Volume 3 no.11 November 1976
Nucleic Acids Research
A restriction endonuclease from Staphylococcus aureus
J.S.Sussenbach, C.H.Monfoort, R.Schiphof and E.E.Stobberingh*
Laboratory for Physiological Chemistry, Vondellaan 24 a, Utrecht,
The Netherlands
Received 15th September 1976
ABSTRACT
A specific endonuclease, Sou 3AI, has been partially purified from
Staphylococcus aureus strain 3A by DEAE-cellulose chromatography. The
enzyme cleaves adenovirus type 5 DNA many times, SVAO DNA eight times but
does not cleave double-stranded <(>X174 DNA. It recognizes the sequence
5' ~G—A—T—C— 3 *
,,
_
_ .. and cleaves as indicated by the arrows. Evidence is presentJ
—L— 1 — A "LIT" J
ed that this enzyme plays a role in the biological restriction-modification
system of Staphylococcus aureus strain 3A.
INTRODUCTION
Recently, a number of endonucleases has been isolated which recognize
specific nucleotide sequences in double-stranded DNA and cleave the DNA at
these sites (1). These so-called restriction endonucleases facilitate the
specific fragmentation of double-stranded DNA and are very useful for DNA
sequence analysis and the unraveling of the genetic organization of viral
chromosomes. Although their name suggests that these enzymes play a role in
restriction-modification it is noteworthy that for most specific endonucleases the physiological function is still unknown and that only for a
few enzymes the actual involvement in biological restriction-modification
has been demonstrated (2, 3, 4 ) .
Staphylococcus aureus is a Gram-positive bacterium, which possesses a
biological restriction-modification system as shown by the analysis of the
propagation of different types of bacteriophages in a number of strains of
this bacterium (5; Stobberingh and Winkler, in press). The presence of a
specific restriction endonuclease, however, has not yet been demonstrated.
This communication describes the partial purification and characterization
of a specific endonuclease from Staphylococcus aureus strain 3A, which is
involved in restriction-modification.
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Nucleic Acids Research
MATERIALS AND METHODS
Materials
Lysostaphin (Schwarz-Mann, New York) was dissolved in 0.05 M Tris-HCl,
0.145 M NaCl, pH 7.4 as a stock solution of 200 U/ml and stored at -20° C.
Strains_of_bacteria_and_bacterioghages
Staphylocoaous aureus strains 3A (NCTC 8319) and 3AR~ (a restrictiondeficient mutant) were examined for the presence of specific endonucleases.
Partial characterization of the enzymes was performed with DNA's isolated
from Staphylococcal phages 6 (NCTC 8403) and 3A (NCTC 8408), adenovirus
type 5, SV40 and <(>X174.
DNA_p_regarations
DNA from adenovirus type 5 was isolated as described previously (6).
Double-stranded cj>X174 DNA and SV40 DNA were generous gifts from Dr. P.D. Baas
(Utrecht) and Dr. J. ter Schegget (Amsterdam), respectively. Staphylococcal
phage DNA was isolated from purified virions. Phage 3A was propagated in
propagating strain (PS) 3A or PS 6 and phage 6 in PS 6 as described by Blair
and Williams (7). Phage suspensions were centrifuged at 2000 x g for 30 min
to remove cellular debris and subsequently sedimented for 2 h at 90,000 x g.
The pellet was resuspended in phosphate buffered saline (PBS) plus 0.02%
EDTA, pH 7.6, then pronase (0.1 ml; 10 mg/ml) was added to 5 ml of the concentrated phage suspension and the mixture was incubated for 1 h at 37
C.
Subsequently 0.8 ml 2% sodium dodecylsulphate solution was added and the
mixture kept for 5 min at room temperature. Finally, the DNA was purified
by extraction with phenol saturated with PBS and remaining phenol was
removed by dialysis against 20 mM Tris-HCl, 0.5 mM EDTA, pH 7.5.
ce
.!I s
The method employed was a modification of a procedure described by
Klesius and Schuhardt (8). Nutrient Broth (Difco; 3600 ml) was inoculated
with 400 ml of an overnight culture of Staphyloooocus aureus. After incubation for 4 h at 37° C, the cells were spun down and washed three times with
0.05 M Tris-HCl, 0.015 M trisodiumcitrate, pH 7.4 and resuspended in 4 ml
of the same solution. Lysostaphin was added to a final concentration of
5 U/ml. After incubation for 15 min at 37° C on a rotating table the cells
were resuspended in 0.01 M Tris-HCl, 0.01 M B-mercaptoethanol, pH 7.4.
