Isolation and Characterization of a Rhodobacter Capsulatus bioB Mutant Using a Novel Method to Screen for Strains Unable to Synthesize Heme.
Date of Award
Doctor of Philosophy (PhD)
Alan J. Biel
The common tetrapyrrole pathway of the photosynthetic bacterium, Rhodobacter capsulatus, ultimately leads to the synthesis of bacteriochlorophyll, heme, vitamin B-12, and siroheme. Molecular analysis of tetrapyrrole biosynthesis has been limited due to a lack of mutants. This study describes the isolation and characterization of a biotin-requiring mutant of R. capsulatus unable to synthesize heme. Growth conditions were established to allow selection and characterization of heme-deficient strains of R. capsulatus. A screening method was developed to isolate strains of R. capsulatus unable to synthesize heme. Tn5 derivatives of a bchB strain that exhibited an altered ability to synthesize the fluorescent porphyrin Mg-2,4-divinyl pheoporphyrin $a\sb5$ monomethyl ester were scored for hemin-dependence. One nonfluorescent, hemin-requiring mutant, strain AJB544, was isolated. This mutant could not grow on aminolevulinate or porphobilinogen whereas protoporphyrin and hemin supported growth. AJB544 was found to be a bio mutant, and it was shown that hemin could partially replace the requirement for biotin. Thin-layer chromatography and fluorimetric analysis revealed that AJB544 accumulated coproporphyrin and protoporphyrin when grown in minimal medium containing hemin or 2.5 nM biotin. Coproporphyrin was the major porphyrin that accumulated when AJB544 was grown in minimal medium containing hemin. Porphyrin formation by cell-free extracts of AJB544 and AJB478 demonstrated that AJB544 could form normal levels of protoporphyrin from protoporphyrinogen but not from aminolevulinate or porphobilinogen. Addition of biotin to extracts of AJB544 resulted in a 2.0 to 3.8-fold increase in protoporphyrin formation from aminolevulinate or prophobilinogen. Extracts of AJB544 grown in minimal medium containing biotin demonstrated an increase in protoporphyrin formation from aminolevulinate or porphobilinogen. These data suggest that biotin is required for the conversion of coproporphyrinogen to protoporphyrinogen. Southern blot analysis demonstrated that strain AJB544 contained a single Tn5 insertion. AJB544 was characterized as a bioB mutant by crossfeeding studies and thin-layer chromatography. The bioB gene was cloned from R. capsulatus by screening a pLAFRI library of R. capsulatus DNA and localized to a 2.85 kb EcoRI-PstI fragment. Introduction of the bioB gene into AJB544 restored the fluorescent phenotype.
Cardin, Rhonda Diane, "Isolation and Characterization of a Rhodobacter Capsulatus bioB Mutant Using a Novel Method to Screen for Strains Unable to Synthesize Heme." (1989). LSU Historical Dissertations and Theses. 4834.