DNA-methyltransferase activities in Streptomyces antibioticus

FEMS Microbiology Letters 55 (1988) 59-64 Published by Elsevier

59

FEM 03268

DNA-methyltransferase activities in Streptomyces antibioticus C o vad o n g a Barbes, Carlos Hardisson, Isabel S. Novella, M. Jesus Y ebra and Jesus Sanchez Departamento de Biologla Funcional, Area de Microbiologla, Facultad de Medicina, 33006, Oviedo, Spain

Received 24 April 1988 Accepted 28 April 1988

Key words: Streptomyces antibioticus, DNA-methyltransferase

1. SUMMARY Several DNA methyltransferases have been detected in Streptomyces antibioticus ETHZ 7451. One of these enzymes was purified and partially characterized from crude extracts of Streptomyces antibioticus ETHZ 7451. This enzyme only methylates the adenine residues of DNA. However, ~ and pBR322 DNAs were not protected 'in vitro' with the methylase, indicating that the enzyme does not seem to form part of a restriction-modification system. Remarkably, the efficiency of the enzyme is significantly higher on the DNA isolated from aerial mycelium than from substrate mycelium in S. antibioticus,

2. INTRODUCTION The occurrence of small amounts of the methylated nucleotide bases, N-6-methyladenine and 5methylcytosine in the DNA of bacteria, plants and animals is well documented [1]. Enzymes that modify these minor bases have been isolated and have been shown to catalyze transfer of the methyl Correspondence to: C. Hardisson, Departamento de Biologla Funcional, Area de Microbiologla, Facultad de Medicina, 33006, Oviedo,Spain.

groups from S-adenosyl-methionine (SAM) to the appropriate nucleotide residues. Sequence specific DNA methyltransferases have been, on the other hand, partially purified from various Gram-negative and Gram-positive bacteria [2]. Most of them are associated with restriction systems and serve to protect host DNA against the action of endogenous nuclease activity [3,4]. Others, with different functions have been found in Escherichia coli: an adenine methylase modifying the GATC sequence, encoded by the dam gene and a cytosine methylase acting on the CC(A / T )G C sequence, encoded by the dcm gene [2,5]. The genus Streptomyces is an interesting group of bacteria presenting a complex developmental cycle with several stages that include the formation of a vegetative or substrate mycelium and a reproductive or aerial mycelium (6-8). On the other hand they produce more than 50% of the known antibiotics [9]. At the present we are studying the restriction-modification systems of several species of this genus and their application to the development of cloning vectors. In relation to this, we have previously detected in several strains of Streptornyces antibioticus different deoxyriboendonucleolytic activities ([101; De los Reyes-Gavil~m, C.G. et al., J. Bacteriol. 170, in press), which could be related to an in vivo restriction mechanism. As no studies exist regarding DNA modifying en-

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6O zymes accompanying restriction endonucleases in Streptomyces, we have initiated the purification and characterization of DNA-methylases from S. antibioticus ETHZ 7451. One enzyme is described, which seems not to form part of the enzymatic restriction system encountered in the same strain, The function of this methylase is currently under investigation,

3. MATERIALS A N D M E T H O D S

Strains and DNA sources. The strain used in this study was Streptomyces antibioticus E T H Z 7451 (Eidgen~ssische Technische Hochschule, Ziirich) and pBR322 DNAs were provided by Boehringer. Denatured D N A was obtained after heating the solutions for 10 rain at 1 0 0 ° C and cooling in ice. Purification of DNA methyltransferases. Streptomyces antibioticus E T H Z 7451 was grown at 30 ° C with aeration for 24 h in a complex medium [10] and ceils were harvested by centrifugation. Approximately 60 g of cells were resuspended in 200 ml of a buffer containing 20 mM Tris-HC1 (pH 8.0), 10 mM MgC12 and 7 mM 2-mercaptoethanol. The cells were disrupted by sonication and the extract was clarified by centrifugation at 10000 r.p.m, for 20 min followed by ultracentrifugation at 195000 × g for 2.5 h at 4 ° C. Nucleic acids were precipitated by the addition of polyethyleneimine and proteins were precipitated by addition of solid ammonium sulphate [11]. The dialysed extract was applied to a column of DEAE-Sephacel (bed volume 60 ml) and eluted with a 600 ml gradient of NaC1 between 75 mM and 800 mM. The enzyme-containing fractions were dialysed against buffer A (20 mM Tris-HC1, p H 8.0, 75 m M NaC1, 7 m M 2-mercaptoethanol, 0.1 mM E D T A and 10% glycerol), applied to a column of heparin-agarose (bed volume 15 rnl) and eluted with a 150 ml gradient of NaC1 between 75 mM and 500 mM. The active fractions from this column were concentrated by dialysis in polyethylenglycol 6000 and stored at - 2 0 ° C in buffer containing 20% glycerol,

