JOURNAL or BACTROLOGY, Apr. 1976, p. 294-299 Copyright C 1976 American Society for Microbiology
Vol. 126, No. 1 Printed in U.SA.
Undermethylated Transfer Ribonucleic Acid from a Relaxed Strain of Bacillus subtilis: Construction of the Strain and Analysis of the Transfer Ribonucleic Acid NICOLE KEISEL AND BARBARA VOLD*
Scripps Clinic and Research Foundation, La Jolla, California 92037 Received for publication 21 January 1976
A strain of Bacillus subtilis is described from which undermethylated transfer ribonucleic acid (tRNA) can be obtained. The tRNA's from a methionine-limited culture were compared with those from a control culture with respect to general nucleoside composition, methylated components, and amino acid acceptor activity. The undermethylated tRNA's had the normal amounts of the four major nucleosides, pseudouridine, and 5-methyluridine (ribothymidine), but were deficient in methylated nucleosides other than 5-methyluridine. These methyldeficient nucleosides can be fully remethylated in the presence of the appropriate methylases. Since the majority of the work characterizing undermethylated tRNA's has been done using Escherichia coli, the work with B. subtilis presents some interesting comparisons and offers an alternative substrate for methylase studies.
The genetic locus for the control of the synthesis of ribonucleic acid (RNA) was first described for a strain of Escherichia coli K-12 strain W-6 (3), a derivative of E. coli K-12 strain 58-161 (13). A mutation in this rel locus caused loss of the normal control of RNA synthesis by amino acids which resulted in the continued synthesis of certain classes of RNA even in the absence of a required amino acid. This property was used to obtain preparations of transfer RNA (tRNA) enriched in undermethylated species by starving a methioninerequiring "relaxed" strain for methionine (1).
Growth of cells and extraction of tRNA and aminoacyl-tRNA synthetases. Cultures were grown on Spizizen minimal salts medium (10), supplemented with 0.5% glucose, 40 gug of i-tryptophan per ml, and L-methionine or casein hydrolysate. Precultures were grown with 0.1% casein hydrolysate and the cells were washed twice with sterile minimal medium before addition to the final culture. Cultures were started at an absorbance of 0.1 measured at 660 nm and were grown with shaking at 37 C. Preparations of mixed tRNA's and aminoacyl-tRNA synthetases were prepared as described previously (14). Amino acid acceptor activity. The amino acid acceptor activity for undermethylated and control tRNA's was measured by the aminoacylation of 2 Relaxed strains have also been obtained in two nmole of tRNA in a 200-Ml reaction in the presence other bacterial species, Salmonella typhimu- of 2 to 5 mM adenosine 5'-triphosphate, 10 mM 2rium (5) and Bacillus subtilis (12). However, mercaptoethanol, 2 nmol of "4C-labeled amino acid, the B. subtilis strain did not require methio- 0.8 nmol each of a mixture of 19 amino acids minus nine and thus could not be used to prepare the appropriate amino acid, saturating amounts of undermethylated tRNA's. The relaxed strain of aminoacyl-tRNA synthetases, and the following adB. subtilis, M58, B. subtilis BR16R, was origi- ditional reaction components, depending on the acid: lysine-250 mM sodium cacodylate, pH nally auxotrophic for lysine and tryptophan. By amino and 10 mM MgCl2; alanine and glycine-250 mM transformation, we have introduced a require- 7.9, cacodylate, pH 7.0, 5 mM KCl, and 10 mM ment for methionine and characterized the re- sodium magnesium acetate; methionine-250 mM HEPES sulting strain for growth and the ability to (N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic produce undermethylated tRNA's. acid), pH 7.5, 5 mM KCl, and 10 mM magnesium acetate; phenylalanine, tryptophan, arginine, vaMATERIALS AND METHODS line, leucine, isoleucine, and serine-250 mM Materials. Rifampin, B grade, and phosphodies- tris(hydroxymethyl)aminomethane, pH 7.5, and 10 terase (Russel viper venom, B grade) were obtained mM MgCl; and aspartic acid, asparagine, proline, from Calbiochem. Bacterial alkaline phosphatase histidine, glutamic acid, and tyrosine- 250 mM Nand pancreatic ribonuclease A were obtained from 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, Worthington. Aminex A-6 was purchased from pH 8.4, 5 mM KCl, and 10 mM magnesium acetate. BioRad. Preparation and analysis of nucleosides. tRNA's 194
VOL. 126, 1976
UNDERMETHYLATED tRNA FROM B. SUBTILIS
were degraded by incubation with pancreatic ribonuclease (EC 2.7.7.16), phosphodiesterase (EC 3.1.4.1), and alkaline phosphatase (EC 3.1.3.1), as described by Singhal and Cohn (9), except that incubation was at 37 C for 15 h. The resultant nucleosides were fractionated on an Aminex A-6 column at 50 C by ion-exclusion chromatography, as described by Singhal (8), using 20 mM ammonium carbonate, adjusted to pH 9.80 with NH40H. Test for relaxed phenotype. Recombinants were tested for the relaxed phenotype by a procedure modified from that of Martin (5). Cultures were grown to exponential phase in Spizizen minimal plus 0.5% glucose, 40 ,ug of tryptophan per ml, and 40 ,tg of methionine per ml (or 40 jLg of lysine per ml for the parent). A culture sample with a total absorbance of 0.04 at 660 nm was transferred onto a sterile membrane fi'lter (Millipore Corp.) and placed in a petri dish on minimal agar plus tryptophan for 2 h at 37 C. The filters were transferred to a minimal agar plate containing tryptophan and 8.8 ,mg of ['4C]uridine per ml and incubated for 1 h at 37 C. Filters were then transferred to a plate with nonradioactive uridine for 30 min and then dried, and the radioactivity was determined in toluene scintillation fluid in a liquid scintillation counter.
