Expression characterization and actual function of the second pucBA in Rhodobacter sphaeroides

Abstract

The puc2BA operon of Rhodobacter sphaeroides is highly similar to the original puc1BA operon. Genetic, biochemical and spectroscopic approaches were used to investigate the function of puc2BA; the puc1BA and puc2BA structural genes were amplified and cloned into the pRK415 vector controlled by the puc promoter from R. sphaeroides, which was then introduced into R. sphaeroides mutant strains. The results indicated that puc2BA was normally expressed and puc2BA-encoded polypeptides were assembled into membrane LHII (light-harvesting II) complexes, although the puc2A-encoded polypeptide was much larger than the puc1A-encoded polypeptide. Semi-quantitative RT-PCR (reverse transcription-PCR) and SDS/PAGE indicated that puc1BA and puc2BA were expressed in R. sphaeroides when integrated into the genome or expressed from vectors. Furthermore, the polypeptides from the puc1BA and puc2BA genes were both involved in LHII assembly, and pucC is also necessary to assemble LHII complexes. Nevertheless, the LHII complexes synthesized from puc2BA in R. sphaeroides have blue-shift absorption bands at 801 and 846 nm.