In vitro DNA methylation. The reaction mixture contained 50 mM Tris-HC1 (pH 8), 7 m M 2mercaptoethanol, 1 mM EDTA, 12 /LM 3H Sadenosyl-L-methyl-methionine (Amersham, specific activity 84 Ci/mmol), 1/xg of X-DNA and 2 ttl of enzyme extract (about 2/~g of protein). Incubation was at 37 ° C for 1-2 h and methylated DNA was detected by two different methods: precipitation of D N A with ethanol and N H 4 acetate after protein extraction with phenol and by separation from low molecular weight radioactive material in a small column of Biogel A-0.5 M [11]. The labelled D N A was detected by liquid scintillation counting. Analysis of methylated bases. Methylated bases were analysed after formic acid hydrolysis of the labelled methyl-3H XDNA. 2 #g of this were dried and incubated at 175°C for 35 min with 0.2 ml of 90% formic acid. The tube content was then evaporated to dryness and the residue redissolved in about 10 #1 of HC1 0.1 N, containing 5 m g / m l of unlabelled optical density markers for the bases T, 5 mC, and N-6 mA. The sample was spotted on the corner of a 20 cm × 20 cm glass of thin layer cellulose. Chromatography was carried out in butanol (86 ml), ammonia (1 ml) plus water to 100 ml followed by isopropanol, HCI and water (85 : 20 : 19, v / v ) in the second dimension. Ultraviolet light absorbing spots were located with a short-wave U.V. lamp, scraped and suspended in 0.45 N perchloric acid. After extraction for 20 min at 8 0 ° C the supernatant was assayed for radioactivity in toluene scintillation medium. Assay for "in vitro" modification. In order to investigate if the methylase protected against the restriction endonuclease ([10]; J. Shnchez and C. Barb&, unpublished data), modification was assayed as follows: 1/~g of D N A and pBR322 DNA were methylated, as described above. After 2-3 h of incubation, control and sample DNA were precipitated and resuspended in S. antibioticus endonuclease buffer (20 mM Tris-HC1, pH 7.8, 10 mM MgC12" 6H20- I mM dithiothreitol, 50 mM NaC1), 2 #1 of the partially purified restriction enzyme was added to both control and sample, and the reaction proceeded for 1 h at 37°C. Agarose electrophoresis was carried out and DNA bands observed in U.V. light.

61

4 Es LTs

m O Q

Purification of Streptomyces DNA methyltransferases. M e t h y l a s e activity eluted in a u n i q u e p e a k at 0.6 M NaC1 in the D E A E - S e p h a c e l c o l u m n . T h e e n z y m e - c o n t a i n i n g fractions were d i a l y z e d a n d a p p l i e d to a s e c o n d c o l u m n o f h e p a r i n agarose; in this case we o b s e r v e d two p e a k s of m e t h y l a s e activity that eluted respectively at 0.25 a n d 0.5 M of N a C I . C o n c e n t r a t e d active fractions o f the first p e a k showed a single b a n d after S D S P A G E (Fig. 1); m o l e c u l a r weight was a b o u t 28 k D a . The second p e a k of activity gave a b a n d c o r r e s p o n d i n g to 32 k D a , however, kinetic d a t a a n d the fact t h a t b o t h c y t o s i n e a n d a d e n i n e were s i m u l t a n e o u s l y m e t h y l a t e d i n d i c a t e d that two or m o r e D N A m e t h y l t r a n s f e r a s e s were p r e s e n t in this p e a k ( d a t a n o t shown). N o further s e p a r a t i o n of these activities was a t t e m p t e d , Properties of DNA methylase. I n o r d e r to e s t a b lish the o p t i m a l c o n d i t i o n s of the r e a c t i o n for the p u r i f i e d enzyme, we have tried different conc e n t r a t i o n s of N a C I f r o m 10 m M to 120 m M . T h e o p t i m a l was 30 m M a n d the limits of r e a c t i o n were b e t w e e n 20 a n d 50 m M . W i t h r e s p e c t to S - a d e n o s y l - m e t h i o n i n e c o n c e n t r a t i o n s , limits of r e a c t i o n were b e t w e e n 6 / * M a n d 1 2 / * M , b e i n g 10 / , M to 12 / , M the o p t i m a l . O n the o t h e r h a n d ,

