JOUNAL OF BACTERIOLOGY, Oct. 1980, p. 465-467 Vol. 144, No. 1 0021-9193/80/10-0465/03$02.00/0 Partial Purification and Characterization of an Exonuclease from Xanthomonas oryzae MICHAEL B. FARBERt AND MELANIE EHRLICH* Department ofBiochemistry, Tulane University School ofMedicine, New Orleans, Louisiana 70112 An exonuclease with a strong preference for single-stranded deoxyribonucleic acid over double-stranded deoxyribonucleic acid has been purified 500-fold from Xanthomonas oryzae. This enzyme liberates 5'-mononucleotides in a reaction which requires Mg2+. Xanthomonas oryzae is a relatively little- DNAs was monitored by electrophoresis on 1% studied bacterium and a plant pathogen in the agarose gels. These DNAs were not substrates family Pseudomonadaceae. The DNases of X. for either of the two restriction endonucleases oryzae were of special interest for two reasons. isolated from X. oryzae (16). Also, no endonucle- First, this bacterium is the host for bacterio- ase activity was detected when up to 75 ,ug of phage XP-12, the only known naturally occur- protein in the DNA-free extracts of X. oryzae ring source of DNA in which all of the cytosine was assayed in the DNA-containing polyacryl- residues are methylated in the 5-position (2, 7). amide gel electrophoresis system of Rosenthal XP-12 DNA has 34 mol% 5-methylcytosine, and Lacks (13) with either calf thymus DNA or whereas X. oryzae DNA has 32 mol% cytosine XP-12 DNA as the substrate. The gels were and <0.5 mol% 5-methylcytosine (2, 7). Second, incubated for 72 h at 370C to try to detect in a study of its restriction endonucleases, the enzyme activity, at which time 0.1 ng of DNase level of general endonuclease activity in crude I was easily detected. extracts ofX. oryzae was found to be surprisingly X. oryzae exonuclease was shown to be a low (16). The present study is the first reported DNase by its solubilization of DNA from several analysis of nucleases other than restriction en- sources but not of [3H]RNA isolated from HeLa donucleases (3, 5, 19) in a member of the genus cells. The enzyme showed approximately 5- to Xanthomonas. 20-fold preference for denatured as opposed to We partially purified the major exonucleolytic native DNA (4). With readdition of enzyme, up DNase from X. oryzae, as described in Table 1. to 80% of single-stranded X. oryzae DNA but This DNase is an exonuclease as shown by its only 4.5% of double-stranded X. oryzae DNA acid-solubilization of DNA without a lag period was made acid soluble. Pretreatment of X. ory- and its inability to convert covalently closed, zae DNA with Si nuclease reduced the exonu- double-stranded simian virus 40 DNA to linear clease activity on this native DNA more than or open circular forms. Also, when denatured or 10-fold. Therefore, most or all of the hydrolysis native DNA was incubated with high concentra- of native X. oryzae DNA can be attributed to tions of the enzyme and chromatographed on a single-stranded regions in the DNA, which could Bio-Gel P-100 column, only a low-molecular- be removed by predigestion with the single weight peak in the position of mononucleotides strand-specific Si nuclease (15, 18). and a high-molecular-weight peak in the ex- An absolute requirement for Mg2", which cluded volume were seen. could not be replaced by Ca2" or Mn2" was The only other detected nuclease activity ca- observed (Table 2). The presence of a sulfhy- pable of solubilizing native, denatured, or exten- dryl-protective reagent in the reaction buffer sively DNase I-nicked DNA was a minor activity enhanced enzymatic activity. Monodeoxynu- not retained by the phosphocellulose column in cleotides did not inhibit the reaction (Table 2). step IV and was not further analyzed. Unlike several characterized bacterial DNases No endonuclease activity was detected in the (1, 6, 11), ATP did not stimulate the reaction DNA-free extract or in the DEAE-cellulose- (Table 2). Only at high concentrations of tRNA chromatographed fractions when X. oryzae, was the exonuclease appreciably inhibited, un- phage XP-12, and phage T7 DNAs were used as like E. coli endonuclease I (9). The exonuclease substrates and the molecular weight of the reaction displayed a relatively broad pH opti- mum from approximately pH 7.0 (in phosphate t Present address: Department of Neurology, University of buffer) to 7.5 (in Tris-hydrochloride buffer). Al- California, San Francisco, CA 94143. though the optimum temperature for growth of 465 466 NOTES J .. BACTERIOL. TABLE 1. Purification ofX. oryzae exonuclease Spact Fractiona Total unitsb Recovery (%) (U/mgtein) of pro- I. Crude low-speed supernatant 730 62 0.306 II. DNA-free extract 1,170 (100) 1.26 III. DEAE-cellulose column eluate 1,040 89 2.32 IV. Phosphoceliulose column eluate 970 83 95.8 V. DEAE-Sephadex A-25 column eluate 710 61 159 a Crude and DNA-free extracts were