RESULTS Construction of methionine-requiring, relaxed strain. A relaxed strain, M58, B. subtilis BR16R, has been described by Swanton and Edlin (12). As originally isolated, the strain required lysine and tryptophan but did not require methionine. The strain, given to us by Edlin, was transformed by James Hoch to lysine prototrophy with a saturating concentration of deoxyribonucleic acid from a strain carrying the metB5 allele. The transformation mixture was spread on plates containing tryptophan and methionine, and methionine-requiring transformants were identified by replica plating to plates containing only tryptophan. Methionine-requiring transformants were cloned twice, checked for auxotrophic requirements, and tested for the relaxed phenotype as described in Materials and Methods. The resultant strains had the genotype trpC2 metB5 rel-. We obtained 23 recombinants of this type. Clone no. 27 had the highest level of incorporation of L'4C]uridine under the conditions of the test for the relaxed phenotype and was selected for the remainder of the work reported here. Growth of the relaxed strain and preparation of tRNA enriched in undermethylated species. A growth curve for this relaxed strain is shown in Fig. 1. The upper curve shows the growth of the strain in excess methionine (25 ,ug/ml). The lower curve represents the growth curve in the presence of 2.5 ,g of methionine
295
Hrs of Growth
FIG. 1. Growth curve for a relaxed, methioninerequiring strain, B. subtilis 168 trpC2 metB5 rel(no. 27). Cells were grown as described in Materials and Methods. After 3.5 h ofgrowth, 3.5 p.g of methionine per ml was added to the culture represented in the lower curve.
per ml, which became limiting about halfway through the exponental growth phase. Starvation for methionine resulted in an inhibition of growth which could be reversed by the addition of methionine to the medium. To prepare tRNA enriched in undermethylated species, a culture, started with 2.5 ,ug of Lmethionine per ml, was grown as shown in Fig. 1, and harvested after 3.5 h. A control culture, grown in the presence of 25 ,g of L-methionine per ml, was grown for the same period of time and harvested. The extraction of tRNA is described in Materials and Methods. Analysis of the tRNA prepared from the methionine-limited and control cultures. The methionine-limited culture would be expected to make normal tRNA during growth until the methionine supply had been exhausted. A tRNA preparation isolated from a starved culture, therefore, is a mixture of normally modified and undermethylated tRNA's. To determine the proportion of tRNA that was undermethylated after 3.5 h of growth, nucleosides of tRNA's from methionine-limited and control cultures were fractionated on an Aminex A-6 column. The amount of material in each fraction was quantitated by methods which will be described in detail elsewhere. The results are given in Table 1. The difference between the amounts of each nucleoside in tRNA's from the control and methionine-limited culture are given in the right-hand column. Although the values for the four major nucleosides, pseudouridine, and 5-methyluridine fall within the esti-
296
KEISEL AND VOLD
J. BACTERIOL.
TABLE 1. Nucleoside composition of tRNA's from control and methionine-limited cultures of strain no. 27 Mole/80 residues Nucleoside
Uridine Guanosine Cytidine Adenosine Pseudouridine 5-Methyluridine 1-Methylguanosine
Methionine-limited
Control
Difference
(%)a
runl
run2
Average
runl
run2
Average
14.1 25.9 22.0 15.3 1.66 0.86 0.06
13.9 25.8 22.1 15.5 1.70 0.85 0.08
14.0 25.9 22.0 15.4 1.68 0.85 0.07
13.7 25.8 22.2 15.7 1.62 0.93 0.02
13.5 26.8 21.7 15.3 1.73 0.94 0.02
13.6 26.3 21.9 15.5 1.68 0.93 0.02
2.8% 1.7% 0.5% 0.8% 0 9.2% 67.3%
0.04
0.04
0.04
57.2%
N6-methyladensosine (from 10.09 0.10 0.09 methyladenosine) a Estimated accuracy is ± 3.2% for major nucleosides and
mated accuracy of this technique, the amounts of both 1-methylguanosine and N"-methyladenosine are significantly reduced in the methionine-limited culture. The amounts by which the latter two nucleosides were reduced indicated that the amount of undermethylated tRNA in the tRNA prepared from the methionine-limited culture was 57 to 67% of the total. The fact that the amount of 5-methyluridine (ribothymidine) remained the same is consistent with the observations of others (2, 6) that 5-methyluridine is not formed from the methyl moiety of methionine in B. subtilis as it is in E. coli. In B. subtilis, a tetrahydrofolate derivative furnishes the methyl group. Amino acid acceptor activity. Whether or not undermethylated tRNA's from relaxed E. coli mutants are altered in amino acid acceptance is a subject of some controversy. Although the amino acid acceptance of undermethylated tRNA from E. coli K-12 strain 58-161 was studied in several laboratories and no gross differences between methylated and undermethylated tRNA were found (11), a considerable decrease in activity was observed by Shugart et al. (7). The latter researchers found that about 38% of the amino acid acceptance of a mixture of 14C-labeled amino acids was lost when the tRNA isolated from a methionine-limited culture was compared with that of a control culture. Shugart et al. also examined the amino acid acceptor activities for phenylalanine, leucine, histidine, and tyrosine, and found all these activities lower by 43 to 68% than the values obtained with the control culture. Therefore, we thought it of interest to examine the amino acid acceptor activities of tRNA's from our relaxed B. subtilis strain grown in a methionine-limited culture to determine if gen-
+
10.8% for minor nucleosides.
