Development of a light-regulated cell-recovery system for non-photosynthetic bacteria

Abstract

Background: Recent advances in the understanding of photosensing in biological systems have enabled the use of photoreceptors as novel genetic tools. Exploiting various photoreceptors that cyanobacteria possess, a green light-inducible gene expression system was previously developed for the regulation of gene expression in cyanobacteria. However, the applications of cyanobacterial photoreceptors are not limited to these bacteria but are also available for non-photosynthetic microorganisms by the coexpression of a cyanobacterial chromophore with a cyanobacteria-derived photosensing system. An Escherichia coli-derived self-aggregation system based on Antigen 43 (Ag43) has been shown to induce cell self-aggregation of various bacteria by exogenous introduction of the Ag43 gene.

Results: An E. coli transformant harboring a plasmid encoding the Ag43 structural gene under a green light-regulated gene expression system derived from the cyanobacterium Synechocystis sp. PCC6803 was constructed. Ag43 was inserted downstream of the cpcG 2 promoter P cpcG2 , and its expression was regulated by green light induction, which was achieved by the functional expression of cyanobacterial CcaS/CcaR by coexpressing its chromophore synthesis gene cassette in E. coli. E. coli transformants harboring this designed system self-aggregated under green light exposure and precipitated, whereas transformants lacking the green light induction system did not. The green light induction system effectively functioned before the cell culture entered the stationary growth phase, and approximately 80 % of the cell culture was recovered by simple decantation.

Conclusion: This study demonstrated the construction of a cell recovery system for non-photosynthetic microorganisms induced by exposure of cells to green light. The system was regulated by a two-component regulatory system from cyanobacteria, and cell precipitation was mediated by an autotransporter protein, Ag43. Although further strict control and an increase of cell recovery efficiency are necessary, the system represents a novel tool for future bioprocessing with reduced energy and labor required for cell recovery.

Fig. 1

Plasmid vectors used in this study. Plasmid shown in (a) encodes a gene necessary for aggregation (ag43) and an entire green light-sensing system (a pBRGLAg). Plasmids shown in (b), (c) encode green light-sensing system but lacking gene encoding CcaR (b pBRGLAgΔR, or gene encoding CcaS (c pBRAgΔS). Plasmid shown in (d) encodes a gene necessary for aggregation (ag43) but not the genes for green light-sensing system (d pBRGLAgΔSR). The plasmid shown in (e) encodes phycocyanobilin (PCB) synthetic genes, pSTVPCB

Fig. 2

Time course of aggregation of cells harboring a green light-inducible aggregation system. White circles show the time course of aggregation of cells grown under green light and black circles show the time course of aggregation of cells grown under red light. OD₆₀₀ or OD₅₉₅ were used as cell density indicators. a Cells harboring a green light-inducible aggregation system; b cells harboring a green light-inducible aggregation system lacking CcaS, the green light-sensor protein; c cells harboring a green light-inducible aggregation system lacking CcaR, the cognate response regulator of CcaS; d cells harboring a green light-inducible aggregation system lacking CcaS and CcaR. The experiment was performed in triplicate. The double dagger indicates statistical difference in the cell density under red light from 0 min incubation (‡P < 0.05). The asterisks indicate statistical differences from the cells exposed to red light (**P < 0.01 and ***P < 0.001 based on Dunnet’s t test)

Fig. 3

Transcriptional analyses of Ag43 under green light induction. Relative units of ag43 transcription were normalized to 16S rRNA amounts. Relative units in cells grown under green light are shown with white circles, and relative units in cells grown under red light are shown with black circles. There is no significant difference in the cell density under red light at each incubation time from at 0 min incubation. The asterisks indicate statistical difference from the cells exposed to red light (*P < 0.05 and **P < 0.01, based on Dunnet’s t-test)

Fig. 4

Evaluation of cell recovery under green light induction at various cell growth phases. a Time course of cell growth. b Cell recovery from cultures exposed to green light for 2 h. Cell recovery is shown as the ratio (%) of recovered cells (wet weight) to total cells (wet weight) in culture in a tube. The asterisks indicate statistical differences from the cells exposed to green light on stationary phase (*P < 0.05 and **P < 0.01 based on Dunnet’s t-test). A Cells exposed to green light at 19–21 h of culture, early log phase; B cells exposed to green light at 23–25 h of culture, middle log phase. C Cells exposed to green light at 25–27 h of culture, late log phase; D cells exposed to green light at 40–42 h of culture, stationary phase