| Abstract [eng] |
Rūta Urbonaitė Investigation of Arthrobacter spp. Plasmids SUMMARY Arthrobacter sp. is being investigated very widely all over the world. Researchers are interested in this genus, because various strains of Arthrobacter sp. are capable to utilise different pesticides, N-heterocyclic compounds and other industrial products (phthalate). So, they can be used in the soil detoxification. It was identified, that Arthrobacter sp. strains capable to utilise above mentioned compounds usually harbour catabolic plasmids, where degradation genes are located. Different strains of this genus harbour plasmids, which size vary from 41 to 380 kb. The main objective of my work was to construct hybrid vector, which had the origin of replication from Arthrobacter sp., as there were no suitable plasmid vectors available for gene cloning and expression purposes for Arthrobacter species until the end of the year 2005, when appeared annoucement about plasmids pART2 and pART3. At first plasmids pVP3 and pPRH were extracted from strains VP3 and PRH1, respectivelly. According to the 16S rDNA analysis, both these strains belong to Arthrobacter rhombi species. Both plasmids were sequenced and it was determined, that pVP3 is 6135 bp size and has GC content of 64,6%, pPRH plasmid is 5000 bp size and it’s GC content is 66%. The GC content of both plasmids are in the range of 59% to 66% for the genus Arthrobacter. Computer analysis revealed 9 ORFs longer than 200 bp in pVP3 and 6 ORFs in pPRH. Only four ORFs (ORF1, ORF3, ORF6 and ORF7) from pVP3 were identified. Five of six ORFs from plasmid pPRH were identified. Searching for homologies we did not find any significant similarity of the potential proteins of pVP3 ORF2, ORF4, ORF5, ORF8, ORF9 and pPRH ORF1 to known bacterial proteins. It was determined, that pPRH replicates via theta replication, but we still did not manage to define the replication mechanism of the plasmid pVP3. We chose plasmid pPRH for the hibrid vector construction, as it has replication proteins. The genetic marker tet (TcR) gene was inserted in the hybrid plasmid pPRHHind4. Constructed plasmid was called pPRHHind4Tc1, it is 9324 bp size. It was determined, that the hybrid plasmid can replicate in Arthrobacter globiformis, Arthrobacter rhombi VP3, Arthrobacter oxydans PY21, Rhodococcus erythropolis SQ1 strains and of course Escherichia coli DH5α. |