eral undermethylation would affect the amino acid acceptor function. The results are shown in Table 2. It had already been established by ultraviolet analysis of the nucleosides that 57 to 67% of the tRNA from the methionine-limited culture was undermethylated. Therefore, a decrease to 57 to 67% of the control would reflect a total loss of acceptance by the undermethylated species. As seen in Table 2, four amino acid acceptor groups showed decreases of 13 to 33% and, somewhat surprisingly, four groups had increases of 28 to 34%. These increases were probably real and not the result of a difference in specific activity between the two tRNA samples since the sum of the specific activities for all tRNA's was very similar for both samples and 10 other amino acceptor groups showed the same acceptance as the control. Therefore, although the amino acid acceptor activities of most tRNA species were unchanged, certain species showed differences in activity either as an effect of undermethylation or because starvation conditions caused a change in the composition of the tRNA population. Analysis of tRNA labeled in vivo with L[methyl-'4C]methionine in the presence of rifampin. It was desirable to establish which methyl groups could be accepted by the undermethylated tRNA prepared from a methioninelimited culture; however, experiments designed to determine this by in vitro methylation always resulted in the lack of methylation of some nucleoside which was normally methylated in vivo. Presumably, our purified enzyme preparations had lost certain methylase activities. Therefore, an experiment was designed to remethylate the undermethylated tRNA in vivo under conditions where synthesis of new tRNA was inhibited by rifampin. Growth
UNDERMETHYLATED tRNA FROM B. SUBTILIS
VOL. 126, 1976
297
TABLE 2. Aminoacylation of tRNA from control and methionine-limited cultures Amino acid (pmol)/A260 tRNA Amino acid
Methionine ited
Control
Asp Pro Thr His Trp Val Ile Met Arg Asn Phe Leu Ser Ala Gly Lys Try Glu
93.2 44.9 65.5 39.0 20.0 53.6 55.9 63.9 114.9 125.5 63.1 160.9 107.3 78.0 91.6 63.3 42.5 22.2
80.7 34.7 54.3 26.1 26.2 68.8 71.0 85.9 123.4 115.3 61.4 153.2 109.1 73.5 96.2 68.2 39.3 22.8
curves are shown in Fig. 2. Two types of experiments are represented by the figure. In one type of experiment, i4methyl- 4C]methionine was added to both methionine-limited cultures, one having added rifampin and one without. In another type of experiment, nonradioactive xmethionine and [3H]uridine were added. The latcontrolI
'11^
c 1.0c 0
a
(D
T
riA - f
rrt , d
f ~ add ~
-1
~ ri+;
/rif
0.5-
0
n
A n
< 0.1-
6
2
4 Hrs
of
Difference as % of
lim-
6
Growth
FIG. 2. In vivo labeling of undermethylated tRNA with L"-methyl-'4C]methionine in the presence of rifampin. The absorbances from three cultures, each containing a total volume of 200 ml, are represented. The control culture was started with 25 pg of Lmethionine per ml, which provides sufficient methionine throughout growth. The two other cultures were started with 2.5 ug of L-methionine per ml, which is exhausted by the cells during the exponential growth phase. Both methionine-limited cultures were given either 0.7 mg (50 pCi) of L-[methyl- 14C]methionine or 0.7 mg of nonradioactive imethionine and 10 pg (2 mCi) of [3H]uridine after 5 h ofgrowth. To one of the methionine-limited cultures, 1 mg of rifampin was added at 4 h 50 min. Cultures were harvested at 7 h.
Difference
(limited-control)
-12.5 ±5.4 -10.2 +2.3 -11.2 +3.0 -12.9 ±1.9 +6.2 ±1.6 +15.2 ±5.0 +15.1 ±4.8 +22.0 +4.6 +8.5 ±3.2 -10.2 ±3.8 -1.7 ±2.0 -7.7 ±4.2 +1.8 ±4.4 -4.5 ±3.4 +4.6 ±2.6 +4.9 ±2.9 -3.2 ±2.0 +0.6 ±4.5
control 13.4 22.7 17.1 33.1 31.1 28.4 27.0 34.4 7.4 8.1 2.7 4.8 1.7 5.8
5.0 7.7 7.5 2.7
ter type of experiment was done as a control to establish that the synthesis of new tRNA had indeed been terminated in the culture possessing rifampin. Incorporation of [3H]uridine into tRNA in the culture grown in the presence of rifampin was 1% of that incorporated into tRNA in the culture without rifampin, showing that the synthesis of new tRNA had been suppressed. The tRNA's extracted from cultures labeled with L-[methyl-'4C]methionine were used to study the type of nucleosides which were capable of being remethylated in the absence of newly synthesized tRNA. These 14Clabeled tRNA's were enzymically degraded to nucleosides, as described in Materials and Methods, and the nucleosides were fractionated on an Aminex A-6 column by cation-exclusion
chromatography. Radioactivity from the 14Cmethyl-labeled nucleosides in the eluted fractions is represented in Fig. 3, which is included to show that the nucleosides have been separated from each other well enough to permit quantitation of the amount of each. The relative amounts of each nucleoside fractionated by the column were as follows, for tRNA's from cultures with or without rifampin, respectively: no. 1, 27.5% and 20.4%; no. 2, 38.6% and 37.2%; no. 3, 4.2% and 5.5%; no. 4 is a degradation product and was not included in the calculations; no. 5, 3.3% and 1.9%; no. 6, 7.5% and 10.3%; no. 7, 17.4% and 22.7%; no. 8, 1.6% and 1.9%. These percentages are not completely representative of the methylated nucleoside composition of B. subtilis tRNA's because of the alkaline instability of some of the compounds.
