, replicons.

VIRION MORPHOLOGY AND STRUCTURAL ORGANIZATION OF POLYVALENT BACTERIOPHAGES TT10-27 AND KEY.

Fine ultrastructure of polyvalent bacteriophages TT10-27 and KEY isolated from affected with fire blight disease plant tissues, was studied using electron microscopy. Phages have isometric heads connected to short complex tail (TT10-27, C1-morphotype) or long non-contractile tail (KEY B-1 morphotype). Maximum diameter of TT10-27 head, measured as the distance between opposite vertices, is 71.3 nm; tail tube of 22 nm in diameter and 9.0 nm in width is framed with 12 appendages that form flabellate structure of 47.0-58.6 nm in diameter. KEY features capsid of 78.6 nm in diameter and flexible non-contractile tail of 172.5 nm long, which ends with a conical tip. Due to a number of features phage TT10-27 was assigned to a group of N4-like phages of Podoviridae family. KEY is a representative of family Siphoviridae, the least freaquent group of Erwinia amylovora phages.

[Polyvalence of bacteriophages isolated from fruit trees, affected by bacterial fire blight].

Phage populations appearing as a result of a pathogenic process caused by Erwinia amylovora have been discovered and described. They accompany bacterial fire blight development in the process of quince, pear and apple trees vegetation in Zakarpattya region of Ukraine. Phage isolates of the affected pear and quince include polyvalent virulent phages able to develop on bacterial strains associated with plants--E. amylovora. E. "horticola" and Pantoea agglomerans. E. amylovora isolated from the plant tissues affected by the fire blight and detected at the same time as phages proved to be resistant to the viral infection. It is hard to explain now this characteristic however it was noticed that resistance to phages can change drastically in case of dissociation, lysogenization and mutagenesis of erwinia in laboratory conditions. Phage population study shows that they are heterogeneous and can obviously include not only polyvalent but also specific viruses. Further studies of biology and molecular genetics of pure lines of isolated phages will help to get closer to understanding the place and role of bacteriophages in the complicated network of relations between bacterial pathogens and plants.

[Electron microscopy and restriction analysis of bacteriophages isolated from quince and pear with symptoms of fire blight].

Phage populations of isolates from quince and pear affected with fire blight disease were studied using electron microscopy, restriction analysis and both agarose gel electrophoresis of particles and host range scoping method. The isolate from quince (pMA1) comprises at least three phage populations and two phage variants that can be detected on different bacterial indicators. After titration of this isolate on Erwinia amylovora the bacteriophage KEY of B1 morphotype with the genome size of 82.4 kb was identified. The isolate pMA1 also includes a unique phage population 4*, which can be identified on the test bacteria Pantoea agglomerans (Pag) g150. Two analogous populations being also present in the isolate pMA1 that appeared to be close phage variants with almost identical Hpal-restriction patterns can be identified using Pag g157 and 9/7-1. The situation is similar in the case of phage isolates from pear, pMG. Three phage populations identified in it using three different indicators represent the same phage of C1 morphotype (TT10-27) with a genome size of 71.4 kb. At least two other phage populations were also detected in the same isolate using P. agglomerans 9/7-2 as an indicator. A model system allowing the most efficient analysis of the isolates for the presence of different phage populations and phage variants in plants infected by fire blight disease has been developed. It provides for using three indicator enterobacterial species closely associated with the plants: E. amylovora, Erwinia "horticola" and Pagglomerans and ignoring of the phage cloning procedure.

New approach for identification of bacteriophage virion structural proteins.

