Evidence for functional drift of bacterial isolates in response to cyanobacterial microcystin-lr and multiple peptide degradation in paucibacter toxinivorans.
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KREITZ, J. 2016. Evidence for functional drift of bacterial isolates in response to cyanobacterial microcystin-lr and multiple peptide degradation in paucibacter toxinivorans. Robert Gordon University, MPhil thesis.
Bacterial bioremediation has been proposed as an efficient, low cost and ecologically safe method to clean vital water reservoirs from cyanobacterial peptide toxins microcystins (MCs). In previous work carried out in 2008 several bacteria were isolated from Scottish freshwaters that effectively degraded MCs. Rhodococcus sp. C1, among the biocatalytic isolates, exhibited particular catabolic capacities as it degraded a range of chemically and structurally diverse prokaryotic and eukaryotic peptides. The work presented here aimed to unravel the universal peptide degradation mechanisms in Rhodococcus sp. C1. However, current biodegradation studies indicated repeated sub-culturing and long-term cryopreservation to have caused changes in the cellular mechanisms involved in MC-LR degradation as MC-LR degradation activity was no longer observed. Therefore, the focus of the study was shifted towards other isolates of the freshwater samples as well as a MC-LR degrading organism of unknown origin. Based on 16S rRNA gene analysis the isolates were identified as Rhodococcus sp., Arthrobacter sp. and Pseudomonas sp., respectively. The different bacterial genera were subjected to MC-LR biodegradation studies including Paucibacter toxinivorans (2007), a MC degrading bacterium from Finnish water previously used as positive control organism. However, it was shown that the three isolates and P. toxinivorans (2007) also lost their MC-LR degradation activity over long-term maintenance under laboratory conditions. This led to the belief that routine maintenance of the bacterial isolates in nutrient rich media such as Luria-Bertani (LB) broth had caused a functional drift that impeded the isolates’ ability to degrade MC-LR. To assess whether nutrient availability has an impact on the bacterial MC-LR degradation activity a simple and rapid 96-well plate based method was developed for testing MC-LR biodegradation in growth media of different nutrient concentration and composition. In addition to the long-term maintained and repeatedly sub-cultured strain of P. toxinivorans (2007) a new P. toxinivorans strain (2015) from the German Collection of Microorganisms and Cell Cultures was included in the nutrient assay. Comparison studies between the two strains supported the occurrence of a physiological drift in the repeatedly sub-cultured strain as cell morphology, oxidase activity and media tolerance of the strains were found to be different. The nutrient assay showed that the use of different growth media had little effect on MC-LR degradation activity of the long-term preserved bacteria. However, the newly obtained P. toxinivorans (2015) effectively removed MC-LR from all media except LB broth. Furthermore, UPLC-PDA-MS analysis revealed MC-LR intermediates in samples exposed to P. toxinivorans (2015). Two of the degradation products were identified as linearised (acyclo-) MC-LR and one as the side chain Adda. Broader investigation of the organism’s catabolic abilities demonstrated P. toxinivorans (2015) is capable of degrading multiple MC variants, nodularin (NOD), anabaenopeptin-type peptides and human peptides. MC variants and NOD were found to be cleaved by hydrolysis indicating a single mechanism to be involved in their degradation. This is the first study to report partial elucidation of the MC and NOD degradation pathway in P. toxinivorans. Further research could include a complete elucidation of the enzymatic degradation pathway in P. toxinivorans (2015) along with studies to determine the genes encoding the enzymes involved.