Sergeeva Z. The expansion and characteristic of Erwinia carotovora's cryptic plasmids

Thesis for Candidate's degree in specialty 03.00.07 - Microbiology. - Zabolotny Institute of Microbiology and Virology of National Academy of Sciences of Ukraine, Kyiv, 2009. PhD work is devoted to the study of the cryptic plasmids of E. carotovora strains isolated from the different sources for the purpose of molecular-biological properties determination. The study of plasmid composition of E. carotovora subsp. carotovora and Е. carotovora subsp. atroseptica strains was carried out. Certain correlation between geographical origin of the strains and plasmid maintenance of these strains was detected. These E. carotovora plasmids belong to the four discrete size classes: 3.8-6.8, 9.8-16.7, 47.7-64.5 and 129 kb. For the first time it was shown that the most widespread size group among erwinias plasmids were 9,8 kb plasmids and their deletion/insertion variants (8.7 - 10.4 kb). The influence of the autonomous genetic elements of these strains of E. carotovora on the sensibility of bacterial cells to bacteriophages was studied. No correlation between the plasmid composition and the carotovoricins killer activity of E. carotovora strains was also detected. The comparative restriction analysis of E. carotovora megaplasmids (129 kb) from three stains of different origin was carried out. And for the first time the comparative restriction analysis of phage and plasmid DNA from a plasmid strain was carried out. It was found out that all the studied megaplasmid DNAs were represented with the unique sequences. Also for the first time it was detected that рСА16-1 megaplasmid and NF16 bacteriophage DNAs isolated from the E. carotovora 33A strain differed in primary sequences. The existence of the primary sequence homology in the most widespread E. carotovora plasmid size class (9.8 kb) was shown for the first time using the results of restriction analysis. The bacterial clone E. carotovora 48А-7/4b was detected during the study of plasmid composition of the E. carotovora 48А strain P1-transductants. The 7/4b clone cells contained extrachromosomal DNA of the enlarged size. The enlargement of plasmid рСА25 in size in the 7/4b clone cells appeared due to the insertion of the Tn9 transposon. Plasmid pCA25 (9.8 kb) belongs to the most widespread size class of erwinias extrachromosomal DNAs. It was supposed earlier that pCA25 plasmid was of a prophage origin. For the first time ever the restriction site mapping of the E. carotovora extrachromosomal element pCA25 and its transposon variant рСА25::Tn9 was performed. The restriction map of the plasmid was created for endonucleases HpaІ, BglI, EcoRI, EcoRV and PstI and the site of the Tn9 transposon incorporation was detected corresponding to the obtained data. Plasmid рСА25::Tn9 can be used as a vector for genetic information transfer between strains in perspective.