The ability of the phage structural polypeptides to undergo post-translational modification makes the task of correlation of the primary nucleotide sequence data with the actual structural proteins of a virion extremely challenging. This study describes an alternative model approach based on two-stage chromatography for allocation of virion structural components and identification of their major polypeptides. Bacteriophage T4D, its amber mutant T4D23 (amH11) and its tail preparations were purified, concentrated and separated by ion exchange chromatograpgy based on fibrous DEAE-cellulose. The major tail fraction was then exposed to size-exclusion chromatography which enabled to separate tail components by size. This method proved itself as a highly efficient and gentle enough to save most of the biological material without changing the basic properties of the native phage. The result also shows that the accumulation of individual phage tails in the course of the amber mutant T4D23 (amH11) propagation on the permissive host Escherichia coli CR63 was resulted by changes in the conditions of reproduction. The ability of bacteriophages to form an excess of tails, capsids and other structures during reproduction on a non-traditional host provides an alternative way for obtaining highly concentrated preparations of virion components for further analysis of their major proteins and determination of the genes responsible for their synthesis.

Plasmid profile, colicinogeny and phage sensitivity as indicators of the dynamics of Escherichia coli populations in the human gut.

A possibility to use such bacterial phenotypes as plasmid profiles, colicinogeny and phage sensitivity as dynamics indicators of enterobacterial population in the human intestine was considered in the present work. All these three phenotypes, considered together with the type of enterobacterial association and age of the patients may reflect the dynamic state of the individual E. coli population that is currently prevailing in the intestinal microflora. The data on plasmid profile structure, colicinogeny and phage sensitivity indicate to considerable quantitative and qualitative diversity of E. coli genetic elements and the intensive interaction between bacteria in the human gut. This diversity is reflected on the formation of the dynamic population of intestinal bacteria, which obviously depends on the host age. The evaluation of the three above mentioned phenotypes confirmed that the E. coli isolates are closely coordinating with other members of the intestinal microbial association. It is noted that the plasmid frequency increases, the colicin range expands and phage sensitivity decreases in E. coli cells simultaneously with the increase of the number of enterobacterial species in the gut. The dynamics of changes in the biological features was observed among E. coli strains from different age groups of patients. The most significant was high frequency of colicinogenic strains in adult patients and di-associated E. coli containing one large plasmid in the youngest patients with dysbiosis.

Identification of the major proteins of the virions of bacteriophage ZF40 Pectobacterium carotovorum.

The vast variety of bacteriophages and the uniqueness of their individual representatives dictate to perform the detailed study of the actual phage-cell interactions, the virion morphogenesis and morphopoiesis in particular. An analysis of the complete genome sequence of the temperate phage ZF40 Pectobacterium carotovorum has shown that it is a representative of a unique group of phages of the Myoviridae family [Comeau A. M, Tremblay D., Moineau S., Rattei T., Kushkina A. I, Tovkach F I., H.M. Krisch, H.W. Ackermann Phage Morphology Recapitulates Phylogeny: The Comparative Genomics of a New Group of Myoviruses // PLoS ONE.--July 2012. - 7. - N 7. - e40102]. Characteristic features of these viruses are a small length of the tail compared with the diameter of the capsid and a complicated pattern of the tail sheath, leading to its criss-cross striation. In the presented article the major proteins were identified by means of the SDS-PAGE method: the head proteins (mp2: 33.9 kDa), the sheath (mp1: 39.2 kDa) and the tail tube ones (mp3: 19.9 kDa). It was proved that the mp2 molecular weight is the same with the gp46, the putative major capsid protein derived from the results of the genome sequencing. Therefore, it is still not determined whether the gp46 (mp2) of the virulent mutant 421 of the phage ZF40 is exposed to post-translational modification in the course of the phage particle maturation during its development in the cells of the strain M2-4/50RI P. carotovorum. To study the morphogenetic development pathways it was proposed to use the phage variants that form an excess of individual components of the virion: capsids, procapsids and separate tails propagated on different hosts.

Long-term preservation of unstable bacteriophages of enterobacteria.