298 3-
KEISEL AND VOLD
J. BACTERIOL.
2 3
2
u
1
4
,
'
7
1 0 0
i Ilb
70
3C
8
90
FRATION NUMBER
FIG. 3. Analysis of methyl-'4C-lo!abeled nucleosides by ion-exclusion chromatograph y on Aminex A6. Aminex A-6 columns were run cas described in Materials and Methods. Radioactiive nucleosides were located in the column eluate by (determining the radioactivity in the column fractio)ns in Aquasol (New England Nuclear) in a liquid scintillation spectrometer.
Nevertheless, it is clear that the undermethylated tRNA produced by our rela:xed strain under conditions of starvation was ]lacking in the methylated nucleosides normall)y present and that these undermodified nucleo.sides could be remethylated to approximately inormal levels with the appropriate methylases. Identification of these nucleosi ricsa wx,a mw by comparing their elution positi(on on Aminex A-6 and Aminex A-28 wit' stan dards and by comparing their Rf values with standards on thin-layer chromatography. Ten tative assignments are as follows (Fig. 3): no . 1, a methylated uridine; no. 2, alkaline degrradation products of 7-methylguanosine and 7--methyladenosine; no. 3, 7-methyladenosine; xno. 4, an unidentified degradation product; n(o. 5, N -methylguanosine; no. 6, 1-methylgua nosine; no. 7, N"-methyladenosine arising mairnly from alkaline rearrangement of 1-methyla denosine; and no. 8, an unidentified nucleoside . There is also a component that was eluted wit]h 50% ethanol when the column was washed aft er elution with normal buffer. This componentt cochromatographed with isopentenyladeno: sine on thinlayer chromatography and is prob)ably a methyl derivative of that nucleoside. DISCUSSION The strain described in this paLper provides a new source for obtaining preparaktions of tRNA
enriched in undermethylated species. At present, a relaxed strain of E. coli is most commonly used for this purpose. The new B. subtilis strain offers an interesting alternative because the undermethylated tRNA's from this B. subtilis strain have a different composition of methylated components than E. coli and have fully modified 5-methyluridine (ribothymidine). Like the undermethylated tRNA from E. coli (4), the undermethylated tRNA from this relaxed B. subtilis strain has normal amounts of the four main nucleoside components and pseudouridine. Unlike the undermethylated tRNA from E. coli, 5-methyluridine is also present in normal amounts in the undermethylated tRNA from B. subtilis since the transmethylation reaction in this organism does not involve Sadenosylmethionine (6). The pattern of nucleoside methylation of the undermethylated tRNA from B. subtilis is different from that of E. coli, since these two organisms differ with respect to the normal composition of methyl components. Amino acid acceptor activities of the tRNA from a methionine-limited culture when compared with that of a control culture showed no statistically significant differences for 10 amino acids; a decrease in activity for aspartic acid, proline, threonine, and histidine; and an increase in activity for tryptophan, valine, isoleucine, and methionine. This pattern is different from that of E. coli undermethylated tRNA's where either no differences were found (11) or decreases in phenylalanine, leucine, histidine, and tyrosine were reported (7). ACKNOWLEDGMENTS I thank Gordon Edlin for providing us with the relaxed strain of B. subtilis BR16R and James Hoch for transforming this strain. The technical assistance of Arlene Carbone is also gratefully acknowledged. This work was supported by Public Health Service research grant GM 17421 and Research Career Development award GM 23,736 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Borek, E. and P. R. Srinivasan. 1966. The methylation of nucleic acids. Annu. Rev. Biochem. 35:275-298. 2. Kersten, H., L. Sandig, and H. H. Arnold. 1975. Tetrahydrofolate-dependent 5-methyluracil-tRNA transferase activity in B. subtilis. FEBS Lett. 55:57-60. 3. Lederberg, J. 1947. Genetic recombination and linked segregations in Escherichia coli. Genetics 32:505-525. 4. Mandel, L. R., and E. Borek. 1963. The nature of the RNA synthesized during conditions of unbalanced growth in E. coli K,2W-6. Biochemistry 2:560-566. 5. Martin, R. G. 1968. Polarity in related strains of Salmonella typhimurium. J. Mol. Biol. 31:127-134. 6. Romeo, J. M., A. S. Delk, and J. C. Rabinowitz. 1974. The occurrence of a transmethylation reaction not
involving S-adenosylmethionine in the formation of ribothymidine in Bacillus subtilis transfer RNA. Bio-
VOL. 126, 1976
UNDERMETHYLATED tRNA FROM B. SUBTILIS
chem. Biophys. Res. Comun. 61:1256-1261. 7. Shugart, L., B. H. Chastain, G. D. Novelli, and M. P. Stulberg. 1968. Restoration of aminoacylation activity of undermethylated transfer RNA by in vitro methylation. Biochem. Biophys. Res. Commun. 31:404-409. 8. Singhal, R. P. 1972. Ion-exclusion chromatography: analysis and isolation of nucleic acid components, and influence of separation parameters. Arch. Biochem. Biophys. 152:800-810. 9. Singhal, R. P., and W. E. Cohn. 1973. Cation-exclusion chromatography on anion exchangers: application to nucleic acid components and comparison with anionexchange chromatography. Biochemistry 12:15321537.
299
10. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. U.S.A. 44:1072-1078. 11. Starr, J. L., and B. H. Sells. 1969. Methylated ribonucleic acids. Physiol. Rev. 49:623-669. 12. Swanton, M., and G. Edlin. 1972. Isolation and characterization of an RNA relaxed mutant of B. subtilis. Biochem. Biophys. Res. Commun. 46:583-588. 13. Tatum. E. L. 1945. X-ray induced mutant strains of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 31:215-219. 14. Vold, B. S. 1974. Preparation of tRNAs and aminoacyltRNA synthetases from Bacillus subtilis cells at various stages of growth and spores. Methods Enzymol. 24:502-510.
