A green-light inducible lytic system for cyanobacterial cells

Abstract

Background: Cyanobacteria are an attractive candidate for the production of biofuel because of their ability to capture carbon dioxide by photosynthesis and grow on non-arable land. However, because huge quantities of water are required for cultivation, strict water management is one of the greatest issues in algae- and cyanobacteria-based biofuel production. In this study, we aim to construct a lytic cyanobacterium that can be regulated by a physical signal (green-light illumination) for future use in the recovery of biofuel related compounds.

Results: We introduced T4 bacteriophage-derived lysis genes encoding holin and endolysin under the control of the green-light regulated cpcG2 promoter in Synechocystis sp. PCC 6803. When cells harboring the lysis genes were illuminated with both red and green light, we observed a considerable decrease in growth rate, a significant increase in cellular phycocyanin released in the medium, and a considerable fraction of dead cells. These effects were not observed when these cells were illuminated with only red light, or when cells not containing the lysis genes were grown under either red light or red and green light. These results indicate that our constructed green-light inducible lytic system was clearly induced by green-light illumination, resulting in lytic cells that released intracellular phycocyanin into the culture supernatant. This property suggests a future possibility to construct photosynthetic genetically modified organisms that are unable to survive under sunlight exposure. Expression of the self-lysis system with green-light illumination was also found to greatly increase the fragility of the cell membrane, as determined by subjecting the induced cells to detergent, osmotic-shock, and freeze-thaw treatments.

Conclusions: A green-light inducible lytic system was constructed in Synechocystis sp. PCC 6803. The engineered lytic cyanobacterial cells should be beneficial for the recovery of biofuels and related compounds from cells with minimal effort and energy, due to the fragile nature of the induced cells. Furthermore, the use of light-sensing two-component systems to regulate the expression of exogenous genes in cyanobacteria promises to replace conventional chemical inducers in many bioprocess applications, impacting the limiting water management issues.

Keywords: Cyanobacteria; Self-lysis; Synthetic biology; T4 bacteriophage; Two-component system.

Figure 1

Regulation of cyanobacterial lytic system by green light. Schematic diagram of a novel system to prepare lytic cyanobacterial cells by physical signal, green-light illumination. The lysis system is composed of the CcaS/CcaR green-light regulated two-component system derived from Synechocystis, combined with T4 phage-derived lysis genes under the CcaS/CcaR-regulated cpcG2 promoter. CcaS is a sensor histidine kinase that activates its cognate response regulator, CcaR, under green light to induce the expression of the cpcG2 gene. Under red light, CcaS is in an ‘off-state’ and expression of the target gene is not activated (A). However under green light, CcaS is in an ‘on-state’, resulting in activation of CcaR by phosphorylation and a concomitant induction of the target gene expression (B). Holin forms a ‘tunnel’ in the plasma membrane, which provides endolysin access to the peptidoglycan and allows it to enzymatically break it down, leading to lysis. Antiholin, which is regulated by a weak constitutive promoter, blocks holin and prevents premature lysis due to low background levels of endolysin.

Figure 2

Growth curves and supernatant fluorescence ofSynechocystiscultures. Growth curves (circles with solid line) and supernatant fluorescence intensity (squares with dashed line; excitation: 620 nm, emission: 650 nm), representing leaked phycocyanin, of Synechocystis/pKT230 (A and B) and Synechocystis/pKTLYS (C and D). The cells were grown under either continuous red-light illumination (A and C) or under red light for 84 hours followed by simultaneous red- and green-light illumination (B and D).

Figure 3

Analysis of T4 holin transcription level by RT-PCR. Total RNA was purified from Synechocystis/pKTLYS at 2, 5, 8, 12, and 24 hours after green-light induction to analyze the relative transcription level of the holin gene under red light (black bars) and green light (white bars).

Figure 4

Characterization of lytic cyanobacterial cells under different stress treatments.Synechocystis/pKT230 (pKT230) and Synechocystis/pKTLYS (pKTLYS) were incubated for 64 hours under only red light (black bars) or under simultaneous red and green light (white bars). Cells were then subjected to freeze-thaw, osmotic shock, or Triton X-100 treatments, followed by measurement of the supernatant fluorescence intensity, representing the released phycocyanin.

Figure 5

Microscopic images of SYTOX green-stainedSynechocystis.Synechocystis/pKT230 grown under continuous red light (A and B) or under red and green light (C and D), and Synechocystis/pKTLYS grown under red light (E and F) or under red and green light (G and H) were observed by visible-light microscopy (A, C, E, and G) and by fluorescence microscopy (B, D, F, and H). All cells were grown for 64 hours and then incubated with 5 μM SYTOX Green nucleic acid stain for five minutes at room temperature. Fluorescence microscopy was performed on a Biozero BZ-8000 (Keyence, Osaka, Japan) (excitation: 485 nm, emission: 520 nm).