Bacteriophages are integral components of bacterial communities. Their practical applications and significance for humans are various. Thus, keeping phage collections along with their specific host bacteria is an urgent and important mission for biologists. The problems of the long-term storage of phages steel are not completely solved. The main difficulties may occur due to the structural instability of virions as well as an accelerated genetic variability in phage genomes both in vivo and in vitro. In the paper the results of 10-years observation over unstable bacteriophage storage process was presented as well the method of their long-term preservation was proposed. It consisted of the optimization of STMG buffer system (200 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM MgCl, 100 microg/ml gelatin) [Serwer P, Pichler ME. Electrophoresis of bacteroiphage T7 capsids in agarose gels//J. Virol. - 1978 - V28, N3. - P.917-928] by higher gelatin concentration or its replacement by ficoll (2 - 6%), and increasing of Mg(2-) concentration to 10 mM. The proposed buffer composition allowed saving structural entirety of unstable phage virions during their long-term storage.

Characteristics of defective phage particles of Pectobacterium carotovorum ZM1.

It is shown for the first time that the expression products of defective prophages are typical of defective lysogenic systems of phytopathogenic Pectobacterium carotovorum. It is established that virus-like particles (LP) such as phage capsids are packing bacterial DNA which size is determined by pulse field gel electrophoresis separation. Based on data about capsid structures which are formed by the virulent mutant ZF40/421, there is made a suggestion about the forming mechanism of defective virions of P carotovorum.

[Dissociation of Pectobacterium carotovorum subsp. carotovorumrelated to the changes in the cell wall lipopolysaccharide].

It is shown for the first time that population heterogeneity of Pectobacterium carotovorum subsp. carotovorum is applicable to a wide range of strains and therefore is a universal characteristic. Using the method of specific selection with the help of carotovoricins which are identical to the phage tails a set of population dissociants of different types was obtained, due to the fact that S-LPS is the part of the cell wall which contains their attachment sites. It was determined that changes in S-lipopolysaccharides lead to the formation of SR-, R-forms of P. carotovorum. A suggestion is made that the changes in the surface structures of dissociants have a significant impact on secretion types II and III--the main pathogenicity factor of some bacteria. The results presented are a prerequisite for studying the direction, the reasons for dissociation process and its impact on the pathogenicity of P. carotovorum.

[Polypeptide content and HPLC-chromatography of the virions of phage ZF40 mutants].

Study of the polypeptide content of erwiniophage ZF40c(5/5) and ZF40-421 virulent mutants has shown that their virions include no less than 10 structural proteins with molecular weights ranging from 16.9 to 96.5 kDa. Three polypeptides belong to a group of major proteins with molecular weights 39.2, 33.1 and 18.5 kDa. They correlate with the polypeptides of phage head, tail sheath and tail core correspondingly. It has been proven that the protein contents of these phages are identical, taking into account that the percent ratio of all polypeptides approaches 1.0. The polypeptide profile of isogenic variant of phage ZF40-421 obtained on EccRC5297 is characterized by another ratio of major proteins. These differences are reflected in the structure of procapsids, that explains low level of stability and viability of the variant. The work shows for the first time the possibility of using HPLC-chromatography for studying native phage particles and their structural components.

[Structural stability of DNA of the transposon derivatives of pCA25 plasmid].

Mutants of Erwinia carotovora subsp. carotovra 48A carrying plasmid pCA25 and its transposon variant and resistant to mitomycin C, nalidixic acid and streptomycin were used in the research. It has been shown that the presence of transposon in plasmid pCA25 (strain 48A-7/4b[pCA25::Tn9]) does not practically affect the frequency of appearance of the stable bacterial mutants under the effect of all three antibiotics. Several variants, the plasmid profile of which differs from the initial one were found only in the case of this strain clones resistant to mitomycin C. Electrophoretic mobility of the plasmid DNA of the mutant clones 31 and 32 decreased and approached mobility of the initial plasmid pCA25, the mutant plasmid 13 had high electrophoretic mobility compared to pCA25::Tn9. Some plasmids are the deletion variants of pCA25::Tn9, one of two IS1-elements of the transposon Tn9 being absent in them. The obtained results indicate that the studied plasmid pCA25 is stable, is not eliminated from the cells under different treatments and confirm once more the hypothesis of the prophage origin of the plasmid. The paper is presented in Russian.