Vol. 126, No. 1 Printed in U.SA.
Undermethylated Transfer Ribonucleic Acid from a Relaxed Strain of Bacillus subtilis: Construction of the Strain and Analysis of the Transfer Ribonucleic Acid NICOLE KEISEL AND BARBARA VOLD*
Scripps Clinic and Research Foundation, La Jolla, California 92037 Received for publication 21 January 1976
A strain of Bacillus subtilis is described from which undermethylated transfer ribonucleic acid (tRNA) can be obtained. The tRNA's from a methionine-limited culture were compared with those from a control culture with respect to general nucleoside composition, methylated components, and amino acid acceptor activity. The undermethylated tRNA's had the normal amounts of the four major nucleosides, pseudouridine, and 5-methyluridine (ribothymidine), but were deficient in methylated nucleosides other than 5-methyluridine. These methyldeficient nucleosides can be fully remethylated in the presence of the appropriate methylases. Since the majority of the work characterizing undermethylated tRNA's has been done using Escherichia coli, the work with B. subtilis presents some interesting comparisons and offers an alternative substrate for methylase studies.
The genetic locus for the control of the synthesis of ribonucleic acid (RNA) was first described for a strain of Escherichia coli K-12 strain W-6 (3), a derivative of E. coli K-12 strain 58-161 (13). A mutation in this rel locus caused loss of the normal control of RNA synthesis by amino acids which resulted in the continued synthesis of certain classes of RNA even in the absence of a required amino acid. This property was used to obtain preparations of transfer RNA (tRNA) enriched in undermethylated species by starving a methioninerequiring "relaxed" strain for methionine (1).
Growth of cells and extraction of tRNA and aminoacyl-tRNA synthetases. Cultures were grown on Spizizen minimal salts medium (10), supplemented with 0.5% glucose, 40 gug of i-tryptophan per ml, and L-methionine or casein hydrolysate. Precultures were grown with 0.1% casein hydrolysate and the cells were washed twice with sterile minimal medium before addition to the final culture. Cultures were started at an absorbance of 0.1 measured at 660 nm and were grown with shaking at 37 C. Preparations of mixed tRNA's and aminoacyl-tRNA synthetases were prepared as described previously (14). Amino acid acceptor activity. The amino acid acceptor activity for undermethylated and control tRNA's was measured by the aminoacylation of 2 Relaxed strains have also been obtained in two nmole of tRNA in a 200-Ml reaction in the presence other bacterial species, Salmonella typhimu- of 2 to 5 mM adenosine 5'-triphosphate, 10 mM 2rium (5) and Bacillus subtilis (12). However, mercaptoethanol, 2 nmol of "4C-labeled amino acid, the B. subtilis strain did not require methio- 0.8 nmol each of a mixture of 19 amino acids minus nine and thus could not be used to prepare the appropriate amino acid, saturating amounts of undermethylated tRNA's. The relaxed strain of aminoacyl-tRNA synthetases, and the following adB. subtilis, M58, B. subtilis BR16R, was origi- ditional reaction components, depending on the acid: lysine-250 mM sodium cacodylate, pH nally auxotrophic for lysine and tryptophan. By amino and 10 mM MgCl2; alanine and glycine-250 mM transformation, we have introduced a require- 7.9, cacodylate, pH 7.0, 5 mM KCl, and 10 mM ment for methionine and characterized the re- sodium magnesium acetate; methionine-250 mM HEPES sulting strain for growth and the ability to (N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic produce undermethylated tRNA's. acid), pH 7.5, 5 mM KCl, and 10 mM magnesium acetate; phenylalanine, tryptophan, arginine, vaMATERIALS AND METHODS line, leucine, isoleucine, and serine-250 mM Materials. Rifampin, B grade, and phosphodies- tris(hydroxymethyl)aminomethane, pH 7.5, and 10 terase (Russel viper venom, B grade) were obtained mM MgCl; and aspartic acid, asparagine, proline, from Calbiochem. Bacterial alkaline phosphatase histidine, glutamic acid, and tyrosine- 250 mM Nand pancreatic ribonuclease A were obtained from 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, Worthington. Aminex A-6 was purchased from pH 8.4, 5 mM KCl, and 10 mM magnesium acetate. BioRad. Preparation and analysis of nucleosides. tRNA's 194
VOL. 126, 1976
UNDERMETHYLATED tRNA FROM B. SUBTILIS
were degraded by incubation with pancreatic ribonuclease (EC 2.7.7.16), phosphodiesterase (EC 3.1.4.1), and alkaline phosphatase (EC 3.1.3.1), as described by Singhal and Cohn (9), except that incubation was at 37 C for 15 h. The resultant nucleosides were fractionated on an Aminex A-6 column at 50 C by ion-exclusion chromatography, as described by Singhal (8), using 20 mM ammonium carbonate, adjusted to pH 9.80 with NH40H. Test for relaxed phenotype. Recombinants were tested for the relaxed phenotype by a procedure modified from that of Martin (5). Cultures were grown to exponential phase in Spizizen minimal plus 0.5% glucose, 40 ,ug of tryptophan per ml, and 40 ,tg of methionine per ml (or 40 jLg of lysine per ml for the parent). A culture sample with a total absorbance of 0.04 at 660 nm was transferred onto a sterile membrane fi'lter (Millipore Corp.) and placed in a petri dish on minimal agar plus tryptophan for 2 h at 37 C. The filters were transferred to a minimal agar plate containing tryptophan and 8.8 ,mg of ['4C]uridine per ml and incubated for 1 h at 37 C. Filters were then transferred to a plate with nonradioactive uridine for 30 min and then dried, and the radioactivity was determined in toluene scintillation fluid in a liquid scintillation counter.
