In situ light responses of the proteorhodopsin-bearing Antarctic sea-ice bacterium, Psychroflexus torques

Abstract

Proteorhodopsin (PR) is a wide-spread protein found in many marine prokaryotes. PR allows for the potential conversion of solar energy to ATP, possibly assisting in cellular growth and survival during periods of high environmental stress. PR utilises either blue or green light through a single amino acid substitution. We incubated the PR-bearing bacterium Psychroflexus torquis 50 cm deep within Antarctic sea ice for 13 days, exposing cultures to diurnal fluctuations in light and temperature. Enhanced growth occurred most prominently in cultures incubated under irradiance levels of ∼50 μmol photons m^-2 s^-1, suggesting PR provides a strong selective advantage. In addition, cultures grown under blue light yielded over 5.5 times more live cells per photon compared to green-light incubations. Because P. torquis expresses an apparently 'green-shifted' PR gene variant, this finding infers that the spectral tuning of PR is more complex than previously thought. This study supports the theory that PR provides additional energy to bacteria under sub-optimal conditions, and raises several points of interest to be addressed by future research.

Figure 1

(a) Mean number (±s.e.m.) of live in situ incubated P. torquis cells. Treatments include exposure to ambient photosynthetically active radiation (), complete darkness (), reduced photosynthetically active radiation (), blue (), green () or red light (). Data points are offset (±3 h) for ease of viewing. Following incubation for 6, 10 or 13 days, 2 ml samples were fixed in 1% formaldehyde (Sigma-Aldrich, Auckland, New Zealand) and stored at −20 °C. After thawing, 1 ml of each was centrifuged (5000 g, 12 min), and the pellets air dried for ∼10 min. Cells were re-suspended in 350 μl of 0.22 μm filtered phosphate buffered saline (Life Technologies, Auckland, New Zealand) diluted 10 × and 350 μl was transferred to a 5 ml tube containing 35 μl of TruCount bead solution (Becton Dickinson). Each sample was incubated at room temperature for 15 min in 3 μm propidium iodide (Life Technologies). Prepared samples were analysed using a FACSCanto II flow cytometer (Becton Dickinson), acquiring data until 1000 TruCount bead events were recorded. Total cell counts were obtained through particle sizing, and intact and membrane-permeablised cells (live and dead at the time of fixation) were differentiated via the uptake of propidium iodide. (b) Mean number (±s.e.m.) of light-generated live cells per μmol photon present in each waveband treatment on day 13. The number of light-generated live cells of each sample was calculated by subtracting the number of live cells present in each light-exposed sample from the mean number of live cells present in dark incubations. The number of live cells under red light was similar to that in darkness, and so the red-light treatment was not included in this analysis. This value was then divided by the irradiance of their corresponding treatment to provide an estimate of the light-generated live cells per μmol photon for each light treatment.