Gene Expression in Rhodopseudomonas Sphaeroides (Photosynthesis, Carboxylase, Membrane Biosynthesis

254 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.A DNA-directed coupled transcription-translation system has been developed in cell-free extracts from the facultative phototroph, Rhodopseudomonas sphaeroides. The in vitro protein synthesizing system was active when prepared from either aerobically or anaerobically grown cells. Optimal activity was dependent upon use of extracts prepared freshly from early exponential phase cells, the method of cell breakage, and the length of preincubation of the extract (S-30), as well as the concentrations of S-30, DNA template, and cations. The cell-free system was used to study the physical and biochemical characterization of an enzymatically active form II ribulose-1,5-bisphosphate carboxylase which was synthesized and assembled in vitro from cloned R. sphaeroides DNA. The predominant enzymatically active oligomeric form of the in vitro synthesized and assembled RuBP carboxylase was a tetramer-hexamer, and assembly of the monomeric subunits to form active enzyme was dependent on the presence of a minimum number of subunits.The cell-free system described here was compared to one prepared from Escherichia coli and transcriptional-translational specificity was observed. In order to test the hypothesis that species specificity of gene expression could be due to differences in DNA base composition of E. coli (48% guanine-plus-cytosine) versus R. sphaeroides (70% guanine-plus-cytosine), cloned DNA templates from a variety of diverse microorganisms known to have a relatively high mole present guanine-plus-cytosine (mol% G+C) DNA base composition (> 65%) were analyzed in the R. sphaeroides cell-free system. Most of the templates tested were expressed in the R. sphaeroides coupled system, implying that the regulatory signals on the DNA for recognition by RNA polymerase may be similar in organisms with a high mol% G+C DNA base composition.Additionally, physiological studies were performed on cells of R. sphaeroides in order to examine the regulation of the synthesis and assembly of components in the photosynthetic membrane associated with light-harvesting and reaction center complexes. The ramifications of these results are discussed in terms of the oxygen- or light-dependent regulation of gene expression in R. sphaeroides and the possible site-specific assembly of pigment-protein complexes during de novo or derepressed synthesis of the photosynthetic membrane of R. sphaeroides.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD