Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins

Abstract

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...

Phenotypic characterization of c-type-cytochrome-deficient mutants of Methylobacterium extorquens AM1 and identification of two chromosomal regions essential for the production of c-type cytochromes

“…There have been recent reports describing c-typecytochrome-deficient mutants of Bradyrhizobium japonicum (Ramseier et al, 1991), P. denitrificans (Page & Ferguson, 1989 and Rhodobacter capsulatus (Kranz, 1989;Biel & Biel, 1990). In the case of P. denitrificans, a mutant lacking all the holo-forms of the c-type cytochromes contained the apo-forms of at least two of these cytochromes in the periplasm, indicating that in this organism haem attachment may occur in the periplasm.…”

“…To date, selection of bacterial mutants which are unable to produce any c-type cytochromes has mainly made use of the Nadi test (Marrs & Gest, 1973;Willison & John, 1979;Biel & Biel, 1990), since a positive result is thought to depend on the presence of a c-type cytochrome, together with an Cy t och rome c mu tan ts of Met h j ?lo hac tuii ~I I I sp .…”

“…By analogy with mitochondria1 c-type cytochromes this is likely to be catalysed by a haem lyase (Page & Ferguson, 1989 but it is unclear whether this enzyme acts before or after translocation to the periplasm. The further elucidation of the steps involved in c-type cytochrome biosynthesis in bacteria is being aided by the isolation and characterization of mutants which are deficient in c-type cytochromes (Kranz, 1989;Page & Ferguson, 1989Biel& Biel, 1990;Ramseier et al, 1991) and in this paper we describe four such mutants of M . extorquens AM1.…”

Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation

“…The use of thioredoxinderived reducing equivalents is a fundamental requirement for a variety of bacterial extracellular and periplasmic activities, such as the activities of the disulfide bond forming (Dsb) system, the crosslinking of spore coat proteins, and CCM. These processes cannot be controlled by concentration dependence alone; and, in the case of CCM, genetic elimination of the terminal thioredoxin-like protein has been shown to specifically abolish maturation of c-type cytochromes for a number of systems (5,14,31,32). Crow et al (21) presented a structure-based theory that ResA (system II CCM) uses redox-coupled conformational changes to select its substrate.…”

“…This study biochemically demonstrates the specificity of this thioldisulfide oxidoreductase and enables us to outline a structural mechanism of regulating the usage of electrons in a thiol-disulfide oxidoreductase system. NMR ͉ thiol-disulfide oxidoreductase ͉ x-ray crystallography T he c-type cytochromes are involved in a number of cellular processes including aerobic and anaerobic respiration, plant and bacterial photosynthesis, detoxification via electron transport (1), apoptosis (2,3), redox metabolism (4), and biosynthesis of heme intermediates (5,6). Maturation of these important cytochromes involves the posttranslational covalent attachment of a heme cofactor to the C-x-x-C-H motif of the apocytochrome polypeptide.…”