JOURNAL OF BACTERIOLOGY, JUlY 1987, P. 2977-2983 Vol. 169, No. 7 0021-9193/87/072977-07$02.00/0 Copyright ) 1987, American Society for Microbiology Genetic and Physiological Characterization of Bacillus subtilis Mutants Resistant to Purine Analogs HANS H. SAXILD AND PER NYGAARD* Enzyme Division, University Institute ofBiological Chemistry B, DK-1307 Copenhagen K, Denmark Received 14 November 1986/Accepted 1 April 1987 BaciUus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosylbransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively. The sporeforming gram-positive bacterium Bacillus sub- sporulation, a shrinkage of the intracellular pools of GDP tilis is able to synthesize purine nucleotides from smaller and GTP occurs under different experimental conditions (8, metabolites via the purine de novo pathway and to reutilize 18, 26). The purpose of the present study was to gain insight nucleosides and nucleobases via the purine salvage path- into purine base and nucleoside utilization. We report the ways (24) (Fig. 1). The preformed purine compounds may be isolation and the physiological and genetic characterization formed endogenously from the turnover of nucleic acids, of mutants of B. subtilis resistant to either 2-fluoroadenine, from nucleic acids taken up, or as degradation products in 8-azaguanine, or 8-azaxanthine. The results obtained have the surroundings from decaying cells. B. subtilis possesses contributed to our knowledge ofthe purine salvage pathways both extracellular and intracellular nucleotidases (6, 24). The and transport systems of B. subtilis. utilization of nucleosides as carbon or nitrogen sources is of minor importance in B. subtilis (9). The purine salvage pathways are well documented in many microorganisms (24) MATERIALS AND METHODS and have been investigated in some detail in B. subtilis (7). The B. used in this While the purine de novo pathway appears to be almost Organisms and media. strains of subtilis identical in the various organisms studied, the salvage path- study are listed in Table 1. The minimal medium used was ways seem far more diverse, as defined by the biochemical Spizizen salts (29) supplemented with 5 F.M MnSO4, 1 ,ug of reactions involved in purine utilization (24). Even among the thiamine hydrochloride per ml, 0.2% L-glutamic acid, and various bacilli there are major differences in how exogenous 0.4% D-glucose. Minimal agar plates contained Spizizen purine compounds are metabolized (11, 24). By comparison, salts, 5 ,uM MnSO4, 1 ,ug of thiamine hydrochloride per ml, less is known about the transport of purine bases and 0.4% glycerol, and 1.6% agar. MG1 medium was Spizizen nucleosides in microorganisms (3, 21). One major obstacle salts, S ,uM MnSO4, 1 p.g of thiamine hydrochloride per ml, has been the problem of separating transport from subse- 0.5% D-glucose, 0.2% yeast extract, and an amino acid quent intracellular conversion. mixture known to enhance competence in B. subtilis (30): L-histidine, L-tryptophan, L-argiline, L-valine, L-lysine, L- Attempts to elucidate purine transport systems in B. threonine, L-glycine, L-asparagine, and L-methionine (all at subtilis have revealed the presence of several systems (3, 50 ,ug/ml). MG2 medium was MG1 supplemented with 2 mM 21). Useful tools for the identification of purine salvage MgCl2 and 0.5 mM CaCl2. As complex media, L-broth and pathways and purine transport systems are the use of brain-heart infusion broth (Difco Laboratories, Detroit, mutants defective in specific steps of the pathways or Mich.) were used. Nutrients required by auxotrophic strains transport systems and of analogs which specifically inhibit were added at a final concentration of 50 ,ug/ml for amino purine metabolism. Purine analogs which are not toxic until acids, 1 p.g/ml for vitamins, and 30 ,ug/ml for punne and they have been converted to nucleotides have been used pyrimidine compounds unless stated otherwise. extensively in microorganisms to select purine salvage and Growth conditions. Cells were cultured overnight at 30°C transport mutants (21, 24). Purine metabolism in B. subtilis is and diluted in the same medium to exponential growth at of particular interest because purine compounds appear to 37°C. Growth was followed by measuring the optical density play a key role in the initiation of sporulation (26, 28). Freese (OD) at 436 nm in an Eppendorf photometer. An optical and colleagues have shown that during the initiation of density of 1 (1-cm light path) corresponds to 0.2 mg (dry weight) of bacteria per ml. Resistance to 2-fluoroadenine (2 * Corresponding author. ,uM), 8-azaguanine (0.5 mM), 8-azaxanthine (2 mM), and 2977 2978 SAXILD AND NYGAARD J. BACTERIOL. ATP---- AICAR-----PRPP GTP Histidine 0 ADP FAICAW~I guaC r.npuuLJI I purE purA grgguaA I AMP *- sAMP *- IMP XMP _-. GMP ujaF Ixpt guaF pupA tapt adeC to guaP guaP Ado - Ade - Hyp * Ino Xan Gua .