Genetic manipulation of the puc-aba gene pair isolated by PCR from Rps. palustris genomic DNA

Abstract

The light-harvesting complexes of Rhodopseudomonas palustris have been studied extensively and are reasonably well characterized. Growth in high-light results in LH2 with characteristic absorption at 800-850 nm (B800-850). When the light is lowered the absorption spectrum shifts to 800 and we are able to isolate a novel low-light adapted B800 complex with a 800/850 nm absorption ratio >20:1. HPLC analysis of this complex shows it to consist exclusively of the a bd peptides. Purification to homogeneity is hampered by the presence of small amounts of the a ba peptide. To overcome this and to discover more about the overall control of assembly we are cloning the a ba gene with a view to performing a gene disruption and site directed mutagenesis. The use of molecular genetic techniques to examine the structure and function of the B800 LH2 has been hampered by the availability of a stable plasmid system. Recently, a series of Rps. palustris/E. coli shuttle vectors has been characterized. Dr H Yukawa from the Research Institute of innovative technology for the Earth, Yoto, Japan kindly gifted vector pMG103. Here we describe the isolation of the gene by PCR and its cloning into the pMG103 vector. The aim is to produce uncontaminated B800 LH2 complex for X-ray diffraction studies and for unambiguous measurement of its energy transfer properties.