Effects of Overexpressed Transketolase Genes on Carbon Metabolism in Rhodopseudomonas palustris

Transketolase is a thiamin diphosphate (ThDP)-dependent enzyme that functions as a key catalyst in the oxidative pentose phosphate pathway of virtually all organisms. It also plays an important role in the Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle of photosynthetic organisms. In Rhodopseudomonas palustris, a purple, non-sulfur photobacterium, there are two isoforms of transketolase, CbbT1 and CbbT2, encoded by transketolase genes, cbbT1 (rpa4643) and cbbT2 (rpa0945), respectively.n this study, we overexpressed cbbT1 and cbbT2 under photoautotrophic conditions, in which CO2 was the only carbon source. We integrated oligo microarray analysis, real-time quantitative PCR (qPCR), bioluminescent ATP assay and absorbance spectra to study the effects of overexpressed transketolase. From our microarray data, overexpressed cbbT1 had more effects on R. palustris than overexpressed cbbT2. In the T1 strain, an increase of the peak amplitudes in the absorption spectrum and increased expression of related photosynthetic genes were observed. On the other hand, we measured the glycolytic ATP synthetic activity by bioluminescent ATP assay and observed an increased amount of ATP in the T2 strain. Moreover, immunogold labeling of HA-tag fusion tranketolase reveals that CbbT1 is mainly located in the intracytoplasmic membrane systems, but CbbT2 in the cytosol and the ICM structures are not abundant in the T2 strain compared with the T1 strain. In this study, we suggest that the two isoforms of transketolase are likely involved in different carbon metabolic pathways and have distinct effects on bacterial physiology.