Isolation and Characterization of a Rhodobacter Capsulatus hemC Mutant.

Rhodobacter capsulatus, a purple, nonsulfur photosynthetic bacterium, uses the tetrapyrrole pathway to synthesize four end products: heme, bacteriochlorophyll, vitamin B12, and siroheme. This laboratory has focused on the regulation of the common pathway leading from aminolevulinate to protoporphyrin IX in R. capsulatus. The common portion of the pathway is regulated up to 100-fold by changes in oxygen tension. Research on the regulation of this pathway has been hampered due to a lack of mutants. Until now, the only mutants isolated which block heme production are hemA mutants, which lack aminolevulinate synthase. Since R. capsulatus lacks a cytochrome-independent growth mechanism, mutations later in the tetrapyrrole pathway would be lethal unless the mutant can use exogenous hemin or protoporphyrin. To overcome this lack of mutants, a method for isolating hem mutants has been devised. First, a growth medium was developed so that the hemA mutant, and presumably other hem mutants, grow well on exogenous hemin. We then took advantage of the recent cloning of several of the R. capsulatus hem genes to make a hem mutation in vitro and move it into R. capsulatus by conjugation. Thus, we were looking for a high probability recombination event rather than a much lower probability transposition event. This study describes the development of this medium as well as the isolation and characterization of a hemC mutant of R. capsulatus . This is the first hem mutant in a photosynthetic bacterium, after hemA, which has a complete block in the pathway. The mutant was constructed by inserting a kanamycin-resistance cartridge into a plasmid containing the R. capsulatus hemC gene. The mutated hemC gene was then recombined into the R. capsulatus chromosome. The resulting mutant requires heme for growth, lacks porphobilinogen deaminase activity and is unable to synthesize bacteriochlorophyll. This mutant was also used to provide direct evidence that the point of oxygen regulation in the tetrapyrrole pathway is located after the formation of porphobilinogen, most likely the usage of porphobilinogen