The Light Responses of Proteorhodopsin-bearing, Antarctic Sea-ice Bacteria
Although homogenous in appearance, Antarctic sea ice forms a complex habitat that is characterised by steep vertical gradients of temperature, irradiance and salinity. Despite these harsh and variable environmental conditions, numerous microbial organisms prosper within Antarctic sea ice. In 2010, bacteria bearing the proteorhodopsin (PR) gene were found within Antarctic sea ice. PR is a photoactive membrane protein that functions as a light-driven proton pump. The hydrogen ion membrane gradient that PR establishes has the potential to drive ATP synthesis, thus allowing PR-bearing bacteria to obtain energy from solar radiation. Although this gene is present in up to 80% of marine bacteria, the active contribution of PR in vivo is debatable. Light induced growth or enhanced survival is generally observed only when PR-bearing bacteria are grown under sub-optimum conditions, such as limited nutrients or carbon, or variations in salinity. This has lead to the general hypothesis that PR has multiple functions, becoming most influential under conditions of stress. In this way, Antarctic sea-ice bacteria may utilise PR to promote survival and enhance energy inputs, when exposed to the harsh conditions of this environment. To explore this hypothesis, potential PR-bearing isolates were cultured from samples of Antarctic sea-ice bacteria. Using 16S rRNA gene sequencing as well as a comparison of phenotypic and environmental characteristics, the isolates were identified as; Psychrobacter nivimaris, Polaribacter dokdonensis, Paracoccus marcusii and Micrococcus sp. These species, along with Psychroflexus torquis (an Antarctic sea-ice bacterium known to possess PR) were examined for the presence of the PR gene. This gene was identified in P. torquis, Ps. nivimaris and Po. dokdonensis. To my knowledge, this is the first time PR has been found in Ps. nivimaris. To assess the influence of irradiance on these species, a series of culture based experiments were undertaken. In 2012, a preliminary field experiment was conducted in which a mixed culture of PR-bearing and non PR-bearing bacteria; Ps. nivimaris, Po. dokdonensis, Pa. marcusii and Micrococcus sp., was incubated in situ in the annual sea ice surrounding Ross Island, Antarctica. The method developed for these experiments is unique, in that cultures of sea-ice bacteria have not before been incubated within their natural environment. No major differences in growth patterns were observed when bacteria were incubated under different wavelengths and light intensities, however, valuable insight into methodological improvement was obtained. Using these refinements, a second in situ incubation experiment was conducted at the same field site, in 2013. Over this 2 week incubation, monocultures of P. torquis grown in full strength media grew most readily under 50%- and blue-light treatments, with red- and green-light yielding lower biomasses, and no growth occurring in the dark. Ambient sea-ice irradiance resulted in highly variable growth, attributed to high irradiance growth-inhibition. These results indicate that P. torquis utilises low levels of light in order to increase its growth in Antarctic sea ice. The influence of light on the growth of P. torquis, Ps. nivimaris and Po. dokdonensis was examined in a laboratory-based experiment, in which media strength and temperature were varied. When cultured at 12°C, Ps. nivimaris grown under constant irradiance reached a higher biomass than in darkness. This trend was most pronounced when this species was cultured in a 10% media concentration. A trend of decreased exponential-growth was observed in light-incubated cultures of Ps. nivimaris, grown at 4°C or -1°C. Elevated maximum growth of Po. dokdonensis was observed under irradiated conditions in the 10% media treatment. This species however, only grew at 12°C; an unexpected result for an Antarctic microbe. P. torquis was not affected by irradiance under any culture conditions and did not grow at -1°C. This last result contrasts the results of the in situ incubations and may have been affected by factors such as culture age. This research demonstrates multiple examples of light-enhanced growth occurring in PR-bearing Antarctic sea-ice bacteria, with the most prominent trends occurring in reduced concentration media. Therefore, this work agrees with the overarching hypothesis that PR is most influential under conditions of stress. The varying effect of temperature on the influence of PR suggests that some species are able to use this protein at low temperatures, whilst others cannot. Therefore, PR likely provides a selective advantage to some species, depending on a variety of physicochemical factors, including nutrient and carbon availability, salinity and temperature.