Vol. 140, No. 2

JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 574-579

0021-9193/79/11-0574/06$02.00/0

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi M. C. GOMEZ-EICHELMANN Departamento de Biotecnologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Autdnoma de Mexico, Mexico 20, D.F., Mexico

Received for publication 9 July 1979

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence -Cc(A)GG- were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC-

(DpnI and MboI) and -ICCQA)GG- (EcoRII). The restriction patterns obtained for this plasmid isolated from transfonned S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at -CC( A )GG- are met hylated by enzymes coded for by the genes dam and dcm, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.

Bacterial DNA, like the DNAs from practically all organisms examined, contains small amounts of methylated bases (30). The methylation occurs at the polynucleotide level and, involves the enzymatic transfer of the methyl group of S-adenosyl-L-methionine to specific bases in DNA (10, 12). The chromosome of Escherichia coli K-12 contains about 0.5 mol% of N6-methyladenine (6-MeA) and 0.25 mol% of 5-methylcytosine (9, 30). A minute fraction, probably less than 2 or 3%, of the 6-MeA is involved in the K restrictionmodification system (23). The methylation of the adenines of the major fraction is carried out by a methylase coded by the gene dam (25). This methylase specifically recognizes the sequence -GATC- (20). The second cytosine in the sequence -CC( )GG- is methylated by an enzyme coded by the gene dcm (27). The purpose of this work was to determine whether in other Enterobacteriaceae, such as Salmonella typhimurium and Salmonella typhi, the 5-methylcytosine and the major fraction of 6-MeA are also present in the sequences -CC( )GG- and -GATC-, respectively. This study was designed as an initial approach to investigate the possible function of methylated bases in the stability of plasmids in bacteria. Three different restriction enzymes, MboI,

DpnI, and EcoRII, were used to digest the small plasmid pBR322, which is a ColEl-like plasmid that specifies ampicillin (Ap) and tetracycline (Tc) resistance (5) and whose complete DNA sequence has been determined (G. Sutcliffe, Cold Spring Harbor Symp. Quant. Biol., in press). MboI and DpnI show complementary specificity with respect to methylation of sites in DNA: whereas MboI cleaves at the sequence -IGATC- (11), DpnI cleaves at the same sequence only when the adenine residues of both strands are methylated (20, 31). EcoRH cuts the nonmethylated sequence-cCC( A )GG- (3). Therefore, by using MboI, DpnI, and EcoRII, it is possible to determnine and compare the methylation patterns in the sequences -GATC- and -CCQA)GG- of pBR322 isolated from different bacterial hosts. MATERIALS AND METHODS Plasmid and bacterial strains. Plasmid pBR322 (Apr Tcr) and E. coli K-12 substrain GM31 thr-1 leu6 thi-l his-4 ara-14 galK2 galT22 lacYl xyl-5 tonA31 tsx-78 mtl-l str-136 were provided by F. Bolivar. E. coli K-12 substrain GM110 thr-I leu-6 proA2 his-4 metBI lacYI galK2 ara-14 tsx-33 thi-1 thyA12 drm-6 supE44 and substrain GM111, with the same genotype as GM110 but dam-3, were gifts of M. Marinus. The two plasmid-free Salmonella strains, S. typhimurium