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Endonuclease §ssay_and_gel_electroghoresis
Digestions were made in 15 mM MgCl 2 , 6 mM Tris-HCl, pH 7.5, 6 mM S-mercaptoethanol, 60 mM NaCl at 30° C. After incubation sodium acetate was added
to 0.1 M and the DNA fragments were deproteinized by extraction with chloroform-isoamylalcohol (24:1). Then DNA was precipitated by addition of 2.7
volumes of ethanol and centrifugation at -5° C in a Spinco SW65 rotor at
35,000 rpm for 30 min. The DNA pellet was dissolved in a small volume of
20 mM Tris-HCl pH 7.5, 1 mM EDTA and subjected to electrophoresis in 1.4%
agarose gels (Sussenbach and Kuijk, submitted).
Se(juence_analYsis_of the_5^-termini_of_Sau_3AI_fragments
Sau 3AI fragments were produced by digestion of 7.5 pg Ad5 DNA with
Sou 3AI. After deproteinization by extraction with chloroform-isoamylalcohol
(24:1) and ethanol precipitation of the DNA, the fragmented DNA was brought
in 100 ul 40 mM Tris-HCl pH 8.5, 10 mM MgCl 2 and 10 mM dithiothreitol. Then
2 ug of bacterial alkaline phosphatase (Worthington, Freehold, N.J.) was
added and the DNA was incubated for 1 h at 37° C. Subsequently the fragmented DNA was deproteinized and precipitated as described above and again
taken up in 100 yl of 40 mM Tris-HCl pH 8.5, 10 mM MgCl 2 and 10 mM dithiothreitol. Then 10 pi (10 U) T4 polynucleotide kinase (Biolabs, Beverly, Ma)
and 5 yl y-32P-ATP (5 yCi; 16 C/mmole) (The Radiochemical Centre, Amersham,
England) was added and the mixture incubacted for 1 h at 37° C (9).
DNA was then precipitated by addition of 2 ml of 10% trichloroacetic
acid, 0.01 M PPi at 0° C and was centrifuged for 25 min at 30,000 rpm in a
Spinco SW65 rotor. The supernatant was discarded and the precipitate washed
with trichloroacetic acid as described above. This procedure was repeated
five times. Finally, trichloroacetic acid was removed by three washings
with 90% ethanol. The pellet was dissolved in 50 pi H 2 0.
For the analysis of the 5'-terminal nucleotide 0.15 yg
32
P-phosphoryl-
ated DNA was digested with 10 yg pancreatic DNase (Worthington, Freehold,
N.J.) in 10 mM Tris-HCl, 10 mM MgCl 2 , pH 7.5 for 2 h at 37° C. Subsequently
pH was raised to 9.0 and 2 yg of snake venom phosphodiesterase (Boehringer,
Mannheim) was added and incubation was continued for another 2 h at 37
C.
Under these conditions DNA is completely digested to 5'-mononucleotides.
Separation of the mononucleotides was performed by paper chromatography on
Whatman no. 1 paper (10). The chromatogram was developed using saturated
ammonium sulphate-1.0 M sodium acetate-isopropanol (80:18:2) as solvent.
Detection of the mononucleotides was facilitated by cochromatography of
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unlabeled mononucleotides. The four nucleotide spots were visualized by
UV-light, cut from the chromatogram and counted in a liquid scintillation
counter.
For the analysis of the recognition site
32
P-phosphorylated DNA was
partially digested with pancreatic DNase. The products were fractionated
according to a standard two-dimensional fractionation procedure developed
by Brownlee and Sanger (11) in a modification according to Volckaert,
Min Jou and Fiers (12). In the 1st dimension fractionation was performed by
electrophoresis at pH 3.5 on cellulose acetate and in the 2nd dimension by
homochromatography on PEI-cellulose using an RNA homomix. The RNA homomix
was prepared by hydrolysis of a 3% RNA (yeast RNA, sodium salt, BDH, Poole,
U.K.) solution at pH 12.8 for 30 min at room temperature.
RESULTS
Isolation_of an_endonuclease
Staphyloaooous aureus 3A was grown as described in Materials and
Methods. About 3 grams of packed cells were treated with lysostaphin and
after centrifugation resuspended in 8 ml 0.01 M Tris-HCl, 0.01 M g-mercaptoethanol, pH 7.4 (see Materials and Methods). It appeared that the length of
the lysostaphin treatment is rather critical for the optimal detection of
endonucleases. Extensive treatment of the cells with lysostaphin causes
lysis of the cells and very low levels of endonuclease.