[Peculiarities of morphogenetical development of erwiniophage ZF40 virulent mutants ].

The distortion of morphopoiesis or tail attachment to the capsid is a characteristic feature of morphogenetical development not only of a reproductive infection but also of the lysogenic induction of the defective bacteriophage Erwinia carotovora subsp. carotovora (Ecc). A model system for studying morphogenetical development and assembling of the virion was created on the basis of the phage ZF40 and its two virulent mutants ZF40-421 and ZF40(5/5), as well as the indicator culture Ecc M2-4/50 R1 being nontraditional host for these phages. It has helped to establish that the diameter of the phage capsid is not a conservative value. The presence of capsids of two types with the average diameters 60.3 and 65.0 nm is characteristic of the virmutant ZF40c(5/5)/50RI, while in the course of morphogenesis the phage ZF40-421/50RI forms only one type of heads of 65 nm in size. These heads are probably not firmly connected to the tails since the degree of the secondary destruction of the virions of the phage Zf40-421/50RI is considerably higher, than that of the virions of the phage ZF40c(5/5)/50RI. The number of capsids being 60.3 nm in diameter prevails considerably in the latter. The both virulent mutants as a whole are essentially more stable than their isogenic partners obtained on Ecc RC5297 which helps to make a conclusion about considerable influence of specific bacterial proteins of the host-cell on morphogenesis and morphopoiesis.

[Transduction as one of the methods of obtaining genetic information in Erwinia].

Transduction is one of the key processes of horizontal transfer of genes in bacteria. It is known that it is involved in distribution of the main factors of pathogenicity among numerous enterobacteria. It is shown that clear mutants and some variants of the temperate bacteriophage ZF40 Erwinia carotovora subsp. carotovora can perform general transduction of chromosome and plasmid genes of the bacterium E. carotovora. The indicators of chromosome transduction frequencies of the markers--arg+, met+, trp+, ura+ have a broad range of values: 7.10-10(-8) - 1.1-10(-1). The authors have succeeded in increasing the transduction efficiency due to infecting the recipient bacteria on the solid medium LB. Such approach is important for the phages similar to ZF40 in which adsorption is accompanied by re-adsorption of phage particles. The mechanism of transfer of bacterial genes by the type of general transduction is connected with cyclic permutation of phage DNA.

[Detection of bacteriophages of siphoviridae family in Erwinia carotovora subsp. carotovora].

It was established that the polylysogenic phage system of culture Erwinia carotovora subsp. carotovora 91P includes: a) defective bacteriophages of Myoviridae family, which are displayed as macromolecular carotovoricins b) valuable highly unstable temperate phage, which can be attributed to the family Myoviridae, and which, perhaps, is an analogue of phage ZF40 [6], and c) resistant to osmotic shock temperate phage of family Siphoviridae. This phage, called TIRI, consists of isometric head 50 nm in diameter and a rigid tail structure 203 nm long. A characteristic feature of the phage tail is an evident transverse striation, which is also indicative for the tail-like particle of the defective temperate phage of the strain 48A-7/4b. In general, the phage system of E carotovora subsp. carotovora is similar to Pseudomonas aeruginosa with its R- and F-bacteriocins, and phages of the families Myoviridae and Siphoviridae.

[Polypeptide composition and killer specificity as indices of multiplicity of carotovoricins].

A method of separation and purification of macromolecular bacteriocins of phytopathogenic bacterium Erwinia carotovora is proposed. It is shown on the basis of polypeptide composition of the particles and their killer specificity, that E. carotovora ESP86 is a complex defective-polylysogenic system which includes no less than three different types of biologically active tails of incomplete temperate bacteriophages.

