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Cloning and Expression of the Rickettsia Prowazekii ADP

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Cloning and Expression of the Rickettsia Prowazekii ADP Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3015-3019, May 1985 Microbiology Cloning and expression of the Rickettsia prowazekii ADP/ATP translocator in Escherichia coli (obligate intracellular parasite/carrier-mediated exchange/recombinant DNA) DUNCAN C. KRAUSE, HERBERT H. WINKLER, AND DAVID 0. WOOD* Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688 Communicated by Saul Roseman, December 17, 1984 ABSTRACT Cosmid clone banks of Rickettsia prowazekii terization, have been unsuccessful (unpublished observa- genomic DNA were established in Escherichia coli and tion). The cloning and expression in Escherichia coli of the screened for expression of the rickettsial carrier-mediated gene(s) responsible for ADP/ATP exchange would permit ADP/ATP translocator. Out of 2700 clones screened, a single circumvention of these problems. Purification of the translo- clone, designated MOB286, accumulated radioactivity when cator would be facilitated immensely, modulation of this ac- incubated with [a-32P]ATP in 100 mM sodium phosphate tivity by various effectors could be studied against a well- buffer. This clone carried a plasmid, pMW286, containing a 9- defined E. coli physiological background, membrane vesi- kilobase-pair insert of rickettsial DNA, as established by cles could be formed, and gene organization and regulation DNADNA hybridizations. Transformation studies with puri- could be assessed. We report here the cloning in E. coli of a fied pMW286 established that the ability of E. coli cells to ac- R. prowazekii genetic locus that directs the expression in E. cumulate radioactivity was mediated by the recombinant plas- coli of the rickettsial ADP/ATP translocator. mid. Results from experiments in which [3H]ATP was substi- tuted for [a-32PJATP strongly suggested that the radiolabeled ATP was transported intact. Furthermore, [3H]ATP was in- MATERIALS AND METHODS corporated into 10% (wt/vol) trichloroacetic acid-precipitable Enzymes and Reagents. Ampicillin, Tris, adenine, adeno- material in a time-dependent manner. Uptake of ATP was also sine, AMP (sodium salt, type II), ADP (sodium salt, grade temperature-dependent, insensitive to atractyloside, N-ethyl- IX), ATP (disodium salt), atractyloside (disodium salt), thia- maleimide, and dinitrophenol, and specific for ADP and ATP. mine, EDTA, and boric acid were purchased from Sigma. N- Efflux of radiolabeled nucleotide was observed in the presence ethylmaleimide, 2,4-dinitrophenol, and trichloroacetic acid of extracellular ADP or ATP but not AMP and was not ob- were purchased from Fisher. Agarose, restriction endonu- served in the absence of extracellular adenine nucleotides. The cleases, dithiothreitol, and T4 DNA ligase were obtained successful cloning and expression of the rickettsial ADP/ATP from Bethesda Research Laboratories. Nitrocellulose filters translocator in E. coli will permit better characterization of were purchased from Schleicher & Schuell. Membrane fil- rickettsial bioenergetics and of the metabolic regulation of ob- ters (HAWPO25, 0.45-ptm pore size) were purchased from ligate intracellular parasitism. Millipore. [2,8,5'-3H]ATP was purchased from ICN. [a- 32P]ATP was purchased from ICN and New England Nucle- Rickettsia prowazekii, the etiologic agent of epidemic ty- ar. phus, is an obligate intracellular parasite. Whereas other in- Bacterial Strains and Culture Conditions. R. prowazekii tracellular bacteria are generally found surrounded by a strain Madrid E was cultivated in yolk sacs of antibiotic-free membrane of host origin, rickettsiae escape quickly from embryonated chicken eggs and purified as described else- phagocytic vacuoles and grow directly within the cytoplasm where (1). The purification protocol included Renografin (and occasionally the nucleus) of their host cell. Rickettsiae density gradient centrifugation to remove contaminating are not leaky, as had been postulated, but possess both usual yolk sac mitochondria (6). The E. coli strains used in this and unusual transport systems (1-4), most notably, a carrier- study were DH1 (gyrA96 recAl relAl(?) endAl thi-J hsdRJ7 mediated ADP/ATP translocator (1). This activity, similar to glnV44 X- F-) and HB101 (7) (hsdS20 rB mB recAl3 xyl-5 that present in mitochondria, permits the exchange of ADP mtl-l ara-14 proA2 lacYJ galK2 leuB6 rpsL20 thi-l glnV44 from the rickettsia for ATP present in the host cell's cyto- X- F-). The former was obtained from B. Bachmann (E. coli plasm. The carrier is specific for ADP and ATP, operates Genetic Stock Center, New Haven, CT); the latter strain equally well in either direction, has no energy requirement, was supplied by D. Kopecko (Walter Reed Army Institute of and is of an obligatory exchange type-i.e., for every mole- Research, Washington, D.C.). E. coli strains were cultured cule of ATP transported into a