Characterisation of Pigmentation in a Novel Isolate of Arthrobacter Recovered from Soils of the Namib Desert
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The high solar irradiation of desert systems is biologically stressful upon their indigenous soil bacteria, and has major influences on the complexity of their irradiation resistomes (Yuan et al. 2012; Paulino-Lima et al. 2013; Pavlopoulou et al. 2016; León-Sobrino et al. 2019). Characterising features of these bacterial resistomes which confer tolerance to irradiation stress develops our understanding of the breadth of survival systems utilised by extremophiles, and is thus valuable to the field of microbial ecology (Matallana-Surget and Wattiez 2013; Pérez et al. 2017). Arthrobacter sp. NamB2 is a pink-pigmented bacterium from surface soils of the Namib Desert (Buckley 2020). This bacterium demonstrates substantial tolerance to ultraviolet irradiation, and has a corresponding multifaceted intrinsic irradiation resistome (Buckley 2020). The contribution of this bacterium’s pink-pigmentation to its irradiation tolerance was the focus of investigation for this thesis, as pigmentation – particularly the red/yellow pigments of the carotenoid class – have been attributed as a major component in the tolerance of bacteria from other desert systems, including the Atacama and Antarctic deserts (Dieser et al. 2010; Silva et al. 2019; Flores et al. 2020). Despite the stable, demonstrably microbiologically-harmful solar irradiance of the Namib Desert, no similar investigations of pigmentation as a component of intrinsic bacterial resistomes in this environment have yet been performed. This thesis thus sought to expand knowledge on the breadth of irradiation tolerance systems utilised by extremophiles from regions of high solar irradiance by characterising the role of pigmentation in the irradiation-resistome of Arthrobacter sp. NamB2. To achieve this, the pigment was first identified, while its contributions to irradiation tolerance were investigated via analyses of its light-inducibility in biosynthesis, and pigment-specific mitigation of lethality arising under ultraviolet-A, -B and -C irradiation. The pigmentation of Arthrobacter sp. NamB2 was extracted and subject to a series of analyses for identification. The extracted pigmentation produced characteristic carotenoid spectral responses under scanning ultraviolet-visible spectroscopy, confirming it was a carotenoid. Chromatographic separation of the pigment extract using thin-layer chromatography and high-performance liquid-chromatography demonstrated the pigment comprised six – eight polarity-discrete carotenoids, each possessing a chromophore thirteen conjugated double bonds in length. Subsequent mass-spectrometry confirmed the presence of the carotenoid bacterioruberin within the extract, and provided evidence for the presence of a number of its commonly co-isolated dehydrated/glycosylated carotenoid variants. The pink-pigment of Arthrobacter sp. NamB2 was thus concluded as carotenogenic in nature, and comprised of a pigment complement specifically based around the carotenoid bacterioruberin. The light-inducibility of pigment production by Arthrobacter sp. NamB2 was investigated as evidence of its function in irradiation-tolerance. The total pigment content produced by cultures grown at a series of light intensities was compared for evidence of light-responsive carotenoid biosynthesis. Results of these assays were supported by in silico examinations of the available Arthrobacter sp. NamB2 genome (GenBank: GCA_005281365.1) for the presence of candidate genes encoding homologues to known light-responsive regulators of carotenogenesis. This was used to establish if there was a genetic basis for light-responsive carotenoid production in Arthrobacter sp. NamB2. There was no significant difference between the pigment content of cultures grown under differing light conditions, indicating a lack of light-responsive carotenogenesis. This conclusion was supported by the absence of relevant homologues to known light-responsive carotenogenesis regulators within the Arthrobacter sp. NamB2 genome. Lack of light-responsive pigmentation in Arthrobacter sp. NamB2 did not discount its photoprotective role, but was a novel contribution to the study of photoinducibility within both Arthrobacter and desert organisms. Finally, pigment-mediated protection against ultraviolet-irradiation was examined through survival comparisons of pigmented Arthrobacter sp. NamB2 cultures and their unpigmented variants generated via carotenoid inhibition. Pigmented and unpigmented cultures were exposed to high intensities of ultraviolet-A, -B and -C irradiation to determine within which waveband(s) the pigment was capable of cellular protection. Pigmented cells had significantly higher survival than unpigmented cells under ultraviolet-A and ultraviolet-B irradiation, with weakening protection afforded under higher doses of ultraviolet-B, and a total loss of protection under ultraviolet-C. Potential ultraviolet-screening protective roles of the pigment were investigated and dismissed from its lack of pronounced absorbance within the ultraviolet waveband. From the known damage modes of ultraviolet-irradiation, these findings indicated that pigmentation protected Arthrobacter sp. NamB2 only from ultraviolet-wavebands and dosages stimulating damage through the production of reactive oxygen species, with no capacity for the mitigation of direct DNA/protein damage. These inferred conclusions would be strengthened by specific investigations of the mechanisms of protection afforded by pigmentation, but did confirm the contribution of pigmentation to the ultraviolet-irradiation tolerance of Arthrobacter sp. NamB2. The findings of this thesis demonstrated that the carotenoid pigmentation of Arthrobacter sp. NamB2 contributes meaningfully to its ultraviolet-irradiation resistome. Use of carotenoid pigmentation in irradiation-protection is consistent with findings in bacteria from other desert systems, while limitation of their protective capacity to the mitigation of damage inflicted by reactive oxygen species matches expectations of carotenoid photoprotection. This thesis demonstrates for the first time the contribution of pigmentation in a novel edaphic Namib Desert bacterium to its irradiation-resistome, and speculates a similar physiological utility in its natural environment.