Biomaterial Database

Cloning and expression of the Rickettsia prowazekii ADP/ATP translocator in Escherichia coli. 1985

D C Krause, and H H Winkler, and D O Wood

Cosmid clone banks of Rickettsia prowazekii genomic DNA were established in Escherichia coli and screened for expression of the rickettsial carrier-mediated ADP/ATP translocator. Out of 2700 clones screened, a single clone, designated MOB286, accumulated radioactivity when incubated with [alpha-32P]ATP in 100 mM sodium phosphate buffer. This clone carried a plasmid, pMW286, containing a 9-kilobase-pair insert of rickettsial DNA, as established by DNA X DNA hybridizations. Transformation studies with purified pMW286 established that the ability of E. coli cells to accumulate radioactivity was mediated by the recombinant plasmid. Results from experiments in which [3H]ATP was substituted for [alpha-32P]ATP strongly suggested that the radiolabeled ATP was transported intact. Furthermore, [3H]ATP was incorporated into 10% (wt/vol) trichloroacetic acid-precipitable material in a time-dependent manner. Uptake of ATP was also temperature-dependent, insensitive to atractyloside, N-ethylmaleimide, and dinitrophenol, and specific for ADP and ATP. Efflux of radiolabeled nucleotide was observed in the presence of extracellular ADP or ATP but not AMP and was not observed in the absence of extracellular adenine nucleotides. The successful cloning and expression of the rickettsial ADP/ATP translocator in E. coli will permit better characterization of rickettsial bioenergetics and of the metabolic regulation of obligate intracellular parasitism.

UI	MeSH Term	Description	Entries
D009713	Nucleotidyltransferases	A class of enzymes that transfers nucleotidyl residues. EC 2.7.7.	Nucleotidyltransferase
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
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
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
D000226	Mitochondrial ADP, ATP Translocases	A class of nucleotide translocases found abundantly in mitochondria that function as integral components of the inner mitochondrial membrane. They facilitate the exchange of ADP and ATP between the cytosol and the mitochondria, thereby linking the subcellular compartments of ATP production to those of ATP utilization.	ADP,ATP Carrier,ADP,ATP Translocator Protein,Adenine Nucleotide Translocase,ADP Translocase,ATP Translocase,ATP,ADP-Carrier,ATP-ADP Translocase,Adenine Nucleotide Carrier (Mitochondrial),Mitochondrial ADP-ATP Carriers,ADP-ATP Carriers, Mitochondrial,Mitochondrial ADP ATP Carriers
D000255	Adenosine Triphosphate	An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter.	ATP,Adenosine Triphosphate, Calcium Salt,Adenosine Triphosphate, Chromium Salt,Adenosine Triphosphate, Magnesium Salt,Adenosine Triphosphate, Manganese Salt,Adenylpyrophosphate,CaATP,CrATP,Manganese Adenosine Triphosphate,MgATP,MnATP,ATP-MgCl2,Adenosine Triphosphate, Chromium Ammonium Salt,Adenosine Triphosphate, Magnesium Chloride,Atriphos,Chromium Adenosine Triphosphate,Cr(H2O)4 ATP,Magnesium Adenosine Triphosphate,Striadyne,ATP MgCl2
D001693	Biological Transport, Active	The movement of materials across cell membranes and epithelial layers against an electrochemical gradient, requiring the expenditure of metabolic energy.	Active Transport,Uphill Transport,Active Biological Transport,Biologic Transport, Active,Transport, Active Biological,Active Biologic Transport,Transport, Active,Transport, Active Biologic,Transport, Uphill
D012283	Rickettsia prowazekii	A species of gram-negative, aerobic bacteria that is the etiologic agent of epidemic typhus fever acquired through contact with lice (TYPHUS, EPIDEMIC LOUSE-BORNE) as well as Brill's disease.