F

1

2

'

3

= =

~

8rnA

~ lo x. E a ~ 6 _~ ~ 4 o ~ 2 ~-~ F---I Contr--~ol Fig. 2. Analysis by thin layer chromatography of the base residues methylated by incubation of ~ DNA with purified Streptomycesmethylase. Only the results obtained after chromatography in the first dimension are shown. The upper part shows the disposition of the bases after migration (from left to right). Lower part, radioactivity obtained after scraping the spots. a d d i t i o n o f M g 2+ to the b u f f e r d i d n o t p r o d u c e s t i m u l a t i o n of the reaction. A n a l y s i s of the kinetics of the r e a c t i o n with respect to i n c u b a t i o n time, DNA concentration and methylase enzyme showed a t y p i c a l s a t u r a t i o n curve (not shown). O n the o t h e r h a n d , cellulose t h i n - l a y e r c h r o m a t o g r a p h y of m e t h y l a t e d ~ D N A s h o w e d a d e n i n e as the o n l y

~

67

D

/13

Table 1

",

2 0 . '!

.'~P

14 • 4

4

Substrate

....... ~+

r

m e t h y l a t e d b a s e (Fig. 2). T h e enzyme, as occurs with m o s t p r o k a r y o t i c m e t h y l a s e s was n o t c a p a b l e of using h e a t - d e n a t u r e d D N A as a s u b s t r a t e (Tab l e 1). It is i n t e r e s t i n g to n o t e that D N A f r o m S. antibioticus was m e t h y l a t e d in vitro b y the enzyme, b e i n g the D N A f r o m aerial m y c e l i u m a significantly b e t t e r s u b s t r a t e ( T a b l e 1).

~

i

G 5mc

Fig. 1. SDS-PAGE of protein fractions in the course of purification of the methylase from S. antibioticus ETHZ 7451. Numbers: l, dialyzed cell-free extract; 2, pooled active fractions after DEAE-Sephacel chromatography; 3, pooled active fractions after Heparin-agarose chromatography; 4, standard markers for molecular mass. Mr (kDa) are indicated in numbers.

DNA Heat denatured ~ DNA Substrate mycelium Aerial mycelium

Methyltransferase activity (c.p.m.) 7500 613 550 4121

Reactions were carried out as described in MATERIALSAND METHODSusing 3 /*1 of concentrated enzyme in glycerol per reaction mixture and 2 /*g of DNA. Mixtures were incubated for 2 h. Results are given in c.p.m. Data are the average of three experiments.

62 Lack of protection against a self-containing restriction system. Most of the methylating enzymes that have been characterized in procaryotes correspond to a restriction-modification system. The strain used in this report, S. antibioticus E T H Z 7451, has a type II restriction endonuclease, whose function in biological restriction is currently being investigated (J.F. Aparicio, C. Barbrs, C. Hardisson and J. Shnchez, in preparation). We have studied if the S. antibioticus methylase shares the specificity of this endonuclease, by performing an in vitro methylation of the substrates before hydrolysis. No protection has been found using pBR322 DNA as substrate (Fig. 3). On the other hand, hydrolysis of the former D N A with the endonuclease, previous to the methylation, did not inhibit this (data not shown), thus, clearly proving that the recognition sequences are different for methylase and the endonuclease activities, A possibility exists that the isolated methylase was a multifunctional enzyme, with a double ac-

1

2

Fig. 3. Agarose gel electrophoresis of methylated and unmethylated pBR322 DNA (1/~g) after enzymaticdigestion with 2 ~tl of a partially purified enzymaticpreparation of S. antibioticus ETHZ 7451 Type II endonuclease. Numbers: 1. Methylated DNA; 2. Non-methylated DNA.

tivity of modification and restriction (something similar to type I or type III restriction-modification enzymes). To test this, several reactions were attempted in the presence of different cofactors, such as ATP (0.7 m M and 1.5 raM), Mg 2+ (7 mM) and SAM (12 /~M), and the possible hydrolytic activity analyzed. No restriction was observed in any of the above conditions.

