A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by TnS mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between the synthesis of c-type cytochromes and the expression of the tetrapyrrole biosynthetic pathway.The photosynthetic bacterium Rhodobacter capsulatus uses a single branched pathway to synthesize two major tetrapyrrole end products, heme and bacteriochlorophyll, and two minor tetrapyrrole end products, siroheme and vitamin B12. Since bacteriochlorophyll levels vary greatly with growth conditions, whereas heme levels remain relatively constant, some mechanism must exist to monitor and independently control the levels of these two tetrapyrroles. Studies in Rhodobacter sphaeroides have suggested that carbon flow over the common tetrapyrrole pathway leading to protoporphyrin is controlled by heme (25), and that heme may feedback inhibit aminolevulinate synthase (36). Recently, there have been some intriguing observations suggesting a link between cytochrome synthesis and pigment formation. Mutants lacking the R. capsulatus cytochrome be complex showed increased pigmentation (12), whereas mutants with reduced capacity to synthesize c-type cytochromes excreted an uncharacterized pigment (17). These observations suggest that the regulation of these two branches may be more complicated than previously thought. We attempted to probe the possible link between cytochrome c biosynthesis and expression of the tetrapyrrole pathway by isolating a mutant that completely lacks c-type cytochromes. Several years ago an R. capsulatus strain, MT113, was isolated (38) and subsequently shown to have reduced levels of all c-type cytochromes (13). This strain was isolated during a screen for normally pigmented mutants that lack cytochrome oxidase activity (38). In this report we describe the use of the cytochrome oxidase assay to isolate a TnS insertion mutant that completely lacks c-type cytochromes. In addition to lacking c-type cytochromes, this strain excretes coproporphyrin and protoporphyrin and synthesizes reduced amounts of bacteriochlorophyll. The isolation of this strain and the cloning of the gene responsible for the pleotropic phenotype will allow us to investigate the connection between cytochrome c synthesis and the expression of the tetrapyrrole biosynthetic pathway. sulfoxide. Escherichia coli was grown in L broth (4), which was modified by omitting glucose and decreasing the sodium chloride concentration to 0.5%. Solid media contained 1.5% agar (Difco). Antibiotics, when necessary, were added at the following final concentrations: kanamycin, 10 ,ug/ml; tetracycline, 1 pug/ml; spectinomycin, 5 ,ug/ml. Pigment determinations. Porphyrins...

MATERIALS AND METHODS Bacterial strains and plasmids. Bacterial strains used in this work are described in Table 1. pRPS404 is a derivative of RP1 containing all of the known bch and crt genes. The plasmid carries a gene for kanamycin resistance and the crtD223 allele. The genes for ampi-686

Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment.

The regulation of bacteriochlorophyll and carotenoid synthesis in purple non-sulfur photosynthetic bacteria has been the subject of intense investigation for over a quarter century. Cohen-Bazire et al. (6), studying Rhodobacter sphaeroides and Rhodospirillum rubrum, demonstrated that oxygen and light prevented the accumulation ofboth bacteriochlorophyll and carotenoids. They hypothesized that the two photopigments were governed by the oxidation state of some component of the electron transport chain. This was based on the assumption that the same mechanism governed regulation by both oxygen and light. Much more recently, Arnheim and Oelze (1) measured intracellular bacteriochlorophyll levels in chemostat cultures with carefully controlled oxygen and light levels. They found that light acted independently of oxygen in the regulation of bacteriochlorophyll accumulation. Further investigation revealed that while both oxygen and light control bacteriochlorophyll accumulation, aminolevulinate synthase responds only to changes in oxygen tension, not to changes in light intensity (11).Two studies have focused on the control of bacteriochlorophyll formation by oxygen in Rhodobacter capsulatus. Clark et al. (4) followed the accumulation of bch gene mRNA after a shift from high to low oxygen tension. They found that transcription of the bch genes increased when the culture was shifted to 2% oxygen. Using bch-lacZ fusions, Biel and Marrs (2) found that transcription of the bch genes increased two to fourfold when the oxygen tension was lowered from 23 to 3%. However, they did not detect a change in bch gene transcription when the light intensity was changed. It was suggested that the lack of response to light was due to the inability of the fusion strains to synthesize bacteriochlorophyll and thus the pigment-protein complexes. This study has focused on the control of bacteriochlorophyll accumulation by light in R. capsulatus growing under low aeration. Exposure to bright light did not reduce transcription from the bch genes, nor did it reduce carbon flow to bacteriochlorophyllide a. These results suggest that in cultures grown under low oxygen, bright light inhibits bacteriochlorophyll accumulation, but does not reduce bacteriochlorophyll synthesis. MATERIALS AND METHODSBacterial strains. The bacterial strains used in this work are described in Table 1. Strain AJB516 was isolated by introducing the conjugative plasmid pRPS404 (10) into strain Y142 and purifying a crtD puf recombinant. Strain AJB522 is a spontaneous bchA' revertant of strain BY1651.Media and growth conditions. R. capsulatus strains used to measure bacteriochlorophyll accumulation were grown overnight in a malate-minimal salts (RCV) medium (18) and subcultured into RCV medium supplemented with 0.6% glucose, 0.5% pyruvate, and 0.5 M dimethyl sulfoxide (RCV+) to yield an initial density of 25 Klett units (red filter). The culture tubes and sparging apparatus have been described previously (2). The cultures were sparged with a mixture of 92% nitrogen-5% carbon...

The effect of exogenous aminolevulinate and porphobilinogen on protoporphyrin accumulation in Rhodobacter capsulatus was measured. Oxygen inhibited protoporphyrin accumulation in strain AJB456, a bchH mutant, even in the presence of exogenous aminolevulinate, suggesting that some step in the formation of protoporphyrin from aminolevulinate is regulated by oxygen. In contrast, in the presence of exogenous porphobilinogen, oxygen did not inhibit protoporphyrin accumulation. The results presented in this study indicate that oxygen regulates the formation of porphobilinogen from aminolevulinate.