rickettsia, a molecule of ADP at 37°C in L broth or on L agar plates (8), except where indi- must be transported from the rickettsia, or vice versa. ADP/ cated. ATP exchange is regulated at least in part by the concentra- Cosmid Clone Bank Formation. The protocol for formation tion of inorganic phosphate (5). of cosmid clone banks of the R. prowazekii genome has been Characterization of the ADP/ATP translocator is funda- published in detail (9). Briefly, rickettsial DNA was extract- mental to gaining an understanding of rickettsial physiology ed according to Myers and Wisseman (10) and purified by and the regulation thereof. This task is complicated enor- CsCl density gradient equilibrium centrifugation. The cos- mously by the difficulty in obtaining large quantities of high- mid vector pHC79 (11) was purified according to Clewell and ly purified rickettsiae due to the required intracellular propa- Helinski (12). Sau3A-digested rickettsial DNA was inserted gation of these bacteria, usually in embryonated hen eggs. into the BamHI site of pHC79. The ligated DNA was pack- Furthermore, attempts to form membrane vesicles from R. aged into capsids by using packaging extracts prepared from prowazekii, which would be very important in this charac- E. coli strains BHB2688 and BHB2690 (13, 14) as described by Davis et al. (15). E. coli DH1 was cultured in L broth The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: kb, kilobase pair(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3015 Downloaded by guest on September 25, 2021 3016 Microbiology: Krause et al. Proc. NatL Acad Sci. USA 82 (1985) containing maltose rather than glucose, infected with the R. prowazekii was included as a positive control in well 1A, packaged recombinant cosmids, and cultured on L agar con- while E. coli DH1 was present in 12G. The intensity of 9B in taining ampicillin at 15 ,ug/ml. Ampicillin-resistant colonies this autoradiograph was significantly greater than that of the were stocked individually in 96-well Microtiter dishes in 200 other clones present, the E. coli negative control, and the /.l of L broth containing ampicillin and approximately 10% rickettsial positive control. Variability in the intensity of the glycerol. positive control from experiment to experiment was a func- In Situ Screening of Clone Banks for ATP Transport. Cos- tion of total rickettsial cell number and viability as indicated mid clones were replica plated to 96-well Microtiter dishes by hemolytic activity (16). containing 200 A.l of L broth with ampicillin (15 pug/ml) per Physical Characterization of the Recombinant Plasmid. well and incubated overnight at 370C. The bacterial suspen- Clone 9B has been designated MOB286, and the recombi- sions were transferred to a 96-well vacuum manifold, in nant plasmid associated with this clone, pMW286. Plasmid which the cells were collected on a nitrocellulose filter (pore DNA was extracted from MOB286 according to Hansen and size 0.45 gum). After two washes with 100 mM sodium phos- Olsen (17) and purified by two successive cesium chloride/ phate buffer, pH 7.2 (PB), suction was removed and 200 g4 ethidium bromide density gradient equilibrium centrifuga- of PB containing [a-32P]ATP (1 p.Ci/ml, 5-25 Ci/mmol; 1 Ci tions. Plasmid pMW286 was digested to completion with re- = 37 GBq) was added per well. The cells were incubated in striction endonuclease HindIlI and analyzed by agarose gel the presence of radiolabeled ATP for 20 min at room tem- electrophoresis (Fig. 2A). HindIII restriction fragments to- perature, after which the vacuum was restored and the filters taling approximately 15.4 kilobase pairs (kb) were observed were washed four times with PB, using approximately 0.5 ml (Fig. 2A, lane d). HindIII digestions of R. prowazekii geno- per wash. The filters were then removed from the manifold mic DNA, E. coli DNA, and chicken egg yolk sac DNA were and placed on blotting paper saturated with PB for an addi- included for comparison. The DNA shown in Fig. 2A was tional 15-20 min to permit removal of radioactivity that transferred to nitrocellulose paper according to Southern might have seeped between the wells. After air-drying, the (18) and probed with pMW286 biotinylated by nick-transla- filters were heated to 80'C for 45 min and exposed to x-ray tion (19), using a kit obtained from Bethesda Research Labo- film (Kodak X-Omat RP) overnight. Autoradiography was ratories. Hybridization was carried out under conditions re- enhanced with an intensifier screen. Transport of ATP and quiring greater than 90% DNA sequence homology. No hy- exchange of ADP and ATP by E. coli clones or R. prowazekii bridization was observed to E. coli or yolk sac DNA. Some were assayed in uptake experiments as detailed elsewhere hybridization to bacteriophage X DNA was observed and (1). Briefly, E. coli cells were cultured to late logarithmic probably can be attributed to the X cos site in the cosmid phase (optical density at 600 nm of 0.6-0.7) in L broth with vector pHC79.
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