Directed mutagenesis and gene fusion analysis of the Rhodobacter capsulatus puc operon

• Main Author: LeBlanc, Heidi
• Format: Others
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/7276

The puc operon of Rhodobacter capsulatus encodes the genes for the pigment binding peptides of the light harvesting (LH) II complex, pucB and pucA, followed by three open reading frames named pucC, pucD and pucE. RNA blot analysis and promoter mapping experiments using translational fusions of lac’Z to the distal gene, pucE, showed that all five genes are primarily transcribed from a promoter upstream of pucB. The primary transcript from this promoter is of low abundance and is processed to give a 1.0 kb transcript containing pucDE sequences. A minor promoter was detected between the 3’ end of pucC and the middle of pucD; a 0.5 kb RNA molecule detected by hybridisation probes to the pucDE region could be the transcription product of this minor promoter. The most abundant transcript detected was the previously characterised 0.55 kb pucBA mRNA. Strain ΔLHII, which lacked the LHII complex, was created by replacement of the five chromosomal puc genes with the spectinomycin cartridge. This strain was complemented by plasmids carrying deletions of the pucCDE genes. The pucC gene was required for the presence of the LHII complex, and deletion of the pucE gene, which encodes ƴthe subunit of the LHII complex, led to a reduced level of the complex. Deletion of the pucD gene had no discernable effect on LHII complex levels. Photosynthetic growth of strain ALHII was similar to that of wild type cells at light levels greater than 30 µE m-2.s-1, but strains containing deletions of pucC or pucE were significantly impaired for photosynthetic growth at all light levels tested. In the case of pucC-deleted cells, chromosomal suppressor mutations frequently arose that restored the LHII complex and/or photosynthetic growth ability. A model for the trans-membrane topology of the PucC protein was derived by theoretical analyses and tested using the genetic system of alkaline phosphatase fusions. The model predicts 12 transmembrane domains, with both the N and C termini on the cytoplasmic side of the membrane. The fusions were also used for a deletion analysis of pucC. None of the truncated alleles allowed the cells to synthesize the LHII complex, but several had unexpected pleiotropic effects on LHI and RC complex levels. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate