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Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal

View Article: PubMed Central - PubMed

ABSTRACT

Cyanobacteria harbor unique photoreceptors, designated as cyanobacteriochromes (CBCRs). In this study, we attempted to engineer the chromatic acclimation sensor CcaS, a CBCR derived from the cyanobacterium Synechocystis sp. PCC 6803. The wild-type CcaS induces gene expression under green light illumination and represses it under red light illumination. We focused on the domain structure of CcaS, which consists of an N-terminal transmembrane helix; a GAF domain, which serves as the sensor domain; a linker region (L1); two PAS domains; a second linker region (L2); and a C-terminal histidine kinase (HK) domain. Truncated versions of the photoreceptor were constructed by removing the L1 linker region and the two PAS domains, and fusing the GAF and HK domains with a truncated linker region. Thus constructed “miniaturized CcaSs” were grouped into four distinct categories according to their responses toward green and red light illumination, with some showing improved gene regulation compared to the wild type. Remarkably, one of the miniaturized CcaSs induced gene expression under red light and repressed it under green light, a reversed response to the light signal compared to wild type CcaS. These characteristics of engineered photoreceptors were discussed by analyzing the CcaS structural model.

No MeSH data available.


Related in: MedlinePlus

Green or red light-regulated gene expression using miniaturized CcaSs.Fluorescence was measured in cells cultured under either green (white circles) or red (gray circles) light exposure. Autofluorescence was measured in cells harboring a light sensing system with the wild-type CcaS and without the rfp gene (a). Characterization of CcaSs was carried out in cells harboring the rfp gene and a light sensing system with either the wild-type CcaS (b), CcaS#3 (c), CcaS#10 (d), or CcaS#11 (e). Data represent means ± SD from independent triplicate experiment from one clone (three experiments).
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f3: Green or red light-regulated gene expression using miniaturized CcaSs.Fluorescence was measured in cells cultured under either green (white circles) or red (gray circles) light exposure. Autofluorescence was measured in cells harboring a light sensing system with the wild-type CcaS and without the rfp gene (a). Characterization of CcaSs was carried out in cells harboring the rfp gene and a light sensing system with either the wild-type CcaS (b), CcaS#3 (c), CcaS#10 (d), or CcaS#11 (e). Data represent means ± SD from independent triplicate experiment from one clone (three experiments).

Mentions: Representative time courses of normalized RFP expression levels of transformants containing the various miniaturized CcaS or wild-type CcaS are shown in Figure 3a to e. Under green light illumination, the transformant harboring wild-type CcaS showed a clear increase in RFP fluorescence intensity after 8 hours of cultivation (Fig. 3b). The maximum RFP expression level (0.4 AU) was observed after 12 hours of cultivation. Under red light illumination, an augmentation in RFP expression was also observed after 10 hours of cultivation, and the maximum expression peaked at 12 hours. The RFP expression level under red light was about 0.1 AU; this is 25% of the level observed under green light. However, the repressed fluorescence level under red light illumination was more than 2-fold the intensity observed by auto-fluorescence (Fig. 3a). Therefore, these results confirmed that wild-type CcaS induced the gene expression of the PcpcG2–linked gene under green light and repressed it under red light illumination; however, the repression level was incomplete and leakage of the gene expression was observed.


Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal
Green or red light-regulated gene expression using miniaturized CcaSs.Fluorescence was measured in cells cultured under either green (white circles) or red (gray circles) light exposure. Autofluorescence was measured in cells harboring a light sensing system with the wild-type CcaS and without the rfp gene (a). Characterization of CcaSs was carried out in cells harboring the rfp gene and a light sensing system with either the wild-type CcaS (b), CcaS#3 (c), CcaS#10 (d), or CcaS#11 (e). Data represent means ± SD from independent triplicate experiment from one clone (three experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121610&req=5

f3: Green or red light-regulated gene expression using miniaturized CcaSs.Fluorescence was measured in cells cultured under either green (white circles) or red (gray circles) light exposure. Autofluorescence was measured in cells harboring a light sensing system with the wild-type CcaS and without the rfp gene (a). Characterization of CcaSs was carried out in cells harboring the rfp gene and a light sensing system with either the wild-type CcaS (b), CcaS#3 (c), CcaS#10 (d), or CcaS#11 (e). Data represent means ± SD from independent triplicate experiment from one clone (three experiments).
Mentions: Representative time courses of normalized RFP expression levels of transformants containing the various miniaturized CcaS or wild-type CcaS are shown in Figure 3a to e. Under green light illumination, the transformant harboring wild-type CcaS showed a clear increase in RFP fluorescence intensity after 8 hours of cultivation (Fig. 3b). The maximum RFP expression level (0.4 AU) was observed after 12 hours of cultivation. Under red light illumination, an augmentation in RFP expression was also observed after 10 hours of cultivation, and the maximum expression peaked at 12 hours. The RFP expression level under red light was about 0.1 AU; this is 25% of the level observed under green light. However, the repressed fluorescence level under red light illumination was more than 2-fold the intensity observed by auto-fluorescence (Fig. 3a). Therefore, these results confirmed that wild-type CcaS induced the gene expression of the PcpcG2–linked gene under green light and repressed it under red light illumination; however, the repression level was incomplete and leakage of the gene expression was observed.

View Article: PubMed Central - PubMed

ABSTRACT

Cyanobacteria harbor unique photoreceptors, designated as cyanobacteriochromes (CBCRs). In this study, we attempted to engineer the chromatic acclimation sensor CcaS, a CBCR derived from the cyanobacterium Synechocystis sp. PCC 6803. The wild-type CcaS induces gene expression under green light illumination and represses it under red light illumination. We focused on the domain structure of CcaS, which consists of an N-terminal transmembrane helix; a GAF domain, which serves as the sensor domain; a linker region (L1); two PAS domains; a second linker region (L2); and a C-terminal histidine kinase (HK) domain. Truncated versions of the photoreceptor were constructed by removing the L1 linker region and the two PAS domains, and fusing the GAF and HK domains with a truncated linker region. Thus constructed “miniaturized CcaSs” were grouped into four distinct categories according to their responses toward green and red light illumination, with some showing improved gene regulation compared to the wild type. Remarkably, one of the miniaturized CcaSs induced gene expression under red light and repressed it under green light, a reversed response to the light signal compared to wild type CcaS. These characteristics of engineered photoreceptors were discussed by analyzing the CcaS structural model.

No MeSH data available.


Related in: MedlinePlus