Expression system construction and activity evaluation of reconstituted LH2 of Rhodopseudomonas palustris

Abstract:

[Background] The peripheral light-harvesting complex (LH2) performs the function of light energy capture and transfer in photosynthesis and is essential for the phototrophic growth of anoxygenic phototrophic bacteria (APB). The α and β subunits of LH2 are usually encoded by multiple copies of pucBA genes, which reach 5–7 copies inRhodopseudomonas. As a result, the heterogeneous synthesis of LH2 by multiple genes makes LH2 difficult to be separated from each other, which greatly limits the full understanding about the contributions of different pucBAgenes to the structures and physiological roles of LH2. [Objective] To develop an expression system and evaluate the light energy transfer activity in vivo andin vitro for the reconstituted LH2 of R. palustris. [Methods] Using Rhodopseudomonas palustris CGA009 (ΔpucBA) with all five pucBA genes deleted as the host bacterium and the co-expression products of pucBAd and mrfp (red fluorescent protein gene) as the markers, we conducted genetic modification of the expression vector and promoter screening. The spectroscopic method was employed to determine the biomass of recombinant bacteria, the spectral characteristics of gene expression products, and the spectral characteristics, photosynthetic pigments and light energy transfer activity of purified LH2. [Results] The T7 promoter (P_T7) combined with T7 RNA polymerase gene promoted the co-expression of pucBAd and mrfpin ΔpucBA. We replaced P_T7 with P_badR, P_pckA, and P_ars, respectively, and the activities of the promoters followed a descending order of P_pckA, P_ars, P_badR, and P_T7. This strategy increased the mrfp gene expression, the synthesis product of pucBAd (d-LH2), and the phototrophic growth rate in the host bacteria. The expression system ΔpucBA(pucBAd) harboring P_pckA promoter had the highest expression activity of reconstituted LH2. Then, we used pucBAa from strain CGA009 to replace pucBAd in ΔpucBA(pucBAd) and successfully obtained the recombinant strain ΔpucBA(pucBAa). Compared with the pucBAaconstruct, the recombinant strain with pucBAd showed significantly accelerated growth under low light and slightly accelerated growth under high light. The purified d-LH2 and a-LH2 showed typical characteristic spectra of B800-only and B800-850, respectively. The fluorescence quantum efficiency of d-LH2 at ~863 nm was higher than that of a-LH2. However, the light energy transfer efficiency from carotenoids (Car) to bacteriochlorophylls (BChl) of d-LH2 measured by the classical method was lower than that of a-LH2. The inconsistency between the two results was due to the fact that the classical method did not consider the photon absorption difference at ~863 nm by different spectral types of LH2. [Conclusion] The reconstituted LH2 expression system ofR.palustris was successfully constructed. The light energy transfer activities of two different spectral types of LH2 in strains with multiple copies of pucBA were evaluated on the levels of gene-pure LH2 and photosynthetic growth rate of the recombinant bacteria. The results showed that the energy transfer activity of abnormal spectral LH2 (d-LH2) was higher than that of typical spectral LH2 (a-LH2). The present work laid a foundation for comprehensively understanding the molecular regulatory mechanisms of the formation and assembly of LH2 in the bacteria with multiple copies of pucBA.

Key words:anoxygenic phototrophic bacteria;Rhodopseudomonas palustris;reconstituted LH2;light energy transfer;quantum efficiency