RESULTS Construction of methionine-requiring, relaxed strain. A relaxed strain, M58, B. subtilis BR16R, has been described by Swanton and Edlin (12). As originally isolated, the strain required lysine and tryptophan but did not require methionine. The strain, given to us by Edlin, was transformed by James Hoch to lysine prototrophy with a saturating concentration of deoxyribonucleic acid from a strain carrying the metB5 allele. The transformation mixture was spread on plates containing tryptophan and methionine, and methionine-requiring transformants were identified by replica plating to plates containing only tryptophan. Methionine-requiring transformants were cloned twice, checked for auxotrophic requirements, and tested for the relaxed phenotype as described in Materials and Methods. The resultant strains had the genotype trpC2 metB5 rel-. We obtained 23 recombinants of this type. Clone no. 27 had the highest level of incorporation of L'4C]uridine under the conditions of the test for the relaxed phenotype and was selected for the remainder of the work reported here. Growth of the relaxed strain and preparation of tRNA enriched in undermethylated species. A growth curve for this relaxed strain is shown in Fig. 1. The upper curve shows the growth of the strain in excess methionine (25 ,ug/ml). The lower curve represents the growth curve in the presence of 2.5 ,g of methionine
295
Hrs of Growth
FIG. 1. Growth curve for a relaxed, methioninerequiring strain, B. subtilis 168 trpC2 metB5 rel(no. 27). Cells were grown as described in Materials and Methods. After 3.5 h ofgrowth, 3.5 p.g of methionine per ml was added to the culture represented in the lower curve.
per ml, which became limiting about halfway through the exponental growth phase. Starvation for methionine resulted in an inhibition of growth which could be reversed by the addition of methionine to the medium. To prepare tRNA enriched in undermethylated species, a culture, started with 2.5 ,ug of Lmethionine per ml, was grown as shown in Fig. 1, and harvested after 3.5 h. A control culture, grown in the presence of 25 ,g of L-methionine per ml, was grown for the same period of time and harvested. The extraction of tRNA is described in Materials and Methods. Analysis of the tRNA prepared from the methionine-limited and control cultures. The methionine-limited culture would be expected to make normal tRNA during growth until the methionine supply had been exhausted. A tRNA preparation isolated from a starved culture, therefore, is a mixture of normally modified and undermethylated tRNA's. To determine the proportion of tRNA that was undermethylated after 3.5 h of growth, nucleosides of tRNA's from methionine-limited and control cultures were fractionated on an Aminex A-6 column. The amount of material in each fraction was quantitated by methods which will be described in detail elsewhere. The results are given in Table 1. The difference between the amounts of each nucleoside in tRNA's from the control and methionine-limited culture are given in the right-hand column. Although the values for the four major nucleosides, pseudouridine, and 5-methyluridine fall within the esti-
296
KEISEL AND VOLD
J. BACTERIOL.
TABLE 1. Nucleoside composition of tRNA's from control and methionine-limited cultures of strain no. 27 Mole/80 residues Nucleoside
Uridine Guanosine Cytidine Adenosine Pseudouridine 5-Methyluridine 1-Methylguanosine
Methionine-limited
Control
Difference
(%)a
runl
run2
Average
runl
run2
Average
14.1 25.9 22.0 15.3 1.66 0.86 0.06
13.9 25.8 22.1 15.5 1.70 0.85 0.08
14.0 25.9 22.0 15.4 1.68 0.85 0.07
13.7 25.8 22.2 15.7 1.62 0.93 0.02
13.5 26.8 21.7 15.3 1.73 0.94 0.02
13.6 26.3 21.9 15.5 1.68 0.93 0.02
2.8% 1.7% 0.5% 0.8% 0 9.2% 67.3%
0.04
0.04
0.04
57.2%
N6-methyladensosine (from 10.09 0.10 0.09 methyladenosine) a Estimated accuracy is ± 3.2% for major nucleosides and
mated accuracy of this technique, the amounts of both 1-methylguanosine and N"-methyladenosine are significantly reduced in the methionine-limited culture. The amounts by which the latter two nucleosides were reduced indicated that the amount of undermethylated tRNA in the tRNA prepared from the methionine-limited culture was 57 to 67% of the total. The fact that the amount of 5-methyluridine (ribothymidine) remained the same is consistent with the observations of others (2, 6) that 5-methyluridine is not formed from the methyl moiety of methionine in B. subtilis as it is in E. coli. In B. subtilis, a tetrahydrofolate derivative furnishes the methyl group. Amino acid acceptor activity. Whether or not undermethylated tRNA's from relaxed E. coli mutants are altered in amino acid acceptance is a subject of some controversy. Although the amino acid acceptance of undermethylated tRNA from E. coli K-12 strain 58-161 was studied in several laboratories and no gross differences between methylated and undermethylated tRNA were found (11), a considerable decrease in activity was observed by Shugart et al. (7). The latter researchers found that about 38% of the amino acid acceptance of a mixture of 14C-labeled amino acids was lost when the tRNA isolated from a methionine-limited culture was compared with that of a control culture. Shugart et al. also examined the amino acid acceptor activities for phenylalanine, leucine, histidine, and tyrosine, and found all these activities lower by 43 to 68% than the values obtained with the control culture. Therefore, we thought it of interest to examine the amino acid acceptor activities of tRNA's from our relaxed B. subtilis strain grown in a methionine-limited culture to determine if gen-
+
10.8% for minor nucleosides.
