The molecular dimension of microbial species: 2. Synechococcus strains representative of putative ecotypes inhabiting different depths in the Mushroom Spring microbial mat exhibit different adaptive and acclimative responses to light

Abstract

Closely related strains of thermophilic Synechococcus were cultivated from the microbial mats found in the effluent channels of Mushroom Spring, Yellowstone National Park (YNP). These strains have identical or nearly identical 16S rRNA sequences but are representative of separate, predicted putative ecotype (PE) populations, which were identified by using the more highly resolving psaA locus and which predominate at different vertical positions within the 1-mm-thick upper-green layer of the mat. Pyrosequencing confirmed that each strain contained a single, predominant psaA genotype. Strains differed in growth rate as a function of irradiance. A strain with a psaA genotype corresponding to a predicted PE that predominates near the mat surface grew fastest at high irradiances, whereas strains with psaA genotypes representative of predominant subsurface populations grew faster at low irradiance and exhibited greater sensitivity to abrupt shifts to high light. The high-light-adapted and low-light-adapted strains also exhibited differences in pigment content and the composition of the photosynthetic apparatus (photosystem ratio) when grown under different light intensities. Cells representative of the different strains had similar morphologies under low-light conditions, but under high-light conditions, cells of low-light-adapted strains became elongated and formed short chains of cells. Collectively, the results presented here are consistent with the hypothesis that closely related, but distinct, ecological species of Synechococcus occupy different light niches in the Mushroom Spring microbial mat and acclimate differently to changing light environments.

Keywords: cyanobacteria; light acclimation; light adaptation; microbial species; photosynthesis.

FIGURE 1

Microscopic images of a Synechococcus strain and heterotrophic contaminants. (A) Fluorescence microscopy photomicrograph of the PE A1 strain (65AY6Li) grown at a scalar irradiance of 50 μmol photons m^-2sec^-1. (B) Same image using differential interference contrast microscopy, showing rod-shaped and filamentous heterotrophic contaminants. (C) Differential interference contrast photomicrograph showing elongated cells of the PE A14 strain grown at a scalar irradiance of 600 μmol photons m^-2sec^-1 after pre-growth at 50 μmol photons m^-2sec^-1. Scale bars are 10 μm.

FIGURE 2

Growth of heterotrophic and Synechococcus cells at different irradiances. Number of cells of the heterotrophic population (black) and the PE A1 Synechococcus strain (65AY6Li; blue) at four irradiance values (25, 125, 250, and 600 μmol photons m^-2sec^-1), over a 7-day period at 52°C, and without any additional dissolved inorganic carbon provided to medium DHAY. The culture used as inoculum was grown at 50 μmol photons m^-2sec^-1. The gray lines are the least-square best fit lines that were used to calculate the growth rate during exponential growth phase. Each point is the average of two measurements and the range is shown by the bars.

FIGURE 3

Growth rates of Synechococcus strains representative of predominant PEs as a function of irradiance when grown at 60°C and sparged with 6% (v/v) CO₂in air. Strains used in this experiment were pre-grown at 50 μmol photons m^-2sec^-1, 52°C and without CO₂ sparging (A) Low irradiances only. (B) All tested irradiances. Each point is the average of two measurements and the range is shown by the bars.

FIGURE 4

Growth of Synechococcus strain representative of PE A14 when pre-grown under different conditions. The inoculating cells were pre-grown either at 50 μmol photons m^-2sec^-1, 52°C and without CO₂ sparging (solid line) or at 500 μmol photons m^-2sec^-1, 60°C, and with 6% CO₂ bubbled in air (dashed line). Range bars are shown.

FIGURE 5

Low-temperature fluorescence emission spectra of whole cells of Synechococcus strains representative of PEs A1 (blue), A4 (orange), and A14 (red). (A) The excitation wavelength was set at 440 nm to excite chlorophyll a selectively. (B) The excitation wavelength was set at 590 nm to excite phycobiliproteins selectively. Cells were grown at 25 μmol photons m^-2sec^-1 (solid lines) or 600 μmol photons m^-2sec^-1 (dashed lines).

FIGURE 6

Microscopic and flow cytometric analyses of the strains representative of PEs A1, A4, and A14 grown at 60°C and bubbled with 6% CO₂ in air, under a low-light (25 μmol photons m^-2sec^-1) and a high-light condition (600 μmol photons m^-2sec^-1). Pre-growth light intensity was 50 μmol photons m^-2sec^-1. (A) Differential interference contrast microscopy of low-light and high-light grown cells that were collected at the end of exponential growth phase and then mixed for comparative analyses. Scale bar is 10 μm. (B) Same images using fluorescence microscopy. (C) Scatter plots from BD-FACSCanto flow cytometer of forward scatter (cell size, horizontal axis) versus fluorescence signal (PerCP-Cy5-5-A, vertical axis) of mixed low-light and high-light samples shown in (A) and (B). Cells grown under low irradiance are represented by the green data points and cells grown under high irradiance are represented by the red data points. (D) Plots of forward scatter (cell size, horizontal axis) versus side scatter (complexity, vertical axis) of mixed low-light and high-light samples shown in (A) and (B). The blue data points represent the fluorescent counting beads that serve as a fluorescence control.