Far-red light photoacclimation in a desert Chroococcidiopsis strain with a reduced FaRLiP gene cluster and expression of its chlorophyll f synthase in space-resistant isolates

Abstract

Introduction: Some cyanobacteria can use far-red light (FRL) to drive oxygenic photosynthesis, a phenomenon known as Far-Red Light Photoacclimation (FaRLiP). It can expand photosynthetically active radiation beyond the visible light (VL) range. Therefore, it holds promise for biotechnological applications and may prove useful for the future human exploration of outer space. Typically, FaRLiP relies on a cluster of ~20 genes, encoding paralogs of the standard photosynthetic machinery. One of them, a highly divergent D1 gene known as chlF (or psbA4), is the synthase responsible for the formation of the FRL-absorbing chlorophyll f (Chl f) that is essential for FaRLiP. The minimum gene set required for this phenotype is unclear. The desert cyanobacterium Chroococcidiopsis sp. CCMEE 010 is unusual in being capable of FaRLiP with a reduced gene cluster (15 genes), and it lacks most of the genes encoding FR-Photosystem I.

Methods: Here we investigated whether the reduced gene cluster of Chroococcidiopsis sp. CCMEE 010 is transcriptionally regulated by FRL and characterized the spectral changes that occur during the FaRLiP response of Chroococcidiopsis sp. CCMEE 010. In addition, the heterologous expression of the Chl f synthase from CCMEE 010 was attempted in three closely related desert strains of Chroococcidiopsis.

Results: All 15 genes of the FaRLiP cluster were preferentially expressed under FRL, accompanied by a progressive red-shift of the photosynthetic absorption spectrum. The Chl f synthase from CCMEE 010 was successfully expressed in two desert strains of Chroococcidiopsis and transformants could be selected in both VL and FRL.

Discussion: In Chroococcidiopsis sp. CCME 010, all the far-red genes of the unusually reduced FaRLiP cluster, are transcriptionally regulated by FRL and two closely related desert strains heterologously expressing the chlF010 gene could grow in FRL. Since the transformation hosts had been reported to survive outer space conditions, such an achievement lays the foundation toward novel cyanobacteria-based technologies to support human space exploration.

Keywords: Chl f synthase; Chroococcidiopsis; FaRLiP; genetic manipulation; space exploration.

Figure 1

Gene expression of the FaRLiP cluster in FRL-acclimated CCMEE 010. Structure of far-red gene cluster (A), color-coding: psa genes of FR-PSI (yellow); psb genes of FR-PSII (green); apc genes of FR-phycobilisomes (blue); rfp genes (red); Relative expression after 48 h-exposure to FRL as revealed by RT-qPCR of the rfpA, rfpB, and rfpC genes and FRL-paralogs of the PSI (B), PSII (C), and phycobilisomes (D); The apcE1 gene was used as a control gene highly expressed only in VL condition. Data represents mean +/- SD. Statistical significance for each gene in FRL- vs. VL-cells, was assessed by t-test and indicated by ***P < 0.001; **P < 0.01; and *P < 0.05.

Figure 2

CLSM-lambda-scan analysis of single cells of FRL-acclimated CCMEE 010. Spectra of FRL-exposed cells for 1, 2, and 3 days (A) and for 7, 14, and 21 days (B). Data points represent normalized mean fluorescence intensity at 653 nm +/– standard error for n = 20 cells as a function of emission wavelength.

Figure 3

Transformation of CCMEE 029, CCMEE 057, and CCMEE 064. Map of pTac-ChlF-pDU1 plasmid (A); Transformants selection under FRL and antibiotic pressure, t = 0 days after electroporation (B); Transformants selection under FRL and antibiotic pressure after 1 month (C); Wild-type strains under FRL (D); Transformants selection under VL and antibiotic pressure (E).

Figure 4

Heterologous expression of chlF⁰¹⁰ gene in Chroococcidiopsis transformants under VL-condition. Transcription levels in transformants of strain CCMEE 029, CCMEE 057, and CCMEE 064.

Figure 5

CLSM image and lambda scans of Chroococcidiopsis transformed with the chlF⁰¹⁰ gene under VL-condition. Spectral fluorescence emissions of 1-month-old transformants CCMEE 029 (A), CCMEE 057 (B), and CCMEE 064 (C), are shown as red lines; emission spectra of wild-type strains (black lines) and CCMEE 010 after 3 days in FRL (dotted lines). Data are mean fluorescence intensity ± standard deviation for n > 15 cells. CLSM images (optical sections) corresponding to the maximum emission peaks of the photosynthetic pigments of transformants excited at 488 nm. Scale bar = 5 μm.