Molecular Genetic Analysis of Rhodobacter Sphaeroides Light-Harvesting Complexes
Kiley, Patricia Jeannette
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https://hdl.handle.net/2142/71187
Description
Title
Molecular Genetic Analysis of Rhodobacter Sphaeroides Light-Harvesting Complexes
Author(s)
Kiley, Patricia Jeannette
Issue Date
1987
Doctoral Committee Chair(s)
Kaplan, Samuel
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The genes for the Rhodobacter sphaeroides light-harvesting, B875-$\beta$ and B875-$\alpha$ polypeptides (pufB and pufA) are closely linked to the genes for the reaction center-L and reaction center-M polypeptides (pufL and pufM) on what has been termed the puf operon (gene order pufB,A,L,M). The DNA sequence of pufB and pufA from wild-type R. sphaeroides 2.4.1 was determined. The relative levels of the B875-$\beta$ and B875-$\alpha$ and the reaction center-L and reaction center-M polypeptides synthesized in a homologous cell-free transcription-translation system were compared with those found in vivo. Analysis of the gene products produced in vitro using plasmids containing deletions upstream of the pufB structural gene identified a region of DNA required for expression of the B875-$\beta$ and B875-$\alpha$ polypeptides. These results support the hypothesis that the mapped 5$\sp\prime$ termini of the large and small puf operon transcripts represent transcription initiation sites.
Two deoxyoligonucleotide probes were synthesized in accordance with the available amino acid sequence of the B800-850-$\beta$ polypeptide from Rhodobacter sphaeroides and were used to isolate a 2.6 kilobase PstI fragment from R. sphaeroides 2.4.1 genomic DNA. Identification of the B800-850-$\beta$ and B800-850-$\alpha$ structural genes, pucB and pucA, was confirmed by DNA sequencing. Northern blot analysis, using a pucB,A-specific restriction endonuclease fragment as a probe, revealed a single puc-operon-specific, highly stable, transcript of approximately 640 bases present in photosynthetically growing cells. In vitro transcription-translation analysis of the puc operon revealed maximum synthesis of the puc operon gene products was achieved using the 2.6 kb PstI fragment as template although a 537 bp XmaIII fragment was sufficient to direct the synthesis of pucB and a pucA fusion product.
Mutants deficient in either B800-850 or B875 spectral activity were characterized. The B800-850$\sp-$ mutant also lacked visibly absorbing carotenoids. Moreover, B800-850-$\alpha$ and B800-850-$\beta$ polypeptides could not be detected in the photosynthetic membranes derived from the B800-850$\sp-$ mutant. Reduced levels of the B875-$\alpha$ polypeptide were present in the photosynthetic membranes from the B875$\sp-$ mutant, although there was no detectable absorption at 875 nm. Both mutants responded similarly to the wild type in their response to changes in incident light-intensity. However, the magnitude of derepression for B800-850 synthesis was greater in the B875$\sp-$ mutant than the wild-type when cells grown at equivalent light intensities were examined.
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