Date of Award

1990

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Alan J. Biel

Abstract

The first step in the common tetrapyrrole pathway in Rhodobacter capsulatus is the condensation of glycine with succinyl CoA to form 5-aminolevulinate which is then used in the synthesis of bacteriochlorophyll, heme, vitamin B-12, and siroheme. To isolate mutants unable to form 5-aminolevulinate, as a first step in studying regulation of the tetrapyrrole pathway, R. capsulatus was mated with E. coli carrying plasmid pSUP2021. pSUP2021 carries transposon Tn5 and is incapable of replicating in R. capsulatus making it an ideal vector for transposon mutagenesis. One colony was found which requires 5-aminolevulinate for growth. This mutant strain, designated AJB529, has a growth rate and bacteriochlorophyll production at a level comparable to those of the parental strain when provided with 5-aminolevulinate. A cosmid library of R. capsulatus DNA was mated into AJB529 via the tetracycline resistant vector pLAFRI. Tetracycline resistant colonies which no longer required 5-aminolevulinate for growth were selected. Cosmids were isolated from these colonies and one was designated pCAP17. pCAP17 was transformed into E. coli and mated back to R. capsulatus to confirm 5-aminolevulinate independence. pCAP17 was digested to completion with EcoRI and was found to contain five insert fragments. Through a series of partial digestions S. Biel found that the 0.6 kb and 1.4 kb insert fragments are both required to confer 5-aminolevulinate independence. Through Southern blot analysis the 1.4 kb fragment was found to span the hemA::tn5 insertion. A translational fusion was made between the hemA and lacZ genes. The hemA-lacZ fusion plasmid was mated into R. capsulatus strain PAS100. The size of the fusion protein was determined to be 147,000 by SDS-polyacrylamide gel electrophoresis. Cell-free extracts were assayed for $\beta$-galactosidase activity after growth under high and low oxygen tensions. Transcription of the hemA gene, measured as $\beta$-galactosidase activity, was two-fold higher under low oxygen tension.

Pages

114

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