- Guo <-tt Interior t 4 4- 4 4 I j(3) (1) Exterior IHpuG 1(2) (4) pbuG 1(2) Ado Ade Hyp Ino Xan Gua Guo FIG. 1. Proposed pathways for purine salvage and transport in B. subtilis. The individual reactions are identified by gene symbols. Symbols in italics indicate mapped mutations. Numbers indicate transport systems identified by Beaman et al. (3). AICAR, Aminoimidazolecarboxamide ribonucleotide; FAICAR, formamidoimidazolecarboxamide ribonucleotide; purA, adenylosuccinate synthe- tase; purE, adenylosuccinate lyase; guaA, IMP dehydrogenase; guaB, GMP synthetase; guaC, GMP reductase; apt, adenine phosphoribosyltransferase; xpt, xanthine phosphoribosyltransferase; guaF, guanine-hypoxanthine phosphoribosyltransferase; adeC, adenine deaminase; pupA, adenosine phosphorylase; guaP, guanosine (inosine) phosphorylase; pbuG, purine base uptake (guanine/hypoxanthine). Numbers: 1, Adenine transport system; 2, guanosine (inosine) transport system; 3, adenosine transport system; 4, xanthine transport system. Other abbreviations: see Table 2, footnote b, and Table 3, footnote a. 4-azaleucine (0.2 mM) was scored on minimal agar plates fresh warmed medium, and suspended in 15 ml of warm containing the appropriate analog. The tsi and dnaD markers medium. After 5 min of incubation, 1 ml of culture was were scored on L-broth plates at 48 and 42°C, respectively. transferred to a tube containing the appropriate "C-labeled The tre marker was scored on minimal plates containing purine compound at a final concentration of 1 ,uM (50 0.4% trehalose as the carbon source. Ci/mol). At 15, 30, 45, and 60 s, 200-,ul samples were Chemicals and isotopes. Fine chemicals were purchased withdrawn, filtered through a membrane filter, and washed from Sigma Chemical Co., St. Louis, Mo. 14C-labeled com- with 2.5 ml of medium. The filter was dried, and radioactiv- pounds were purchased from Amersham International, ity was determined by liquid scintillation counting. For Amersham, England. 2-Fluoroadenine was kindly donated uptake measurements in purine auxotrophic strains, the cells by M. W. Taylor (Indiana University, Bloomington, Ind.). were suspended in minimal medium lacking purine, and Carrier-free [32P]phosphoric acid was from Fors0gsanlaeg, uptake was determined immediately thereafter. Ris0, Denmark. Nucleoside triphosphate and PRPP pools. ATP, GTP, and Enzyme assays. Enzyme analyses were carried out at 37°C. PRPP pools were determined as described before (14) in cells Protein was determined by the method of Lowry et al. (20). cultured in low-phosphate medium (23) in the presence of Phosphoribosylpyrophosphate (PRPP) amidotransferase and 32P, (10 Ci/mol). Incorporation of [8-14C]adenine and [8- phosphoribosylglycinamide (GAR) synthetase activities "4C]adenosine into ATP and GTP was performed with cells were measured as described by Houlberg and Jensen (10). growing in minimal medium containing histidine (100 ,uglml). Activities of adenine, guanine, hypoxanthine, and xanthine At an OD436 of 0.5, the labeled compound was added to give phosphoribosyltransferases, adenosine deaminase, and pu- a final concentration of 50 ,uM (5 Ci/mol). At an OD436 of 1, rine nucleoside kinases were assayed as described before 25-,ul samples were withdrawn for nucleotide pool analysis (15, 25). Purine nucleoside phosphorylase activity was de- (14). termined by the method of Jensen (13). AMP deaminase Transduction and phage production. The generalized trans- activity was determined as described by Murakami (22), and ducing phage AR9, which is related to the phage PBS1, was GMP synthetase activity as described by Lambden and used (19). Fifty microliters of producer lysate (propagated in Drabble (16). Adenase activity, the deamination of adenine Bacillus pumilus) and 50 ,ul of bacteria grown in L-broth to hypoxanthine, was measured in whole cells concentrated were added to 5 ml of brain-heart infusion broth and incu- to an OD436 of 3 to 5 in 0.1 M Tris hydrochloride (pH 7.6) bated for 24 h with shaking and then for 24 h without shaking containing 0.5 mM [8-14C]adenine (5 Ci/mol).