5. DISCUSSION We describe some characteristics of a DNA methylase isolated from S. antibioticus. This is, to our knowledge, the first enzyme of this type isolated from Streptomyces. Ionic requirements for the activity (NaC1 and Mg 2+) were similar to those described for most of the prokaryotic DNA methylases, i.e., maximal activity in absence of NaC1 or at low concentrations (20-50 mM) and no requirement for Mg 2+ ions. This cation is not necessary, in general, for activity of Type II DNA methylases in bacteria, being stimulatory in some isolated examples, as in Bacillus subtilis [12] and inhibitory in others, such as M. B a m H I [12]. The enzyme from S. antibioticus does not methylate denatured DNA. Some bacterial DNA methylases are active on single-stranded DNA, but this property is not general in prokaryotic enzymes [3]; both H a e I I I and HpaII methylases (and also the dam methylase of E. coli) have been found to methylate denatured salmon sperm and T7 DNAs although at slightly lower rates [13,14]. B. subtilis, however, efficiently methyIates heat-denatured DNA, whereas other enzymes from Bacillus, such as M. B a m H I , require duplex D N A as a substrate [3]. Eukaryotic mammalian D N A methylases are equally active on native and denatured D N A [3]. No function has been assigned to S. antibioticus D N A methyltransferase. The enzyme does not belong to the hypothetical restriction-modification system from which the only Type II endonuclease isolated from the same strain should be a component. It is interesting to point out that the second peak of D N A methylase activity encountered in the same strain (see RESULTS) does not seem to protect D N A substrates either from the activity of

63 that endonuclease (C. Barb6s, data unpublished). The same occurs after simultaneous methylation with both activity peaks. In addition, no type I nor type I I I restriction activities are associated with the methylase activity. Obviously, another R-M system or systems could remain undetected in this strain; however, the fact that the methyltransferase methylated the D N A from the same strain supports the idea of a functional difference from R-M systems. There are significant examples in bacteria, where D N A methylase activities lack the counterpart of a restriction endonuclease: Bacillus subtilis [15], E. coli [16], Haemophilus influenzae |17], B. amyloliquefaciens and B. brevis [18] and Neisseria gonorrhoeae [19]. One of the possible functions for the S. antibioticus methylase could be one similar to that of DNA-methylases coded by gene darn or dcrn in E. coli [2,5]. D N A methylase specificities analogous to darn or dcrn have not been encountered in B. subtilis or S. aureus [20]. Streptomyces, as other actinomycetes, also lacks a dam and dcm system (J. Sanchez, unpublished results). On the other hand, D N A from differentiating aerial mycelium of S. antibioticus seems to be hypomethylated in relation with the D N A from substrate mycelium (Table 1). This fact gives rise to the suggestion of a hypothetical involvement of D N A methylation in Streptornyces development. In other bacteria, as in B. subtilis [12,21,22] or Caulobacter bacteroides [23], a correlation of the appearance of D N A methylase with the physiological state of cultures has been observed. A different pattern of D N A methylation in spore D N A and vegetative cell D N A was also observed in B. coagulans [24]. In the wall deficient bacteria Spiroplasrna, the extent of D N A methylation in logarithmic and stationary phase varies [25]. An analogous phenomenon occurs in several fungi and yeast, with respect to the dormant sclerotia or conidia, yeast form and metabolically active mycelia [26-28]. All these data suggest the possibility of different roles (perhaps related to the transcriptional gene activity) for the distinct enzymes in processes leading to the perpetuation of D N A methylation patterns in the above organisms. The existence of a developmental modulation of D N A methylases in differentiating S. antibioticus is currently under investigation.

ACKNOWLEDGEMENTS C. Barb6s was a recipient of a EMBO short-term fellowship at the Microbiology Department, Biozentrum, Basel. We wish to thank Dr. T.A. Bickle for his invaluable encouragement and advice during this work and also for the critical reading of the manuscript. I.S. Novella was supported by a fellowship from 'Iniciaci6n a la Investigaci6n', F o n d o de Investigaciones Sanitarias de la Seguridad Social, Spain, and M.J. Yebra by a fellowship of the 'Plan de Formaci6n de Personal Investiga, dor', Ministerio de Educaci6n y Ciencia, Spain. Research was supported by grant P1385-0403 from the CAICYT, M.E.C., Spain.

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