Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus

Abstract

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed "phototaxis," enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJ_Se (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis Plate-based phototaxis assays indicate that UTEX 3055 uses PixJ_Se to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJ_Se controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

Keywords: GAF domain; Synechococcus elongatus; cyanobacteria; photoreceptor; phototaxis.

Fig. 1.

Biofilm formation and phototaxis phenotypes in S. elongatus UTEX 3055. (A) Biofilm formation in S. elongatus UTEX 3055 and PCC 7942. The purple color after crystal violet staining indicates the formation of biofilm by UTEX 3055. (B) Phototaxis assays with S. elongatus UTEX 3055 and PCC 7942. Cells were spotted onto BG-11 soft agarose pads and provided with directional white fluorescent light illumination (28 µmol photons⋅m⁻²⋅s⁻¹), indicated by the light bulb symbol. (C) Formation of finger-like projections by UTEX 3055. Cells were grown under directional light for 10 h on BG-11 soft agarose pads before imaging. (D) End-to-end displacement of cells moving in a projected light gradient from 0 to 20 µmol photons⋅m⁻²⋅s⁻¹ and showing no significant directional bias (Left) or moving directionally when illuminated from an LED light source (broadband, peak 470 nm; Schott LLS) at a 45° angle with intensity of 50 µmol photons⋅m⁻²⋅s⁻¹ (Right). Sample size (n) and end-to-end mean resultant length (r) values are indicated. A circular Kuiper test showed the r values from the two measurements are significantly different (P < 0.001). (E) Frame-to-frame change of mean resultant length (r) (overall movie average) from a Rayleigh test over time from the two plots in D; r = 0 indicates perfect nondirectionality, and r = 1 indicates maximal clustering in one direction. The apparent oscillation pattern may represent noise in the measurements.

Fig. 2.

Chemotaxis-like operon tax1 is responsible for UTEX 3055 phototaxis. (A) Gene organization of chemotaxis-like operons in PCC 7942. Short black bars over genes indicate SNPs in UTEX 3055 (tax1 ORF: UTEX3055_0945-0950). The domain organization of MCP-like proteins is depicted below each operon. Gray rectangles represent a transmembrane domain. A, CheA; HAMP, domain found in histidine kinases, adenylyl cyclase, methyl-accepting proteins, and phosphatases; HP, hypothetical protein; MA: methyl-accepting chemotaxis-like domains; W, CheW; Y, CheY. (B) Phototaxis phenotypes of tax1 and tax2 mutants. A solid triangle indicates a Tn5-insertion mutant, and an open triangle indicates a deletion mutant. (C) Complementation of tax1 phototaxis mutants. Phototaxis phenotypes of pixG, pixH, pixJ, and pixL mutants expressing the respective gene integrated at NS1, as indicated after a slash and with a plus sign. Wild-type UTEX 3055 strains expressing an SpSm cassette and lacking pixJ served as positive and negative controls, respectively. See SI Appendix, Fig. S6A for the experimental setup.

Fig. 3.

Blue/green light sensing by PixJ_SeGAF2 and blue/green-induced motility in UTEX 3055. (A) Absorbance spectra of PixJ_SeGAF2 after illumination with blue or green light (color-coded lines). Green-light–exposed protein showed a peak of blue-light absorption (Pb); blue-light–exposed protein showed a peak of green-light absorption (Pg). Absorption of buffer without protein, measured as a negative control (red curve), showed no absorption at tested wavelengths. (Inset) Exposure to blue light caused the protein to become visibly magenta (Pg, absorbs green light). Subsequent exposure of the same protein to green light changed the protein color to yellow (Pb, absorbs blue light); this process is reversible. (B) Difference spectrum of purified PixJ_SeGAF2 produced by subtracting the green spectrum from the blue spectrum in A. (C and D) Dark reversion of PixJ_SeGAF2 after exposure to blue (C) or green (D) light, measured at the indicated time points. (E) Phototactic movement of UTEX 3055 cells exposed to blue and green light. Cells were placed at four distances from the LED bulbs (represented by colored triangles below the images) to create a range of light fluences (indicated by the triangle at right). See SI Appendix, Fig. S11A for detailed experimental setup and lighting conditions.

Fig. 4.

Amino acid substitution to prevent bilin binding, in GAF4 alone or in combination with GAF5, reverses the direction of phototactic movement of UTEX 3055. PixJ_Se variants were expressed from NS1 in the pixJ-deletion mutant as represented by sketches of the five GAFs (g1–g5). Intact GAF domains with bound chromophores (blue shapes) are shown in yellow, and mutated GAF domains (Cys → Ala) are shown in gray. The direction of the light and the resulting movement are indicated.

Fig. 5.

Polar localization of photoreceptor PixJ_Se in UTEX 3055. (A) Phototaxis of UTEX 3055 that expresses PixJ_Se-YFP on soft agarose plates without (Left) or with (Right) 1 mM IPTG. (B) Fluorescent images indicating cellular localization of PixJ_Se-YFP in cells grown without or with 1 mM IPTG. (Top) TRITC autofluorescence from photosynthetic pigments. (Middle) YFP channel. (Bottom) Merge of TRITC and YFP channels. (Scale bars: 2 µm.)

Fig. 6.

Lensing effect in rod-shaped S. elongatus. (A) S. elongatus UTEX 3055 cell imaged under oblique illumination from the left at the indicated cell-body orientation relative to the light direction. (Scale bars: 3 µm.) (B) FDTD simulation of light (460 nm) passing through rod-shaped UTEX 3055 cell at the orientation shown in A, using the actual cell size measured in SI Appendix, Fig. S2. The color indicates relative light intensity.