After the short lysostaphin treatment the cells were disrupted by
sonication (6 x 1 min with a Branson Sonifier) at 0° C and centrifuged for
1J h at 40,000 rpm in a Spinco SW41 rotor at 4° C. The supernatant was collected and streptomycin sulphate was added for the precipitation of nucleic
acids (1.8 ml of a 10% streptomycin sulphate solution in 0.01 M Tris-HCl,
0.01 M B-mercaptoethanol, pH 7.4). The precipitate was removed by centrifugation at 35,000 rpm for 30 min in a Spinco SW41 rotor at 4° C and the
supernatant was dialyzed against 0.01 M Tris-HCl, 0.01 M B-mercaptoethanol,
pH 7.4. During the dialysis a precipitate arose which was removed by lowspeed centrifugation.
The crude enzyme preparation (9 ml) was brought on a DEAE-cellulose
column (30 ml) which was equilibrated with 0.01 M KPOi, buffer, 0.01 M
B-mercaptoethanol, 0.0001 M EDTA, pH 7.4. Elution was performed with a KC1
gradient (200 ml; 0.0-0.6 M KC1). Fractions of 5 ml were collected and tested for the presence of endonuclease as described in Materials and Methods.
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Figure 1
DEAE-cellulose chromatography of Sou 3AI. Extract of Staphyloooaaus aureus
strain 3A were prepared as described in Materials and Methods and applied
to a 30 ml DEAE-cellulose column and eluted with 200 ml 0-0.6 M KC1 gradient. Fractions of 5 ml were collected and tested alternately for endonucleolytic activity. The gradient starts as indicated by the arrow.
Elution is from left to right.
Endonucleolytic activity was eluted between 0.20 and 0.33 M KC1 (Fig. 1).
Concentration of the enzyme containing fractions was performed by
dialysis against 50% glycerol in 0.01 M KPOi, buffer, pH 7.4, 0.01 M B-mercaptoethanol, 0.0001 M EDTA. The enzyme preparations were stored at -20° C
and no detectable loss in endonucleolytic activity was detected after
storage for several months.
The yield of purified enzyme from 1 gram of packed cells was equal to
about 1000 units (1 unit is the amount of enzyme required for complete
digestion of I ug of adenovirus type 5 DNA in 2 h at 30° C ) .
Characterization of_Sau_3Al
The observation that extracts of Staphyloaocau8 aureuB strain 3A
possess endonucleolytic activity led us to a further characterization of
this specific endonuclease which was called Sou 3AI according to the proposed nomenclature for this category of enzymes (13).
Sou 3AI was characterized by its action on adenovirus type 5 DNA, SV40
DNA and double-stranded <J>X174 DNA. The results are shown in Fig. 2. It
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Figure 2
Gel electrophoresis of DNA's of different sources cleaved by Sou 3AI. DNA's
were cleaved and subjected to gel electrophoresis in 1.4% agarose gels as
indicated in Materials and Methods. Lane 1 shows digested SV40 DNA and
lane 2 digested adenovirus type 5 DNA. Lane 3 and 4 display digested and
undigested 4>X174 RF DNA, respectively. The faster moving band represents
component I and the slower moving band component II.
appears that adenovirus type 5 DNA is cleaved at least 35 times, while
double-stranded ij>X174 DNA is not cleaved by this enzyme. In the latter case
there is a conversion of the closed circular form (component I) to the open
circular form (component II) detectable but this is probably due to an
aspecific contamination in the Sou 3AI preparation. SV40 DNA is cleaved into
eight fragments of 27.2, 25.2, 17.9, 10.4, 6.7, 6.4, 4.2 and 2.0% of genome
size, respectively.
To investigate whether this enzyme was actually involved in biological
restriction-modification, the action of Sou 3AI on phage 3A DNA propagated
in PS 3A, on phage 6 DNA propagated in PS 6 and phage 3A DNA propagated in
PS 6 was determined. Furthermore, the action of crude extracts of a restriction-deficient mutant 3AR~ was studied for the presence of endonucleolytic
activity (Fig. 3 ) . Sau 3AI cleaves phage 6 DNA many times as well as phage
3A DNA propagated in PS 6, but does not cleave phage 3A DNA propagated in
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1
2
Figure 3
Digestion of different staphylococcal phage DNA's with enzyme from Staphyloooaous aureus strains 3A and 3AR . Electrophoresis was in 1.4% agarose as
described in Materials and Methods. Lane 1: phage 6 DNA * extract 3AR";
lane 2: phage 6 DNA x Sou 3AI; lane 3: phage 3A DNA x Sau 3AI; lane 4: DNA
from phage 3A propagated in PS 6 x Sau 3AI.
