Title: Cloning and Overexpression of theRhodobacter capsulatus hemHGene
Abstract:In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized fro...In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized from protoporphyrin by the product of thehemHgene, ferrochelatase. We have cloned theR. capsulatus hemHgene by complementation of anEscherichia coli hemHmutant. When a plasmid carrying thehemHgene is returned to R. capsulatus, ferrochelatase activity increases, aminolevulinate synthase activity decreases, and bacteriochlorophyll levels are dramatically lowered. This is the first in vivo evidence to suggest that heme feedback inhibits aminolevulinate synthase inR. capsulatus, thereby reducing porphyrin synthesis. Inthepurplenonsulfurphotosyntheticbacteria,tetrapyrrole biosynthesis is regulated by at least three factors: oxygen, heme, and c-type cytochromes (2, 4, 5, 7). In Rhodobacter sphaeroides, a model has been developed to tie regulation by heme and oxygen into a single mechanism (13). The model suggests that oxygen prevents the conversion of protoporphyrin into magnesium protoporphyrin. The accumulating protoporphyrin is converted into heme, which causes feedback inhibition of aminolevulinate synthase, thus reducing carbon flow over the common tetrapyrrole pathway. Feedback inhibition of aminolevulinate synthase by heme has been demonstrated in this organism (4). However, this model does not appear to be true for Rhodobacter capsulatus; therefore, the role that heme might play in regulating tetrapyrrole biosynthesis in this organism is unclear (1). One way to study that role is to increase heme synthesis by providing a second copy of the hemHgene, which encodes ferrochelatase. We report here on the effects that the presence of a second hemH gene has on tetrapyrrole biosynthesis. Cloning of the R. capsulatus hemH gene. The R. capsulatus hemHgene was cloned by complementation of anEscherichia coli hemHdeletion mutant, DVisA (12).R. capsulatuschromosomal DNA was isolated, digested with PstI, and cloned into pUC18. The library was then transformed into the E. coli mutant. This mutant requires hemin for growth in the absence of a fermentable carbon source. Transformants were selected for growth on L agar without hemin. A Hem 1 , ampicillinresistant colony was purified, and the plasmid was designated pCAP122. This plasmid has a 3.1-kb PstI fragment (Fig. 1). TheE. colistrain DVisA lacks ferrochelatase activity (specific activity, 0.17 pmol of deuteroheme formed per min per mg of protein). The presence of pCAP123, a subclone of pCAP122 (Fig. 1), in thehemHmutant increases ferrochelatase specific activity 500-fold to 86 pmol of deuteroheme formed per min per mg of protein, a level three times higher than that in theRead More
Publication Year: 1995
Publication Date: 1995-01-01
Language: en
Type: article
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