Cloning, sequencing, and oxygen regulation of the Rhodobacter capsulatus alpha-ketoglutarate dehydrogenase operon

Abstract

The Rhodobacter capsulatus sucA, sucB, and lpd genes, which encode the ␣-ketoglutarate dehydrogenase (E1o), the dihydrolipoamide succinyltransferase (E2o), and the dihydrolipoamide dehydrogenase (E3) components of the ␣-ketoglutarate dehydrogenase complex (KGD), respectively, were cloned, sequenced, and used for regulatory analyses. The KGD enzymatic activity was greater in cells grown under aerobic, respiratory growth conditions than under anaerobic, photosynthetic conditions. Similarly, the sucA gene was transcribed differentially, leading to a greater accumulation of sucA mRNAs under respiratory growth conditions than under photosynthetic conditions, although differential rates of mRNA decay could also contribute to the different amounts of sucA mRNAs under these two growth conditions. The sucA promoter was located about 4 kb upstream of the 5 end of the sucA gene, and transcripts greater than 9.5 kb hybridized to a sucA probe, suggesting the presence of an operon that produces a polycistronic mRNA. Thus, these genes seem to be expressed as an unstable primary transcript, and we speculate that posttranscriptional processes control the stoichiometry of KGD proteins.Rhodobacter capsulatus is a purple nonsulfur phototrophic bacterium capable of growth by a number of different metabolic processes, including aerobic respiration and anaerobic photosynthesis (21,22). During aerobic respiratory growth, R. capsulatus uses the citric acid cycle (CAC) to produce reducing equivalents for ATP synthesis by oxidative phosphorylation (4, 12). When the oxygen tension in the growth environment drops below a critical concentration, cells of R. capsulatus respond by modification of metabolic pathways and synthesis of differentiated membrane structures for photosynthetic energy transduction (14). Although the photosynthetic physiology has been much studied, little is known of the regulation of aerobic metabolism in R. capsulatus.The KGD enzyme complex catalyzes the oxidative decarboxylation of ␣-ketoglutarate to succinyl coenzyme A (succinylCoA), which links the tricarboxylic acid branch (conversion of oxaloacetate plus acetyl-CoA to ␣-ketoglutarate) to the dicarboxylic acid branch (conversion of succinyl-CoA to oxaloacetate) of the CAC. It has been reported that the activities of KGD and other enzymes involved in respiration, although present, are lower in cultures of R. capsulatus grown under anaerobic photosynthetic growth conditions than under aerobic conditions (4, 12). These changes in KGD and other respiratory enzyme activities suggest that although the CAC functions under photosynthetic growth conditions, its activity is reduced because it is required only for the generation of biosynthetic precursors.The KGD enzyme complex has been purified from several eukaryotes and prokaryotes and is invariably composed of three proteins: ␣-ketoglutarate dehydrogenase (E1o), dihydrolipoyl succinyltransferase (E2o), and dihydrolipoamide dehydrogenase (E3). These three proteins form a large complex with a molecular weight in excess of 1...

Molecular Characterization of thesucBGene Encoding the Immunogenic Dihydrolipoamide Succinyltransferase Protein ofBartonella vinsoniisubsp.berkhoffiiandBartonella quintana

“…The DNA sequence of the B. vinsonii subsp. berkhoffii cloned insert showed a similar gene arrangement as that described in Rhodobacter capsulatus and E. coli (17,44).…”

Cloning, Nucleotide Sequence, and Expression of the Brucella melitensis sucB Gene Coding for an Immunogenic Dihydrolipoamide Succinyltransferase Homologous Protein

“…3. This organization appears to be similar to that of Rhodobacter capsulatus and Rhizobium leguminosarum, in which the lpd gene is also located downstream of the sucB gene (5,19). This organization is different from that of E. coli, in which sucC and sucD are located between the sucB and lpdA genes (12,14).…”

“…Recently, two-dimensional electrophoresis, immunoblotting, and N-terminal microsequencing have considerably facilitated the identification of immunogenic proteins in ovine brucellosis (15,16). Among the proteins identified by these methods, one with an apparent molecular mass of 45 kDa was recognized by sera from Brucella-infected sheep, and its N-terminal sequence showed homology to a dihydrolipoamide succinyltransferase (SucB) described in many bacteria (2,5,9,13,19). A monoclonal antibody (MAb) was raised against this protein (16) to allow easy screening of genomic libraries to clone the corresponding gene.…”

The oxidant‐responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)‐NADP(H) reductase

“…When illuminated in anaerobiosis, these microorganisms adopt a photosynthetic lifestyle, generating adenosine triphosphate (ATP) through an anoxygenic electron transport around a single photosystem [1]. On exposure to air, they shift to a respiratory metabolism after de novo synthesis of terminal oxidases and dehydrogenases [2], whereas expression of the pigment binding proteins encoded by the puf and puc operons is repressed, leading to progressive disappearance of reaction centers and antennae [3].…”