[Using the laboratory strains of Escherichia coli for studying colicinogenicity].

The indicator system which includes laboratory strains of Escherichia coli K12, K12-C600, BE, and C-Ia is offered for studying colicinogenicity. It has helped to establish that 32 of 100 patients with dysbacterioses of colon carry a colicin-producing strain of E. coli. A tendency is discovered to the increase of occurrence of colicinogenic strains of escherichias with patients' age. Only 24% of E. coli strains form active colicins in a group of one-year old children. Frequency of colicinogenic strains occurrence increases to 33 and 42 %, respectively, in teenagers and adult patients. A strict decrease of total activity of colicins is the main peculiarity of polymicrobe associations in which the prevailing strain of E. coli is accompanied by 3-5 strains of other enterobacteria. As to their sensitivity of colicins the indicator strains are arranged in the following order: K12-C600 (84%), KR (69%), BE (63%) and C-Ia (47%). In spite of that, the low-sensitive strains can be effective for identification of very specific colicins. Since the laboratory strains of E. coli K12, BE and C-Ia are the hosts for specific bacteriophages of E. coli, the indicator system on their basis may be useful for studying the interrelation between colicins and coliphages, as well as plasmids and restriction-modification systems. The paper is presented in Russian.

[Abortive infection in Erwinia carotovora, as a source of nanoparticles of phage nature].

A possibility to obtain nanoparticles of phage nature using abortive phage infection was shown for the first time. It was found out that the nonspecific host Erwinia carotovora subsp. carotovora J2 being infected by phage ZF 40-RT80, the cells form a 100-fold surplus of capsid structures. Using the electron microscope the authors have found two types of phage capsids which differ from each other and have different modal diameters--47.5 and 71.5 nm. The found capsids pack the phage DNA which releases them under treatment of the preparations by DNAse I. A simple method of purification of capsid structures from mature virions which are formed in inconsiderable quantity in the process of abortive phage infection is proposed. The obtained results create preconditions for obtaining capsid nanoparticles as well as for studying the stages of morphogenesis and morphopoiesis of phage ZF40 without attracting special phage mutants.

[The characteristic of the colispecific bacteriocins of Erwinia carotovora subsp. carotovora Ec153].

It has been shown for the first time that macromolecular carotovoricins from Erwinia carotovora subsp. carotovora Ec153 strain are active to Escherichia coli and posses endonuclease activity. It has been established that 19.1% of strains isolated from the patients are sensitive to these bacteriocins. The type and the form of these particles have been also determined and they show resemblance with phage objects.

[The association of pigment-containing lipid and low-molecular bacteriocin of Erwinia carotovora].

In contrast to lysates of uninduced cells those of E. carotovora J2/S2 induced by nalidixic acid contain the component which has the additional maximum of UV-absorption at 331 nm. Bacteriocin samples, concentrated by ammonium sulfate and purified on DEAE-sepharose column in saccharose gradient do not lose this substance. This component is supposed to represent the precursor of carotenoid synthesis of E. carotovora. The chloroform treatment of the bacteriocin preparation did not affect its killer activity against both E. carotovora and Escherichia coli. Its chloroform extract has the greatest killer activity against E. coli BE cells and as a result has two additional maximums of UV-absorption at 315 and 331 nm. The pronase treatment at 60 degrees C for 5 min resulted in the destruction of the complex and the loss of the killer activity by bacteriocin. Lipase A destroys the complex lipid-bacteriocin. Such disruption of the complex increases with the concentration of lipase A. Chromatography of the disrupted preparations has been performed on the plate Silufor VU 254 using the mixture chloroform: ethanol. As a result the mobility of disrupted preparation was three times higher than that of the native complex. Thus, we have discovered a new type of carotovoricins, which are a stable complex which consists of protein, lipid and pigment, presumably the precursor of carotinoid synthesis.