Figure 1.
Schematic representation of the experimental design.
Potential cbb genes that might affect autotrophic growth were constructed then assessed for their actions on growth ability. The effects of these candidates on autotrophic growth were studied in a variety of ways including measurement of their actions on enzyme activity, and observation of their subcellular localization and absorption spectra. Finally, differentially expressed proteome profiles in strains in which these genes had been overexpressed were compared to ascertain the mechanisms that might regulate autotrophic growth.
Figure 2.
The effects of transketolase overexpression on photoautotrophic growth.
(A) The effects of transketolase I and transketolase II overexpression on autotrophic growth in R. palustris compared with the negative control strain. Biomass analysis was performed to ascertain the CO2-fixing ability of the different strains. The initial number of cells in each strain culture was 108. Each value represents the mean of three replicate cultures grown under identical conditions over the course of 9 days. (B) Growth curves of the transketolase-overexpressing strains of R. palustris. cbbT1 indicates the transketolase I-overexpressing strain; cbbT2, the transketolase II-overexpressing strain; NC, the negative control. *, p<0.05; **, p<0.005.
Figure 3.
Physiological differences between the R. palustris strains in which overexpression of one of the two isoforms of transketolase had been induced.
(A) qPCR analysis of the photosynthetic genes that encode the subunits of the light harvest complex. The ratios indicate the gene expression level of transketolase overexpression relative to negative control strain. The error bar represents SD (n = 3) between the transketolase I and transketolase II-overexpressing strains. *, p<0.05; **, p<0.001. (B) Absorption spectra of cells grown under photoautotrophic conditions. LH I absorbed at 880 nm; LH II complexes at 802 nm and 862 nm; LH IV at 802 nm.
Figure 4.
The different localizations of transketolase I (cbbT1) and II (cbbT2) in R. palustris.
(A) The in situ localization of HA-CbbT1 in the cbbT1-overexpressing strain grown under photoautotrophic conditions. Localization of HA-CbbT1 was detected using 10 nm immunogold labeled anti-HA antibody in ultra-sections. (B) The in situ localization of HA-CbbT2 in the cbbT2-overexpressing strain grown under photoautotrophic conditions. Localization of HA-CbbT2 was detected using 10 nm immunogold-labeled anti-HA antibody in ultra-sections. (C) The ICM distributions of the transketolase isoforms in transketolase I and transketolase II-overexpressing strains. More than 300 longitudinal-section micrographs from 5–6 grids of each strain were evaluated to quantify the density of cells with significant ICM structure in cbbT1- and cbbT2-overexpressing strains. The dark arrows indicate the location of HA-CbbT1 and HA-CbbT2 in the ICMs and cytoplasm. (D) The distribution of immunogold-labeled HA-CbbT fusion protein in the ICM and cytoplasm revealed in ultrathin section micrographs of 30 different bacterial cells of the transketolase I and II-overexpressing strains and the NC strain. cbbT1 indicates transketolase I overexpression; cbbT2, transketolase II overexpression; NC, negative control.
Figure 5.
Two dimensional electrophoresis (2DE) of transketolase-overexpressing R. palustris.
(A) 2DE of R. palustris CGA010 strains overexpressing transketolase I. (B) 2DE of R. palustris CGA010 strains overexpressing transketolase II. (C) 2DE of the negative control (NC) R. palustris CGA010 strains. Proteins indicated in these maps were considered to be differentially expressed and were further identified by MS. The bold arrow indicates the protein spot left by phosphoenolpyruvate carboxykinase (PEPCK). (C) 2D and 3D views of PEPCK expression levels. The expression of PEPCK was increased in transketolase I and II-overexpressing strains. Arrows indicate the spot corresponding to PEPCK. cbbT1 indicates transketolase I overexpression; cbbT2, transketolase II overexpression; NC, negative control.
Figure 6.
Protein-protein interaction networks (PIN) in transketolase I and II-overexpressing strains.
The differentially expressed proteins in transketolase I and II-overexpressing strains and their interacting partners were used to construct the network with the STRING database. All proteins displayed in the network were further analyzed for clustering with an MCL clustering algorithm. (A) The protein interaction networks of the transketolase I-overexpressing strain. (B) The protein interaction networks of the transketolase II-overexpressing strain.
Table 1.
Activities of transketolase and PEPCK in transketolase I, transketolase II and wild type with empty vector (NC) over-expression R. palustris strains.