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LT2 substrain Su695 trpA52 cysB12pyrF146 ilvA178 and S. typhi strain JM1382 Cys- Nalr, phage type Vi degraded (2), were obtained from G. Alfaro. Media. The complex medium used was Luria broth (22). The minimal medium was based upon M9 salts medium (1) supplemented with 0.2% glucose, essential amino acids (20 ,g/ml), thymine (4 jig/ml), and thiamine (0.2 ,ug/ml). When Casamino Acids, tetracycline, or ampicillin was added, the final concentration was, respectively, 0.4%, 25 ,ug/ml, or 50 jug/ml. Preparation of plasmid DNA. Plasmid DNA was prepared from exponentially growing cultures in Luria broth or from cultures in minimal medium supplemented with Casamino Acids by amplification with chloramphenicol (170 ,ig/ml) (7). Clear lysates were obtained by centrifugation after lysis of the cells with lysozyme and Triton X-100 (13). The lysates were extracted once with an equal volume of a 70% aqueous phenol solution and once with an equal volume of chloroform-70% phenol solution (9:1). The aqueous phase was made 0.2 M in NaCl and was precipitated with 2 volumes of absolute ethanol at -20°C. Further plasmid purification by chromatography on a Bio-Gel A-50m agarose column and by CsCl-propidium diiodide density gradient was carried out as described previously (4). Transformation with pBR322 DNA. E. coli cells were made competent for transformation with calcium chloride and then subjected to a heat pulse to enable DNA uptake (8). For S. typhimurium and S. typhi a modified transformation procedure with a higher calcium concentration was used (21). Restriction endonuclease assays. One or two micrograms of plasmid DNA in 20 to 30 pl of buffer was digested with a restriction enzyme according to the following conditions for each endonuclease: EcoRI, 100 mM Tris-hydrochloride-5 mM MgCl2-100 mM NaCl, pH 7.6; EcoRII, 100 mM Tris-hydrochloride-5 mM MgCl2, pH 7.5; DpnI, 50 mM Tris-hydrochloride40 mM NaCl-5 mM MgCl2, pH 7.6; MboI, 10 mM Trishydrochloride-75 mM NaCl-10 mM MgCl2-1 mM dithiothreitol, pH 7.4. Endonuclease reactions were incubated at 37°C for 2 h and were stopped by the addition of 10 pl of 10 mM urea containing 0.05% bromophenol blue. Acrylamide gel electrophoresis. Native or restricted plasmid DNA samples (1 to 2 jg) were applied to 7.3% acrylamide-0.2% bisacrylamide slab gels (13 by 11.5 by 0.15 cm) and were run in Tris-EDTA-borate buffer (4) at 200 V for 1 h. Gels were stained in ethidium bromide (10 jig/ml) for 10 to 20 min. DNA bands were visualized with a short-wave UV lamp and photographed by using Polaroid type 55 film with a yellow filter (Wratten no. 9). Chemicals and enzymes. Antibiotics, lysozyme, RNase, ethidium bromide, and propidium diiodide were purchased from Sigma Chemical Co., St. Louis, Mo. Bio-Gel A-50m agarose and all reagents for acrylamide gels were obtained from Bio-Rad Laboratories, Richmond, Calif. EcoRI and MboI were from New England BioLabs, Beverly, Mass. DpnI was kindly provided by S. Lacks, and EcoRII was the gift of F. Bolivar, M. L6pez, and G. Sober6n.

575

RESULTS

Transformation of E. coli, S. typhimurium, and S. typhi by pBR322 DNA. The pBR322 DNA used for the transformation of E. coli, S. typhimurium, and S. typhi was amplified in GM31 and was purified as described in Materials and Methods. Table 1 shows the frequencies of transformation obtained by use of two different procedures, one developed to transform E. coli cells (8) and the other developed to transform S. typhimurium (21). The frequencies of transformation of each strain were approximately the same by either procedure. The frequencies for GM111 (dam-3) were about 10 times lower than the values obtained for GMl10 (dam'). Using the method for S. typhimurium, a frequency of i0o3 was expected for Su695 (21); however, a much lower value (10-8) was obtained. For S. typhi, the frequencies of transformation were about 102 times lower than those obtained for E. coli. A possible explanation for such low values may be that pBR322 DNA from E. coli was restricted by the Salmonella strains. Study of adenine methylation at sequence -GATC- in S. typhimurium and S. typhL The study of adenine methylation at sequence -GATC- in S. typhimurium and S. typhi was carried out by comparing the DpnI and MboI restriction patterns of pBR322 isolated from both the transformed Su695 and the transformed JM1382 with those obtained by using this plasmid isolated from E. coli K-12. Two different E. coli strains were taken as references: GM110, a dam+ strain, and GM111, a dam-3 mutant. In the dam+ strain all adenines at -GATC- are methylated. Therefore, pBR322 isolated from that strain will be cleaved by DpnI but not by MboI. The dam-3 cells have only 15% of the wild-type level of 6-MeA in their DNA (25). A small proportion of this 15% is probably due to the K modification methylase (23). The TABLE 1. Frequency of transforrnation of E. coli, S. typhimurium, and S. typhi with pBR322 Transformation frequencya Recipient cells E. coli methodb

S. typhdmu'rum methodC

8.2 x 10-4 9.5 x 10-5 1.0 x lo-, 1.5 x 10-8 Su695 4.2 x 10-6 2.6 x 10-6 JM1382 a Transformation frequency is expressed as the av-

GM110 (dam+) GM111 (dam-3)

9.4 x 10-4 1.0 X 10-4

erage number of transformants per viable cell per microgram of plasmid DNA. b Cohen et al. (8). c Lederberg and Cohen (21).