eral undermethylation would affect the amino acid acceptor function. The results are shown in Table 2. It had already been established by ultraviolet analysis of the nucleosides that 57 to 67% of the tRNA from the methionine-limited culture was undermethylated. Therefore, a decrease to 57 to 67% of the control would reflect a total loss of acceptance by the undermethylated species. As seen in Table 2, four amino acid acceptor groups showed decreases of 13 to 33% and, somewhat surprisingly, four groups had increases of 28 to 34%. These increases were probably real and not the result of a difference in specific activity between the two tRNA samples since the sum of the specific activities for all tRNA's was very similar for both samples and 10 other amino acceptor groups showed the same acceptance as the control. Therefore, although the amino acid acceptor activities of most tRNA species were unchanged, certain species showed differences in activity either as an effect of undermethylation or because starvation conditions caused a change in the composition of the tRNA population. Analysis of tRNA labeled in vivo with L[methyl-'4C]methionine in the presence of rifampin. It was desirable to establish which methyl groups could be accepted by the undermethylated tRNA prepared from a methioninelimited culture; however, experiments designed to determine this by in vitro methylation always resulted in the lack of methylation of some nucleoside which was normally methylated in vivo. Presumably, our purified enzyme preparations had lost certain methylase activities. Therefore, an experiment was designed to remethylate the undermethylated tRNA in vivo under conditions where synthesis of new tRNA was inhibited by rifampin. Growth
UNDERMETHYLATED tRNA FROM B. SUBTILIS
VOL. 126, 1976
297
TABLE 2. Aminoacylation of tRNA from control and methionine-limited cultures Amino acid (pmol)/A260 tRNA Amino acid
Methionine ited
Control
Asp Pro Thr His Trp Val Ile Met Arg Asn Phe Leu Ser Ala Gly Lys Try Glu
93.2 44.9 65.5 39.0 20.0 53.6 55.9 63.9 114.9 125.5 63.1 160.9 107.3 78.0 91.6 63.3 42.5 22.2
80.7 34.7 54.3 26.1 26.2 68.8 71.0 85.9 123.4 115.3 61.4 153.2 109.1 73.5 96.2 68.2 39.3 22.8
curves are shown in Fig. 2. Two types of experiments are represented by the figure. In one type of experiment, i4methyl- 4C]methionine was added to both methionine-limited cultures, one having added rifampin and one without. In another type of experiment, nonradioactive xmethionine and [3H]uridine were added. The latcontrolI
'11^
c 1.0c 0
a
(D
T
riA - f
rrt , d
f ~ add ~
-1
~ ri+;
/rif
0.5-
0
n
A n
< 0.1-
6
2
4 Hrs
of
Difference as % of
lim-
6
Growth
FIG. 2. In vivo labeling of undermethylated tRNA with L"-methyl-'4C]methionine in the presence of rifampin. The absorbances from three cultures, each containing a total volume of 200 ml, are represented. The control culture was started with 25 pg of Lmethionine per ml, which provides sufficient methionine throughout growth. The two other cultures were started with 2.5 ug of L-methionine per ml, which is exhausted by the cells during the exponential growth phase. Both methionine-limited cultures were given either 0.7 mg (50 pCi) of L-[methyl- 14C]methionine or 0.7 mg of nonradioactive imethionine and 10 pg (2 mCi) of [3H]uridine after 5 h ofgrowth. To one of the methionine-limited cultures, 1 mg of rifampin was added at 4 h 50 min. Cultures were harvested at 7 h.
Difference
(limited-control)
-12.5 ±5.4 -10.2 +2.3 -11.2 +3.0 -12.9 ±1.9 +6.2 ±1.6 +15.2 ±5.0 +15.1 ±4.8 +22.0 +4.6 +8.5 ±3.2 -10.2 ±3.8 -1.7 ±2.0 -7.7 ±4.2 +1.8 ±4.4 -4.5 ±3.4 +4.6 ±2.6 +4.9 ±2.9 -3.2 ±2.0 +0.6 ±4.5
control 13.4 22.7 17.1 33.1 31.1 28.4 27.0 34.4 7.4 8.1 2.7 4.8 1.7 5.8
5.0 7.7 7.5 2.7
ter type of experiment was done as a control to establish that the synthesis of new tRNA had indeed been terminated in the culture possessing rifampin. Incorporation of [3H]uridine into tRNA in the culture grown in the presence of rifampin was 1% of that incorporated into tRNA in the culture without rifampin, showing that the synthesis of new tRNA had been suppressed. The tRNA's extracted from cultures labeled with L-[methyl-'4C]methionine were used to study the type of nucleosides which were capable of being remethylated in the absence of newly synthesized tRNA. These 14Clabeled tRNA's were enzymically degraded to nucleosides, as described in Materials and Methods, and the nucleosides were fractionated on an Aminex A-6 column by cation-exclusion
chromatography. Radioactivity from the 14Cmethyl-labeled nucleosides in the eluted fractions is represented in Fig. 3, which is included to show that the nucleosides have been separated from each other well enough to permit quantitation of the amount of each. The relative amounts of each nucleoside fractionated by the column were as follows, for tRNA's from cultures with or without rifampin, respectively: no. 1, 27.5% and 20.4%; no. 2, 38.6% and 37.2%; no. 3, 4.2% and 5.5%; no. 4 is a degradation product and was not included in the calculations; no. 5, 3.3% and 1.9%; no. 6, 7.5% and 10.3%; no. 7, 17.4% and 22.7%; no. 8, 1.6% and 1.9%. These percentages are not completely representative of the methylated nucleoside composition of B. subtilis tRNA's because of the alkaline instability of some of the compounds.