PS 3A. Further, the crude extract of the restriction-deficient mutant 3AR
did not contain Sau 3AI activity as indicated by the fact that phage 6 DNA
was not cleaved. These results indicate that Sau 3AI is required for restriction in PS 3A and that PS 3A and PS 6 contain modification enzymes with
different specificities. Sau 3AI cleaves DNA's with the PS 6 modification
but is not able to cleave DNA's with the PS 3A modification.
The recognition site of Sau 3AI was characterized by determination of
the 5'-nucleotide sequence of the DNA fragments produced with this enzyme.
For this purpose adenovirus type 5 DNA cleaved with Sau 3AI was first digested with bacterial alkaline phosphatase to remove 5'-phosphate groups,
and subsequently labeled with ^2P-phosphate using T4 polynucleotide kinase
and y-32P-ATP leading to introduction of a terminal 5'-32P-phosphate group
at the ends of the fragment strands (see Materials and Methods). Complete
digestion of this
32
P-labeled DNA to 5'-mononucleotides with pancreatic
DNase and snake venom phosphodiesterase followed by paper chromatography of
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G
\A
II
Figure 4
Two dimensional fractionation of oligonucleotides obtained after partial
pancreatic DNase digestion of adenovirus type 5 DNA fragments produced by
cleavage with Sau 3AI. The 5'-tenninal nucleotides of the fragment strands
were phosphorylated with 32P-phosphate using T4 polynucleotide kinase. In
the first dimension (I) fractionation is achieved by electrophoresis at
pH 3.5 on cellulose acetate and in the second dimension (II) by homochromatography on PEI-cellulose. The nucleotide sequence was derived from
the known relative mobilities (14).
the nucleotides showed that 97% of the radioactivity was present in pG and
3% in pA. For a further analysis of the 5'-terminal nucleotide sequences of
the DNA fragment 5'-terminal
32
P-labeled DNA was partially digested with
pancreatic DNase. The digestion products were separated two-dimensionally
by electrophoresis on cellulose acetate (1st dimension) followed by homochroma tography on PEI-cellulose (2nd dimension). The autoradiogram displayed
unique mono-, di-, tri- and tetranucleotide spots and multiple penta- and
hexanucleotide spots (Fig. 4 ) . Analysis showed that the fragment strands
terminated specifically with the sequence 5' G-A-T-C-N-N-.
DISCUSSION
Staphyloaoocus auveus strain 3A possesses a biological restrictionmodification system as shown by phage typing experiments (5; Stobberingh
and Winkler, in press). Extracts of Staphylocoecus auveus 3A appear to contain a specific endonucleolytic activity which can b e partially purified by
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DEAE-cellulose chromatography. This enzyme, Sou 3AI, is absent in extracts
of a restriction-deficient mutant, indicating that it is involved in biological restriction-modification. This notion is supported by the observation
that Sou 3AI does not cleave DNA from phage 3A, which has been propagated
in PS 3A, while passage of phage 3A in PS 6 makes phage 3A DNA accesible
for Sou 3AI cleavage. These observations justify the conclusion that Sou 3AI
is a restriction endonuclease which plays a role in the restriction-modification system of this bacterium.
Partial purification of Sou 3AI can easily be achieved by DEAE-cellulose chromatography while even crude extracts prepared as described in
Results are almost free of aspecific nucleases. The enzyme is very stable
at 4° C in crude extracts and at -20° C in 50% glycerol.
Analysis of the 5'-termini of Sou 3AI fragments revealed the sequence
5' G-A-T-C-N-N-. This suggests that the enzyme recognizes the sequences
5' -G-A-T-C- 3'
it n T A /-. ti
J
an
d cleaves as indicated by the arrows. An identical recog-
— U— 1— A—txT J
nition site and cleavage has also been found for an endonuclease from
Moraxella bovis {Mbo I) (R. Gelinas, G.A. Weiss, R.J. Roberts, A. Morrison
and K. Murray, in preparation). Comparison of DNA fragments of adenovirus
type 5 and SV40 obtained by cleavage with Sau 3AI and Mbo I, respectively
confirmed that these enzymes recognize the same nucleotide sequence. A specific restriction endonuclease from DipZococcus pneumoniae (Dpn II) also
recognizes the above sequence (S. Lacks, personal communication).
ACKNOWLEDGEMENTS
The authors thank J.M. Vereijken, A.D.M. van Mansfeld, P.H. Steenbergh
and M.G. Kuijk for assistance and Profs. K.C. Winkler and H.S. Jansz for
interesting discussions and critical reading of the manuscript.
This investigation was supported in part by the Netherlands Foundation for
Chemical Research with financial aid from the Netherlands Organization for
the Advancement of Pure Research.
"Laboratory of Microbiology, State University of Utrecht, Utrecht,
The Netherlands
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