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GOMEZ-EICHELMANN

remaining 6-MeA could be attributed either to a third methylase or to "leakiness" of the dam3 mutation. Consequently, if there is a third methylase, pBR322 isolated from GMlll will be restricted by MboI but not by DpnI. If the dam3 mutation is leaky, the plasmid DNA will not be completely restricted by MboI and probably will be cut at two or three sites by DpnI (pBR322 has a total of 22 -GATC- sites). Although the DpnI restriction patterns of amplified and unamplified pBR322 from GM31 were identical (data not shown), pBR322 was not amplified when isolated from GM11O, GM111, Su695, or JM1382. The DpnI and MboI restriction fragments (Fig. 1) obtained by digesting pBR322 isolated from Su695 and JM1382 (Fig. lf through i) were identical to those resulting from the DpnI and MboI restriction of pBR322 from E. coli GM11O (dam') (Fig. lb and c) but were the inverse of those from GM111 (dam-3) (Fig. ld and e). These results may be interpreted to mean that S. typhimurium and S. typhi have a methylase similar to the E. coli K-12 methylase coded by the gene dam. A second conclusion can be drawn from the results shown in Fig. 1. EcoRI digestion of pBR322 from GM31 was added as a control (Fig. lj). This enzyme cuts the plasmid at one site (5), producing a linear molecule of DNA which displays an electrophoretic mobility different from that of the intact molecule (Fig. la). The com-

.J. BACTERIOL.

parison of EcoRI-digested pBR322 (Fig. lj) with the restriction of pBR322 from GM1ll (dam-3) by DpnI (Fig. ld) shows that DpnI was unable to cut pBR322 at all. Thus, it can be concluded that the dam-3 mutation is not leaky and that the remaining methylation of adenine may correspond to a methylase activity different from those coded for by the genes hsdM and dam. This conclusion is also supported by the data of Vovis and Lacks (31). Study of cytosine methylation at sequence -CC( T )GG- in S. typhimurium and S. typhi. The experimental approach of the study of cytosine methylation at sequence -CC(A)GG- in S. typhimurium and S. typhi was basically the same as the one used for the study of adenine methylation described above. The EcoRII restriction patterns of pBR322 isolated from both the transformed Su695 and the transformed JM1382 were compared with those obtained with pBR322 from E. coli strains GM11O (dcm+) and GM31 (dem). In E. coli cells that are dcm+, the second cytosine at -CC(t)GG- is methylated (27). This methylation does not take place in cells which are dem (24). EcoRII recognizes the unmethylated -CC( )GG- sequence and cleaves the DNA at that site (3). Therefore, EcoRII digests

.1 !: -1~ ~~~~~~~ ~ ~~~~~~~~~~~~~~~~~~~~ J

b. "A_

9

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1.360.66

0.35 0.20'Ili.-). 00 07FIG. 1. Acrylamide slab gel electrophoresis ofpBR322 DNA restricted with DpnI, MboI, or EcoRI. Plasmid DNA was purified from various bacterial strains, and portions of each were treated with an endonuclease and were subjected to acrylamide gel electrophoresis as described in the text. The bacterial strains and enzymes were: (a) GM31, no enzymes; (b) GM110 (dam+), DpnI; (c) GM110 (dam+), MboI; (d) GM111 (dam-3), DpnI; (e) GM111 (dam-3), MboI; (f) Su695, DpnI; (g) Su695, MboI; (h) JM1382, DpnI; (i) JM1382, MboI; (j) GM31, EcoRL The sizes of the fragments, in kilobases, are listed at the left.

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DNA METHYLATION IN ENTEROBACTERIACEAE

577

pBR322 from GM31 but not from GM110. the modification enzyme of the K host specificity Whereas pBR322 from GM31 (dcm) was re- system accounts for only 2 to 3% of the total 6stricted by EcoRII (Fig. 2c), pBR322 isolated MeA present in the E. coli chromosome (23). In from either Su695 (Fig. 2d) or JM1382 (Fig. 2e), S. typhimurium LT2, a higher percentage of 6as that from GM110 (dcm+) (Fig. 2b), was not MeA might be involved in the L, SA, and SB restricted. From these results it is proposed that host specificity systems (14, 17), whereas in S. S. typhimurium and S. typhi have an enzyme typhi these systems have not been studied in which methylates the second cytosine at the detail. In general, it can be said that in E. coli K-12, sequence -CCQA)GG- and which is similar S. typhimurium LT2, and S. typhi the major to the methylase of E. coli K-12 coded for by portions of 6-MeA and 5-methylcytosine are lothe gene dcm'. The presence of a dcm-like calized in the same sequences; however, the 6methylase in S. typhimurium has also been sug- MeA ofthe restriction-modification systems spegested by Hattman et al. (17). cific to each strain must be contained in different DNA sequences. This implies that the bacteria DISCUSSION studied in the present communication have, on The results described in this paper demon- the one hand, common methylation patterns for strate that, in S. typhimurium LT2 and S. typhi, 6-MeA and 5-methylcytosine and, on the other, the methylation of the DNA which is not in- a distinct methylation pattem which is characvolved in the restriction-modification host spec- teristic of the DNA of each strain. The latter is ificity systems shows the same patterns as those carried out by the DNA adenine methylase proof E. coli K-12. In these three strains, the ade- duced by the hsd gene cluster and accounts for nine in the sequence -GATC- and second a small fraction of the total of the methylated present in the genomes of these bacteria. cytosine in -CC(A)GG- are methylated. bases The biological role played by the dam-speciThese findings imply that both S. typhimurium fied 6-MeA in E. coli is still unclear. One funcand S. typhi have two enzymatic activities sim- tion might be to maintain the integrity of the ilar to those of the E. coli K-12 methylases bacterial chromosome by conferring resistance which are coded for by the genes dam and dcm to nuclease digestion (25). It has also been re(24). In E. coli K-12 the methylation determined ported that phage propagation might depend on by these two genes accounts for the major frac- the display of a certain strategy in the DNA tion of DNA adenine and DNA cytosine meth- methylation of the host and, sometimes, specifylation. The DNA adenine methylation due to ically in the adenine methylation at the sequence -GATC-. For example, bacteriophages T3 and T7 carry genes whose products block all DNA a b c d e methylation carried out by the host as well as the action of the host restriction endonucleases (19, 28, 29). In contrast, phage 4X174 does not 2.63contain any -GATC- sequence (26), and 1.44phage T2 carries a gene that codes for an enzyme similar to the host dam' methylase (18). 1.05The biological role for the 5-methylcytosine of the E. coli genome also remains unclear. However, it is known that the hsdII (RII) host specificity controlled by certain fi- R-factors is mediated by a cytosine-specific methylase (16, 32). This methylase is functionally similar to the enzyme produced by the E. coli gene dcm (27). An interesting proposal on the possible funcFIG. 2. Acrylamide slab gel electrophoresis of tion of the dam- and dcm-coded methylases has EcoRII-digested pBR322 DNA. Plasmid DNA was been raised by Lacks and Greenberg (20). These isolated from various bacterial strains, incubated authors suggest that dam- and dcm-coded methwith EcoRII, and subjected to acrylamide gel electro- ylation might allow the bacterial cell to accomphoresis as described in the text. Plasmid pBR322 isolated from the following bacterial strains was modate plasmids, such as N-3 carrying the hsdII tested: (a) GM31 (dcm), no enzyme; (b) GM110 (dcm+); restriction-modification system, or the P1 pro(c) GM31 (dcm); (d) Su695; (e) JM1382. The sizes of phage carrying the hsd-1 system. The N-3 restriction endonuclease recognizes and cleaves the fragments, in kilobases, are listed at the left.

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GOMEZ-EICHELMANN

the nonmethylated sequence -CC( A )GG(3). Regarding the P1 restriction endonuclease, it has been reported that the sequence recognized by this enzyme is -AGATCT- (6); however, there exists recent evidence (15) which suggests that the P1 endonuclease site is not -AGATCT- but is another sequence (-AGACPy-) that would not be protected by the dam-specified methylation. The screening for methylation similar to that coded for by dam and dem among some Enterobacteriaceae occasionally coexisting in the same habitat was carried out by means of endonucleases that recognize the sequences that are modified by the dam- and dcm-coded methylases. The small (4,365 base pairs), well-known pBR322 plasmid was used to monitor the host methylation in the sequences -GATC- and -CCQA)GG-. With use of such a monitor, clear-cut results were obtained. This study was carried out as a first approach to a better understanding of the relationships between dam- and dcm-coded methylation and host specificity methylation (hsd) with respect to the stability of a given plasmid in different Enterobacteriaceae. ACKNOWLEDGMENTS I thank Estela Garcia for technical assistance and G. Alfaro for reading the manuscript. I also thank Veronica Y. Greenhouse for editorial assistance.

1. 2.

3.

4.

5.

6.

7. 8.

9.

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6-methylaminopurine in deoxyribonucleic acids. Biochem. J. 68:627-636. Fujimoto, D., P. R. Strinivason, and E. Borek. 1965. On the nature of the deoxyribonucleic acid methylases. Biological evidence for the multiple nature of the enzymes. Biochemistry 4:2849-2855. Gelinas, R. E., P. A. Myers, and R. J. Roberts. 1977. Two sequence-specific endoncleases from Moraxella bovis. J. Mol. Biol. 114:169-179. Gold, M., and J. Hurwitz. 1964. The enzymatic methylation of ribonucleic acid and deoxyribonucleic acid. V. Purification and properties of the deoxyribonucleic acid-methylating activity of Escherichia coli. J. Biol. Chem. 239:3858-3865. Guerry, P., D. J. LeBlanc, and S. Falkow. 1973. General method for the isolation of plasmid deoxyribonucleic acid. J. Bacteriol. 116:1064-1066. Hattman, S. 1971. Variation of 6-methylaminopurine content in bacteriophage P22 deoxyribonucleic acid as a function of host specificity. J. Virol. 7:690-691. Hattman, S., J. E. Brooks, and M. Masurekar. 1978. Sequence specificity of the P1 modification methylase (M. EcoPl) and the DNA methylase (M. Eco dam) controlled by the Escherichia coli dam gene. J. Mol. Biol. 126:367-380. Hattman, S., E. Gold, and A. Plotnik. 1972. Methylation of cytosine residues in DNA controlled by a drug resistance factor. Proc. Natl. Acad. Sci. U.S.A. 69:187-

190. 17. Hattman, S., S. Schlagman, L. Goldstein, and M. Frohlich. 1976. Salmonella typhimurium SA host specificity system is based on deoxyribonucleic acid-adenine methylation J. Bacteriol. 127:211-217. 18. Hattman, S., H. van Ormondt, and A. de Waard. 1978. Sequence specificity of the wild-type (dam') and mutant (damh) forms of bacteriophage T2 DNA adenine methylase. J. Mol. Biol. 119:361-376. 19. Kriiger, D. H., C. Schroeder, S. Hansen, and H. A. Rosenthal. 1977. Active protection by bacteriophages T3 and T7 against E. coli B- and K-specific restriction of their DNA. Mol. Gen. Genet. 153:99-106. 20. Lacks, S., and B. Greenberg. 1977. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114:153-168. 21. Lederberg, E. M., and S. N. Cohen. 1974. Transformation of SalmoneUa typhimurium by plasmid deoxyribonucleic acid. J. Bacteriol. 119:1072-1074. 22. Lennox, E. 5. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190206. 23. Mamelak, L, and H. W. Boyer. 1970. Genetic control of the secondary modification of deoxyribonucleic acid in Escherichia coli. J. Bacteriol. 104:57-62. 24. Marinus, ML G., and N. R. Morris 1973. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J. Bacteriol. 114:1143-1150. 25. Marinus, M. G., and N. R. Morris. 1974. Biological function for 6-methyladenine residues in the DNA of Escherichia coli K-12. J. Mol. Biol. 86:309-322. 26. Sanger, F., A. R. Coulson, T. Friedmann, G. M. Air, B. G. Barrell, N. L. Brown, J. C. Friddes, C. A.

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The nucleotide sequence of bacteriophage 6X174. J. Mol. Biol. 125:225-246. 27. Schlagman, S., S. Hattman, M. S. May, and L. Berger. 1976. In vivo methylation by Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase protects against in vitro cleavage by the RH restriction endonuclease (R-EcoRII). J. Bacteriol. 126:990-996. 28. Studier, F. W. 1975. Gene 0.3 of bacteriophage T7 acts to overcome the DNA restriction system of the host. J. Mol. Biol. 94:283-295.

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29. Studier, F. W., and N. R. Movva. 1976. SAMase gene of bacteriophage T3 is responsible for overcoming host restriction. J. Virol. 19:136-145. 30. Vanyushin, B. F., A. N. Belozersky, N. A. Kokurina, and D. X. Kadirova. 1968. 5-Methylcytosine and 6methylaminopurine in bacterial DNA. Nature (London) 218:1066-1067. 31. Vovis, G. F., and S. Lacks. 1977. Complementary action

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of restriction enzymes EndoR.DpnI and EndoR.DpnII bacteriophage fl DNA. J. Mol. Biol. 115:525-538. 32. Watanabe, T., T. Takano, T. Arai, H. Niehida, and S. Sato. 1966. Episome-mediated transfer of drug resistance in Enterobacteriaceae. X. Restriction and modification of phages by f- R factors. J. Bacteriol. 92:477486. on