298 3-
KEISEL AND VOLD
J. BACTERIOL.
2 3
2
u
1
4
,
'
7
1 0 0
i Ilb
70
3C
8
90
FRATION NUMBER
FIG. 3. Analysis of methyl-'4C-lo!abeled nucleosides by ion-exclusion chromatograph y on Aminex A6. Aminex A-6 columns were run cas described in Materials and Methods. Radioactiive nucleosides were located in the column eluate by (determining the radioactivity in the column fractio)ns in Aquasol (New England Nuclear) in a liquid scintillation spectrometer.
Nevertheless, it is clear that the undermethylated tRNA produced by our rela:xed strain under conditions of starvation was ]lacking in the methylated nucleosides normall)y present and that these undermodified nucleo.sides could be remethylated to approximately inormal levels with the appropriate methylases. Identification of these nucleosi ricsa wx,a mw by comparing their elution positi(on on Aminex A-6 and Aminex A-28 wit' stan dards and by comparing their Rf values with standards on thin-layer chromatography. Ten tative assignments are as follows (Fig. 3): no . 1, a methylated uridine; no. 2, alkaline degrradation products of 7-methylguanosine and 7--methyladenosine; no. 3, 7-methyladenosine; xno. 4, an unidentified degradation product; n(o. 5, N -methylguanosine; no. 6, 1-methylgua nosine; no. 7, N"-methyladenosine arising mairnly from alkaline rearrangement of 1-methyla denosine; and no. 8, an unidentified nucleoside . There is also a component that was eluted wit]h 50% ethanol when the column was washed aft er elution with normal buffer. This componentt cochromatographed with isopentenyladeno: sine on thinlayer chromatography and is prob)ably a methyl derivative of that nucleoside. DISCUSSION The strain described in this paLper provides a new source for obtaining preparaktions of tRNA
enriched in undermethylated species. At present, a relaxed strain of E. coli is most commonly used for this purpose. The new B. subtilis strain offers an interesting alternative because the undermethylated tRNA's from this B. subtilis strain have a different composition of methylated components than E. coli and have fully modified 5-methyluridine (ribothymidine). Like the undermethylated tRNA from E. coli (4), the undermethylated tRNA from this relaxed B. subtilis strain has normal amounts of the four main nucleoside components and pseudouridine. Unlike the undermethylated tRNA from E. coli, 5-methyluridine is also present in normal amounts in the undermethylated tRNA from B. subtilis since the transmethylation reaction in this organism does not involve Sadenosylmethionine (6). The pattern of nucleoside methylation of the undermethylated tRNA from B. subtilis is different from that of E. coli, since these two organisms differ with respect to the normal composition of methyl components. Amino acid acceptor activities of the tRNA from a methionine-limited culture when compared with that of a control culture showed no statistically significant differences for 10 amino acids; a decrease in activity for aspartic acid, proline, threonine, and histidine; and an increase in activity for tryptophan, valine, isoleucine, and methionine. This pattern is different from that of E. coli undermethylated tRNA's where either no differences were found (11) or decreases in phenylalanine, leucine, histidine, and tyrosine were reported (7). ACKNOWLEDGMENTS I thank Gordon Edlin for providing us with the relaxed strain of B. subtilis BR16R and James Hoch for transforming this strain. The technical assistance of Arlene Carbone is also gratefully acknowledged. This work was supported by Public Health Service research grant GM 17421 and Research Career Development award GM 23,736 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Borek, E. and P. R. Srinivasan. 1966. The methylation of nucleic acids. Annu. Rev. Biochem. 35:275-298. 2. Kersten, H., L. Sandig, and H. H. Arnold. 1975. Tetrahydrofolate-dependent 5-methyluracil-tRNA transferase activity in B. subtilis. FEBS Lett. 55:57-60. 3. Lederberg, J. 1947. Genetic recombination and linked segregations in Escherichia coli. Genetics 32:505-525. 4. Mandel, L. R., and E. Borek. 1963. The nature of the RNA synthesized during conditions of unbalanced growth in E. coli K,2W-6. Biochemistry 2:560-566. 5. Martin, R. G. 1968. Polarity in related strains of Salmonella typhimurium. J. Mol. Biol. 31:127-134. 6. Romeo, J. M., A. S. Delk, and J. C. Rabinowitz. 1974. The occurrence of a transmethylation reaction not
involving S-adenosylmethionine in the formation of ribothymidine in Bacillus subtilis transfer RNA. Bio-
VOL. 126, 1976
UNDERMETHYLATED tRNA FROM B. SUBTILIS
chem. Biophys. Res. Comun. 61:1256-1261. 7. Shugart, L., B. H. Chastain, G. D. Novelli, and M. P. Stulberg. 1968. Restoration of aminoacylation activity of undermethylated transfer RNA by in vitro methylation. Biochem. Biophys. Res. Commun. 31:404-409. 8. Singhal, R. P. 1972. Ion-exclusion chromatography: analysis and isolation of nucleic acid components, and influence of separation parameters. Arch. Biochem. Biophys. 152:800-810. 9. Singhal, R. P., and W. E. Cohn. 1973. Cation-exclusion chromatography on anion exchangers: application to nucleic acid components and comparison with anionexchange chromatography. Biochemistry 12:15321537.
299
10. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. U.S.A. 44:1072-1078. 11. Starr, J. L., and B. H. Sells. 1969. Methylated ribonucleic acids. Physiol. Rev. 49:623-669. 12. Swanton, M., and G. Edlin. 1972. Isolation and characterization of an RNA relaxed mutant of B. subtilis. Biochem. Biophys. Res. Commun. 46:583-588. 13. Tatum. E. L. 1945. X-ray induced mutant strains of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 31:215-219. 14. Vold, B. S. 1974. Preparation of tRNAs and aminoacyltRNA synthetases from Bacillus subtilis cells at various stages of growth and spores. Methods Enzymol. 24:502-510.
