Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803

Abstract

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.

FIG. 1.

(A) Construct used for generating ΔtaxD1 deletion mutant. Black rectangles represent the remaining coding region of taxD1. The kanamycin resistance cassette is represented by the open rectangle (indicated by Km). Black arrows indicate the direction of transcription. Relative locations of the primers taxD1-1 and taxD1-2 with respect to the taxD1 gene are shown. (B) Analysis of phototactic movement with the glass slide-based assay. The cell track is indicated by the solid line. O, origin; E, end. Dx, displacement along the x axis; Dy, displacement along the y axis; arrow, direction of the incident light. (C) Comparison of the glass slide-based and agar surface-based phototaxis assays. Top panel, average displacement (Dx) of cells in unidirectional light with the glass-based phototaxis assay. Phototaxis in a population of Synechocystis sp. strain PCC6803 cells was recorded for 175 s and quantified. The light source was a low-noise illuminator, as in Fig. 1. Fluence rate was 19.3 μmol m⁻² s⁻¹. Each bar represents the average of the mean displacement from three independent populations of the wild-type (WT), ΔtaxD1, pilT1, and pilA1 mutant cells. Error bars represent 1 standard deviation from the mean. Positive displacement = towards light; negative displacement = away from light. Bottom panel, average displacement (Dx) of cells in unidirectional light with the agar-based phototaxis assay. Cells were applied to the surface of 0.5% agar with a micropipette and placed in front of a unidirectional light source for 48 h. Circles indicate the original positions of applied cells. Arrow indicates the direction of light. The light source was a Philips Cool White fluorescent lamp (FT20T12/CW). Fluence rate was 5.3 μmol m⁻² s⁻¹.

FIG. 2.

(A) Average phototactic displacement (Dx) with respect to fluence rate in wild-type and ΔtaxD1 mutant cells of Synechocystis sp. strain PCC6803 in response to broad-spectrum light. Each point represents the average of the mean displacement from three independent populations of cells. Error bars represent 1 standard deviation from the mean. Positive displacement = towards light; negative displacement = away from light. (B) Mean total distance traveled by wild-type (WT, open bars) and ΔtaxD1 cells (shaded bars) at different fluence rates.

FIG. 3.

(A) Method for calculation of quantum effectiveness of a particular wavelength of light in inducing phototaxis. Shown is a fluence rate-response curve for a particular wavelength. The fitted line is extrapolated to the criterion response. The fluence rate at the criterion response (Fc) is inversely proportional to the quantum effectiveness of the light used. Therefore, the inverse of Fc (1/Fc) is expressed as a percentage of the strongest response and then plotted against the wavelength to give the final action spectrum. (B) Action spectrum of positive phototaxis in wild-type (WT) Synechocystis sp. strain PCC6803 cells at low light intensity. (C) Action spectrum of negative phototaxis in wild-type Synechocystis sp. strain PCC6803 cells at high light intensity. At the wavelengths 394, 424, 523, 552, 574, and 595 nm, only positive phototaxis was observed at the highest possible fluence rates (maximum output from the illuminator) used, which were 16.29, 79.65, 422.1, 498.5, 304.9, and 1,046 μmol m⁻² s⁻¹, respectively. Therefore, the negative phototactic responsiveness of cells at these wavelengths was considered to be 0.

FIG. 4.

Antagonistic effect of infrared light on positive phototaxis in wild-type cells. (A) Front view of the experimental setup, showing the assay chamber and directions of the condenser and lateral light. (B) Various light treatments are shown in the top table. The bar graph below the table shows the mean displacement of wild-type cells under the various light treatments. Each bar represents the average of the mean displacement from three independent populations of cells. Error bar represents 1 standard deviation from the mean. “Dark” indicates that no lateral illumination was used. The peak emissions for the lateral and condenser light sources are provided. Fluence rates used are as follows: Lateral, 760 nm, 0.6712 μmol m⁻² s⁻¹; lateral, 645 nm, 0.5266 μmol m⁻² s⁻¹; condenser, 760 nm: A, 0.4651 μmol m⁻² s⁻¹; B, 1.552 μmol m⁻² s⁻¹.

FIG. 5.

Action spectrum of negative phototaxis in the ΔtaxD1 mutant of Synechocystis sp. strain PCC6803 cells at low light intensity.