prepared from X. oryzae as previously described (4) except that in this case uninfected cells were used. Chromatography on DEAE-cellulose, phosphoceliulose, and DEAE-Sephadex chromatography was performed as in previous study of an XP-12 phage-induced nuclease (4) except that a 0 to 1.0 M KCI gradient in 2 mM 2-mercaptoethanol-0.1 mM EDTA-10% glycerol-10 mM potassium phosphate (pH 7.4) was used to elute the X. oryzae exonuclease activity from the phosphocellulose column. b The standard nuclease assay mixture consisted of 10 mM MgCl2, 0.1 mM EDTA, 0.5 mg of bovine serum albumin per ml (Miles, Pentex Fraction V), 1 mM dithiothreitol, 50 mM Tris-hydrochloride (pH 7.45,at 37°C), and 2.3 nmol (nucleotide) of denatured X. oryzae [3H]DNA typically containing 5,000 to 10,000 cpm in a total volume of 125 Il and approximately 0.7 U of enzyme. The assay is linear from 0.2 to 5 U of enzyme per assay. The reaction mixtures were incubated for 1 h at 37°C, and then acid-soluble radioactivity was assayed (4). One unit of enzyme solubilizes 1 nmol of nucleotide residue in 1 h under standard assay conditions. TABLE 2. Requirements for activity of the nuclease X. oryzae is 300C, the exonuclease was more Activity active at 37 or 420C than at 30°C (Table 2). relative We examined the low-molecular-weight prod- to that ucts of digestion of denatured X. oryzae [3H]- Reaction under un- content standard DNA upon incubation with the exonuclease condi- til 33% of the radioactivity was made acid solu- tiona ble. After treatment with 5'-nucleotidase (14), (%) essentially all of the low-molecular-weight, ra- Standard ...... 100 dioactively labeled material migrated upon thin- layer chromatography (4) as mononucleosides. Assay at: Without incubation with 5'-nucleotidase, 67% of 300C ...... 64 the low-molecular-weight products chromato- 420C ...... 95 graphed as mononucleotides, but 23% co-chro- 500C ...... 3 matographed as nucleosides. The latter products MgCl2 omitted ..<...... <2 could be due to phosphatase or nucleotidase MgCl2 reduced to 2 mM ...... 70 activity in the exonuclease preparation, since MgCl2 reduced to 5 mM . 115 prolonging the incubation of the DNA with the MgCl2 increased to 20 mM ...... 80 exonuclease resulted in an increase in the ratio 10 mM CaCl2 substituted for MgCl2 <2 of nucleosides to mononucleotides. The exonu- 10 mM MnCl2 substituted for MgCl2 <2 clease, therefore, liberates 5'-mononucleotides Dithiothreitol omittedb. 78 from single-stranded DNA. DNA partially di- Dithiothreitol omittedb and 10 mM N- gested with DNase I (to 1% or greater acid ethylmaleimide added ...... 1 solubility) also can serve as a substrate for X. Dithiothreitol omittedb and 0.4 mM p-chlo- oryzae exonuclease (4; Table 2) due to the pro- romercuribenzoate added .<2 duction of single-stranded, 5'-phosphoryl or 3'- hydroxyl ends (17). However, a comparable ex- 5'-ATP added to 100,uM ..... 100 tent of digestion with micrococcal nuclease, 5'-dTMP added to 400uM .. 115 which produces 5'-hydroxyl, 3'-phosphoryl ter- DNA to tRNAC added: mini (12), decreased the ability of serve 0.2 jg ...... 103 as a substrate for the exonuclease. With respect 1.5 ug ...... 76 to the above properties, this exonuclease firom 15.4 jg ...... 21 X. oryzae resembles E. coli exonucleaae 1 (8). 53.8 Lg ...... 6 Denatured -or DNase 1-nicked DNA from phage XP-12, in which all of the a Reactions were performed under standard condi- Xanthomona8 tions as described in Table 1. cytosine residues are methylated at the 5-posi. b Although no dithiothreitol was present in the re- tion (2, 7), is a substrate for the exonuclease but action buffer, there was 0.07 mM 2-mercaptoethanol is a several-fold poorer one than are correspond- present due to 2 mM 2-mercaptoethanol in the enzyme ing preparations ofX. oryzae or phage T7 DNA, sample. (4), which 'are normal, not highly modified CE. coli tRNA (type XXI, Sigma). DNAs (2, 7, 10). In contrast to this host exonu- VOL. 144, 1980 NOTES 467 clease, the exonuclease induced upon infection The deoxyribonucleases ofEscherichia coli. III. Studies of X. on the nature of the inhibition of endonuclease by oryzae by phage XP-12 hydrolyzes XP-12 ribonucleic acid. J. Biol. Chem. 237:829-833. DNA and X. oryzae DNA equally well (4). 10. Lunan, K. D., and R. L. Sinsheimer. 1956. A study of the nucleic acid of bacteriophage T7. Virology 2:455- This work was supported in part by Public Health Service 462. grant CA 19942 from the National Cancer Institute and a 11. Miller, R. V., and A. J. Clark. 1976. Purification and grant from the Edward G. Schlieder Foundation to M.E. properties of two deoxyribonucleases of Pseudomonas aeruginosa. J. Bacteriol. 127:794-802. LITERATURE CITED 12. Ohsaka, A., J.-I. Mutai, and M. Laskowski. 1964. The 1. Anai, M., T. Hirahashi, and Y. Takagi. 1970. 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