Biomaterial Database

Cloning and expression in Escherichia coli of sdhA, the structural gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex. 1985

K Magnusson, and L Hederstedt, and L Rutberg

Bacillus subtilis cytochrome b558 is a transmembrane protein which anchors succinate dehydrogenase (SDH) to the cytoplasmic membrane and is reduced by succinate. The structural gene for this cytochrome was cloned and expressed in Escherichia coli. Random BamHI or BglII fragments of B. subtilis 168 DNA were cloned in the BamHI site of plasmid pHV32. The derived plasmids were used to transform B. subtilis SDH mutants to chloramphenicol resistance by integration of the plasmid via DNA homology. Of some 3,000 transformants tested, 6 were SDH positive and had pHV32 integrated close to the sdh operon. Two plasmids, pKIM2 and pKIM4, with an insert of B. subtilis DNA of 5.7 and 3.4 kilobases, respectively, were generated by transforming E. coli with DNA from the SDH-positive transformants after cleavage with EcoRI or BglII and ligation. In E. coli carrying either of the two plasmids, about 4% of total membrane protein was B. subtilis cytochrome b558. E. coli (pKIM2) also contained antigen which reacted with antibodies specific for the flavoprotein and the iron-sulfur protein subunit of B. subtilis SDH. Enzymatically active, membrane-bound B. subtilis SDH could not be demonstrated in E. coli (pKIM2). The B. subtilis DNA insert in pKIM2 could transform B. subtilis sdhA (cytochrome b558), sdhB (flavoprotein), and sdhC (iron-sulfur protein) mutants to the wild type. The results suggest that pKIM2 carries the whole B. subtilis sdh operon. The data confirm the gene order and the proposed direction of transcription of the B. subtilis sdh operon. Most likely the sdh genes in E. coli(pKIM2) are controlled by their natural promoter.

UI	MeSH Term	Description	Entries
D009876	Operon	In bacteria, a group of metabolically related genes, with a common promoter, whose transcription into a single polycistronic MESSENGER RNA is under the control of an OPERATOR REGION.	Operons
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D003573	Cytochrome b Group	Cytochromes (electron-transporting proteins) with protoheme (HEME B) as the prosthetic group.	Cytochromes Type b,Cytochromes, Heme b,Group, Cytochrome b,Heme b Cytochromes,Type b, Cytochromes,b Cytochromes, Heme,b Group, Cytochrome
D004251	DNA Transposable Elements	Discrete segments of DNA which can excise and reintegrate to another site in the genome. Most are inactive, i.e., have not been found to exist outside the integrated state. DNA transposable elements include bacterial IS (insertion sequence) elements, Tn elements, the maize controlling elements Ac and Ds, Drosophila P, gypsy, and pogo elements, the human Tigger elements and the Tc and mariner elements which are found throughout the animal kingdom.	DNA Insertion Elements,DNA Transposons,IS Elements,Insertion Sequence Elements,Tn Elements,Transposable Elements,Elements, Insertion Sequence,Sequence Elements, Insertion,DNA Insertion Element,DNA Transposable Element,DNA Transposon,Element, DNA Insertion,Element, DNA Transposable,Element, IS,Element, Insertion Sequence,Element, Tn,Element, Transposable,Elements, DNA Insertion,Elements, DNA Transposable,Elements, IS,Elements, Tn,Elements, Transposable,IS Element,Insertion Element, DNA,Insertion Elements, DNA,Insertion Sequence Element,Sequence Element, Insertion,Tn Element,Transposable Element,Transposable Element, DNA,Transposable Elements, DNA,Transposon, DNA,Transposons, DNA
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005796	Genes	A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms.	Cistron,Gene,Genetic Materials,Cistrons,Genetic Material,Material, Genetic,Materials, Genetic
D000942	Antigens, Bacterial	Substances elaborated by bacteria that have antigenic activity.	Bacterial Antigen,Bacterial Antigens,Antigen, Bacterial
D001412	Bacillus subtilis	A species of gram-positive bacteria that is a common soil and water saprophyte.	Natto Bacteria,Bacillus subtilis (natto),Bacillus subtilis subsp. natto,Bacillus subtilis var. natto
D013385	Succinate Dehydrogenase	A flavoprotein containing oxidoreductase that catalyzes the dehydrogenation of SUCCINATE to fumarate. In most eukaryotic organisms this enzyme is a component of mitochondrial electron transport complex II.	Succinic Oxidase,Fumarate Reductase,Succinic Dehydrogenase,Dehydrogenase, Succinate,Dehydrogenase, Succinic,Oxidase, Succinic,Reductase, Fumarate
D019255	NADPH Oxidases	A family of membrane-associated flavoprotein NADPH-dependent oxidoreductases that catalyze the univalent reduction of OXYGEN to create SUPEROXIDES. Structurally, they are characterized by six N-terminal transmembrane ALPHA-HELICES, a FLAVIN-ADENINE DINUCLEOTIDE (FAD)-binding region, and a C-terminal NADPH-binding region. They are expressed primarily by EPITHELIAL CELLS in gut, kidney, colon, and smooth muscle tissues, as well as GRANULOCYTES and function to transfer electrons across membranes to molecular oxygen. Defects in the production of superoxide ions by some NADPH oxidases result in GRANULOMATOUS DISEASE, CHRONIC.	NADPH Oxidase,NAD(P)H Oxidases,NAD(P)H oxidase,Nox Proteins,Oxidase, NADPH,Oxidases, NADPH