Bakterialni degradace chlorbenzoovych kyselin

Abstract

This PhD. thesis describes the isolation, characterization and identification of bacterial isolates that are capable of chlorobenzoate (CB) degradation. Chlorobenzoates are intermediates of polychlorinated biphenyls (PCB) biodegradation metabolic pathway in nature. Due to the low range of substrate specificity of formerly isolated strains (A7, A8, A18 and A19), new bacterial strains were obtained from PCB polluted area within the industrial plant Colorlak, Ltd. (Uherske Hradiste, Czech Republic). Four of them (UH133, UH222, UH1411 and UH1613) were found to be Gram-negative and the only one was characterized as a Gram-positive strain (UH82). Newly isolated strains together with the strains A7, A8, A18 and A19 that have been previously classified according to their biochemical properties as the strains of the genus Pseudomonas, were precisely genotypically characterized. In spite of 16S rDNA analysis the strains A7, A8, A18 and A19 were reclassified into the genus Alcaligenes. Gram-negative strains UH133, UH222, UH1411 and UH1613 were taxonomically identified as the genus Pandoraea. Gram-positive isolate UH82 was classified as being the strain of the genus Arthrobacter. For the precise characterization of biodegradation capabilities of Alcaligenes and Pandoraea strains, localisation of the genes responsible for chlorobenzoate degradation was performed. These genes are in the case of the strains belonging into the genus Alcaligenes localised on plasmid. The size of the plasmids was determined by agarose gel electrophoresis to be around 80 kbp. During the cultivation of these strains on nutrient rich medium, the plasmids were cured. This was accompanied by the loss of their biodegradation ability. On the other hand, at the strains belonging into the genus Pandoraea no plasmid was detected and also no mutant strains without the ability to degrade chlorobenzoates were found. The stability of this capability at Pandoraea strains leads into the conclusion that the genes are localized on the chromosome. Consequently, newly isolated strains of the genus Pandoraea were characterized in terms of their three enzymes of degradation pathway for chlorobenzoates - ortho-chlorobenzoate dioxygenase, chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase. Phylogenetic comparison performed by Southern hybridization with two DNA probes (reductase, alpha-subunit) of the multienzyme complex of OHBD of Pseudomonas aeruginosa JB2 and DNA isolated from newly isolated strains showed high level of homology. Multienzyme complex OHBD of Pandoraea strains is thus similar to the reference strain JB2. Its similarity to Alcaligenes strains A7, A8, A18 and A19 has already been described. On the contrary, the genes for chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase show closer similarity to the genes of chlorobenzenes degraders Pseudomonas chlororaphis RW71 and Pseudomonas sp. P51 and 3-chlorobenzoate degrader Ralstonia eutropha NH9, since their similarity to Pseudomonas aeruginosa JB2 genes was only 72%Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi