Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state

Abstract

Rhodobacter sphaeroides 2.4.1 has the ability to synthesize a variety of tetrapyrroles, reflecting the metabolic versatility of this organism and making it capable of aerobic, anaerobic, photosynthetic, and diazotrophic growth. The hemA and hemT genes encode isozymes that catalyze the formation of 5-aminolevulinic acid, the first step in the biosynthesis of all tetrapyrroles present in R. sphaeroides 2.4.1. As part of our studies of the regulation and expression of these genes, we developed a genetic selection that uses transposon mutagenesis to identify loci affecting the aerobic expression of the hemA gene. In developing this selection, we found that sequences constituting an open reading frame immediately upstream of hemA positively affect hemA transcription. Using a transposon-based selection for increased hemA expression in the absence of the upstream open reading frame, we isolated three independent mutants. We have determined that the transposon insertions in these strains map to three different loci located on chromosome I. One of the transposition sites mapped in the vicinity of the recently identified R. sphaeroides 2.4.1 homolog of the anaerobic regulatory gene fnr. By marker rescue and DNA sequence analysis, we found that the transposition site was located between the first two genes of the cco operon in R. sphaeroides 2.4.1, which encodes a cytochrome c terminal oxidase. Examination of the phenotype of the mutant strain revealed that, in addition to increased aerobic expression of hemA, the transposition event also conferred an oxygen-insensitive development of the photosynthetic membranes. We propose that the insertion of the transposon in cells grown in the presence of high oxygen levels has led to the generation of a cellular redox state resembling either reduced oxygen or anaerobiosis, thereby resulting in increased expression of hemA, as well as the accumulation of spectral complex formation. Several models are presented to explain these findings.Rhodobacter sphaeroides 2.4.1, by virtue of its metabolic versatility, would be expected to possess a diversity of regulatory circuits and mechanisms of gene control that enable it to rapidly adapt to changing environmental conditions. A snapshot of the metabolic versatility inherent to this organism is the presence of the four major physiologically active tetrapyrroles, i.e., hemes, bacteriochlorophylls (Bchls), vitamin B 12 , and siroheme, which aptly illustrates the variety of growth conditions to which this organism is capable of adapting. Underlying the presence of these important metabolites is the tetrapyrrole biosynthesis pathway which displays a variety of regulatory features, notably oxygen and light control (see reference 20).The first step in the tetrapyrrole pathway in R. sphaeroides 2.4.1 is the condensation of succinyl-coenzyme A and glycine to 5-aminolevulinic acid (ALA), catalyzed by the enzyme ALA synthase (succinyl-coenzyme A:glycine C-succinyl transferase [decarboxylating] [EC 2.3.1.37]). Unlike all other prokaryotic organisms in wh...

Adaptation to Oxygen

“…When either cbb 3 or bd terminal oxidases were inactivated in R. gelationsus, the resulting mutants were less pigmented and produced lower amounts of photosynthetic complexes. In R. sphaeroides a direct role of the cbb 3 in photosynthesis was also reported; but in contrast to R. gelatinosus, the cbb 3 Ϫ mutant from R. sphaeroides was more pigmented than the wild type under aerobic conditions (25). In R. capsulatus, the bc 1 Ϫ mutant like the cbb 3 Ϫ mutant is also more pigmented that the wild type (26,27).…”

Generalized approach to the regulation and integration of gene expression

“…The first clear indication that the cbb 3 cytochrome c oxidase encoded by the ccoNOQP operon is the source of an inhibitory signal under aerobic conditions regulating the activity of the membrane-localized PrrB sensor kinase emerged from the findings that inactivation of the cbb 3 oxidase leads to derepression of PS genes under highly aerobic conditions (Zeilstra-Ryalls and Kaplan, 1996;O'Gara and Kaplan, 1997). Inactivation of the PrrBA twocomponent regulatory system (RegBA in R. capsulatus) in a Cco null mutant is dominant to the Cco-minus phenotype , suggesting that the PrrBA system resides downstream, but within the same signal transduction pathway as the cbb 3 oxidase (Fig.…”

Global Regulation of Photosynthesis and Respiration by FnrL

“…A model derived from early studies mainly based on analyses of the Fnr mutants and promoter activity measurements in R. sphaeroides was proposed (31,32). From these studies it was inferred that Fnr acts as a positive regulator of genes for heme biosynthesis, 5-aminolevulinic acid synthase (hemA) and the coprophorphyrinogen III oxidases (hemZ and hemN), for bacteriochlorophyll synthesis bchEJG operon, and for the pucBA operon encoding the polypeptides of the light-harvesting complex LHII (11,14,(32)(33)(34). In this organism the fnrL gene was shown to regulate respiratory genes including the ccoNOQP operon encoding the microaerobic cbb 3 cytochrome oxidase and the rdxBHIS genes involved in cytochrome cbb 3 biogenesis (35,36).…”