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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.


Characterization of the miniaturized CcaSs by normalized fluorescence level.Bars show the fluorescence of cells grown under green light (white bars) or red light (gray bars). Bars indicate relative fluorescence intensity to normalized fluorescence level of wild type. Data represent means ± SD from independent triplicate experiment from each of three clones (nine experiments). Asterisks indicate statistically significant differences between fluorescence level of cells under green light and red light. (Dunnet’s test; *P < 0.001).
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f2: Characterization of the miniaturized CcaSs by normalized fluorescence level.Bars show the fluorescence of cells grown under green light (white bars) or red light (gray bars). Bars indicate relative fluorescence intensity to normalized fluorescence level of wild type. Data represent means ± SD from independent triplicate experiment from each of three clones (nine experiments). Asterisks indicate statistically significant differences between fluorescence level of cells under green light and red light. (Dunnet’s test; *P < 0.001).

Mentions: Among the transformants harboring the 11 miniaturized CcaSs, six (CcaS#1, CcaS#2, CcaS#5, CcaS#6, CcaS#8, and CcaS#9) showed no or negligible fluorescence levels under both red or green light illumination (Fig. 2). These miniaturized CcaSs were therefore either inactively produced or are in a constant state of repression of gene expression. Five miniaturized CcaSs (CcaS#3, CcaS#4, CcaS#7, CcaS#10, and CcaS#11) showed RFP-derived fluorescence under various light conditions. The CcaS#3 and CcaS#10 transformants displayed similar high fluorescence intensities to wild-type CcaS under green light illumination and repressed levels under red light illumination. Therefore, miniaturized CcaS#3 and CcaS#10 showed similar gene regulation pattern as wild-type CcaS. Under both green and red light illumination, the CcaS#7 transformant displayed similarly high fluorescence levels as wild-type transformants grown under green light. Therefore, unlike wild-type CcaS, miniaturized CcaS#7 was constantly in an activated state. Interestingly, the transformant harboring CcaS#4 and CcaS#11 displayed fluorescence only under red light illumination, but it was repressed under green light. Therefore, the miniaturized CcaS#4 and CcaS#11 showed the opposite regulation pattern compared the wild-type CcaS, although both harbor the same PCB chromophore.


Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal
Characterization of the miniaturized CcaSs by normalized fluorescence level.Bars show the fluorescence of cells grown under green light (white bars) or red light (gray bars). Bars indicate relative fluorescence intensity to normalized fluorescence level of wild type. Data represent means ± SD from independent triplicate experiment from each of three clones (nine experiments). Asterisks indicate statistically significant differences between fluorescence level of cells under green light and red light. (Dunnet’s test; *P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Characterization of the miniaturized CcaSs by normalized fluorescence level.Bars show the fluorescence of cells grown under green light (white bars) or red light (gray bars). Bars indicate relative fluorescence intensity to normalized fluorescence level of wild type. Data represent means ± SD from independent triplicate experiment from each of three clones (nine experiments). Asterisks indicate statistically significant differences between fluorescence level of cells under green light and red light. (Dunnet’s test; *P < 0.001).
Mentions: Among the transformants harboring the 11 miniaturized CcaSs, six (CcaS#1, CcaS#2, CcaS#5, CcaS#6, CcaS#8, and CcaS#9) showed no or negligible fluorescence levels under both red or green light illumination (Fig. 2). These miniaturized CcaSs were therefore either inactively produced or are in a constant state of repression of gene expression. Five miniaturized CcaSs (CcaS#3, CcaS#4, CcaS#7, CcaS#10, and CcaS#11) showed RFP-derived fluorescence under various light conditions. The CcaS#3 and CcaS#10 transformants displayed similar high fluorescence intensities to wild-type CcaS under green light illumination and repressed levels under red light illumination. Therefore, miniaturized CcaS#3 and CcaS#10 showed similar gene regulation pattern as wild-type CcaS. Under both green and red light illumination, the CcaS#7 transformant displayed similarly high fluorescence levels as wild-type transformants grown under green light. Therefore, unlike wild-type CcaS, miniaturized CcaS#7 was constantly in an activated state. Interestingly, the transformant harboring CcaS#4 and CcaS#11 displayed fluorescence only under red light illumination, but it was repressed under green light. Therefore, the miniaturized CcaS#4 and CcaS#11 showed the opposite regulation pattern compared the wild-type CcaS, although both harbor the same PCB chromophore.

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 &ldquo;miniaturized CcaSs&rdquo; 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.