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Title: Peripheral antenna complexes From Rhodopseudomonas acidophila : structure, function and genetic manipulation
Author: Gardiner, Alastair Thomas
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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All members of the Rhodospirillaceae adapt to a reduced level of light available for photosynthesis by synthesising a greater amount of intra-cytoplasmic membrane (ICM) per cell and, wherever possible, increasing the number and size of photosynthetic units (PSD). It was already known that certain strains of Rhodopseudomonas acidophila have a further adaptative response (Cogdell et ai, 1983), in that they are able to alter the composition of their PSU by replacing the pehpheral B800-850 complex with B800-820. This work delineates the relationship between these two complexes with respect to the light-harvesting capability of this species when grown under different (non-optimal) conditions and sets up a genetic system to introduce cloned DNA into Rhodospirillaceae, enabling the molecular organisation of these genes and their regulation to be investigated. As a prerequisite to any molecular genetic experiments a thorough characterisation of the conditions under which 8800-820 is inserted into the ICM was necessary. Three wild type isolates of Bps. acidophila were cultured under three different growth regimes: (1) progressively decreasing light intensity, (2) progressively decreasing temperature at high light, and (3) progressively decreasing temperature at low light. Chromatophores prepared from each series were analysed using absorption spectroscopy, Deriphat-PAGE and reversed phase HPLC. The conclusions reached concerning each of the strains are as follows: Rps. acidophila 7750: This strain produces B800-820 complex in response to low light and low temperature, however, the light intensity 'switch' takes precedence over its temperature counterpart. No evidence was obtained from Deriphat-PAGE that there was any change in the B890/Reaction Centre (RC) conjugate, specifically, in its carotenoid composition; HPLC revealed that the low light 8800- 820 complex contains relatively more unglucosylated rhodopin than the high light 8800-850 complex. Rps. acidophila 7050: This strain produces 8800-820 complex in response to low light; no evidence could be found of any temperature regulation. From Deriphat- PAGE, the carotenoid composition of both the peripheral and fixed complexes altered under low light. The main change in the peripheral antenna was the replacement of orange coloured rhodopin and its glucoside with purple rhodopinal glucoside. Furthermore, this carotenoid change was not synchronous with, rather preceded, the switch in the peripheral antenna. Rps. acidophila 10050: No evidence was obtained that this strain was able to produce B800-820 under any of the conditions tried. Furthermore, the carotenoid compositions of the peripheral and fixed complexes also remained unchanged. Data was then presented to explain, with regard to the light-harvesting capability of the cell, the insertion of B800-820 in place of B800-850 when the cell is cultured at non-optimum conditions. Fluorescence emission experiments showed that once excitons have arrived in 8890 they can be transferred back to B800-850 but not to B800-820. Singlet-singlet annihilation experiments revealed a B890 domain size of approximately 50 +/- 20 Bchls when the peripheral antenna was B800-820, and at least 200 for the corresponding measurement with B800-850. Thus, B800-820 is produced at non-optimum conditions because it is more efficient at funnelling excitons into B890 and on to the RC. A genetic system based on bacterial conjugation (Simon et al, 1983) was then developed which allowed the introduction of cloned DNA into Rps. acidophila, and two other species with interesting peripheral antennae, Rps. palustris and R. rubrum. The results proved that Inc-P and Inc-Q cloning vectors could be mobilised into these species and that transposon mutagenesis was also feasible. An EMBL3 genomic library had previously been constructed from Rps. acidophila 7050 DNA and a preliminary attempt made to characterise clones that contained peripheral antenna complex genes (MacKenzie, 1990). One of these clones, lambda6, was known to carry a beta/alpha gene pair on a 2.1 kb Sail fragment. This fragment was sequenced and found to contain not one but three beta/alpha gene pairs, designated (5 → 3') sxaBA. sxbBA and sxcBA. which when translated were exceedingly homologous to the alpha, beta1 and beta2 polypeptides from strain 7750. To verify that the above system to introduce cloned DNA was feasible in an experimental context, the 2.1 kb Sall fragment was cloned into a mobilisable vector and introduced into R. rubrum. No constitutive expression of any of the genes from this cluster was apparent, although there was a carotenoid pleiotropic effect which was not able to be further investigated. This preliminary experiment, nevertheless, established the feasibility of using this system in the future to investigate at a molecular genetic level the organisation and regulation of peripheral antenna genes from Rps. acidophila.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available