Cloning, sequencing and expression of the PUC genes of two strains of Rubrivivax gelatinosus

Simmons, Adrian Edward (1995) Cloning, sequencing and expression of the PUC genes of two strains of Rubrivivax gelatinosus. PhD thesis, University of Glasgow.

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An initial observation that the two strains 149 and 151 of Rv. gelatinosus synthesise differing amounts of LH2 (B800-850) when cultured under the same conditions was confirmed. Strain 151 produces approximately one third more LH2 than strain 149 when cultured at any given light intensity, it was also found that both strains vary the level of LH2 to a similar extent in response to changing irradiance levels, both synthesising twice the amount of LH2 under low irradiance than was synthesised at high irradiance. Southern analysis indicated the presence of a single copy of the pucBA genes in each strain, but suggested there may be a difference between them with regard to the pucC gene. Genomic libraries were constructed from the two strains of Rv. gelatinosus using the lambda replacement vector lambda GEM-11. The pucBA genes coding for the alpha and beta-polypeptides of the LH2 complex were cloned and sequenced from both strains, as was the pucC gene from strain 151 and a substantial region of sequence probably involved in transcriptional control. The predicted amino acid sequence of the strain 151 and 149 LH2 alpha and beta-polypeptides matched that achieved by protein sequencing (Brunisholz et al., 1994), whilst the predicted sequence of the strain 151 PucC protein was shown to have high sequence and structural homology to other PucC proteins. The arrangement of the Rv. gelatinosus puc genes was found to be somewhat different from that found in other bacteria from which they have been cloned. With the pucC gene being present downstream of the pucBA genes, but in the opposite orientation. Analysis of the sequence upstream of the pucBA and pucC genes has identified possible E. coli O70 like promoter elements, two upstream of pucBA and one upstream of pucC. Near palindromes similar to the Rhodohacter Pps oxygen sensitive repressor binding sites were also found, two in the promotor more proximal to pucB, and a single one straddling the start of the pucC gene. Northern analysis was carried out on cultures of the two strains grown under a variety of conditions, these indicate that the Rv. gelatinosus pucBA genes are repressed in the presence of oxygen, and are expressed more under low irradiance conditions than at high irradiance. Thus Rv. gelatinosus appears to respond to light and oxygen in a similar manner to other photosynthetic bacteria. The northern analysis also indicates a transcript size of around 600 bp for the puc genes of Rv gelatinosus.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Cogdell, Professor Richard
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-71528
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 14:23
Last Modified: 16 Aug 2021 15:07
Thesis DOI: 10.5525/gla.thesis.71528

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