FEMS Microbiology Letters 138 (I 996) 123- 127
Characterization of a X/z01 isoschizomer in Streptomyces aureofaciens after actinophage infection Andrej Godhy
a’*, Peter Pristag b, Bibiana Oktavcov5 a, Jarmila FarkoSovskh a, Monica Ziffov6 a, Beatrice Sevc’ikovB a
ofSciences, Dlibravskb cesta 21, BratislaL,a, Slovak Republic ’ Institute of Animal Physiology Slol,ak Academy of Sciences, ioltt%ocej 4-6, KoSice, Slovak Republic
a Institute of Molecular Biology Slol,ak Academy
Received 1I December 1995; revised 15 February 1996; accepted 20 February 1996
Abstract After infection of tetracycline producing strains of S. uureofaciens with actinophages ~1/6 and B 1 some phage resistant colonies were obtained in each experiment. These colonies expressed a new restriction-modification (RM) system of type II,
which was different from the common RM system (SauLPI) of these strains recognizing the sequence GCCGGC. This new RM system was not detected before in parental strains. The new endonuclease was purified from a phage resistant strain of S. aureofaciens B96, using two step column chromatography to the grade without non-specific nucleolytic activity. SauLPI endonuclease recognized and cleaved the palindromic hexanucleotide sequence S-C/TCGAG-3’, thus it was a true isoschizomer of XhoI. KeJw0rd.c: Streptomycetes; Phage infection; Restriction endonuclease: XhoI
1. Introduction Restriction-modification (RM) systems were found in more than 3000 bacterial species. It is generally accepted that their primary role is to protect host bacteria against phage infection [I]. Thus phages are very useful tools for biological detection of RM systems, above all when their expression is insufficient for biochemical detection. The regulation of expression of RM systems seems to be very important for cells surviving. It is not yet well understood for any RM system. Control genes (C-genes) of the RM system have been demonstrated
* Corresponding author.
partially only in cloned PwII and BumHI RM systems [2,3]. Bacteria of genus Streptomyces spp. are filamentous soil bacteria of industrial importance. It has been shown that they are a good source of restriction endonucleases . Possibly, the RM systems also play an important role in recombination and subsequently in enhancing genetic diversity within a population , since some streptomycetes are known to be very unstable. There are limited data on the regulation of activity of RM systems in streptomycetes as only a single system has been cloned and analyzed at the molecular level so far [6-81. Recently we have shown that tetracycline (TC)producing strains of S. aureofuciens express a common RM system (SauLPI) of type II recognizing the
037% 1097/96/$12.00 0 1996 Federation of European Microbiological Societies. All rights reserved PII SO378-1097(96)00077-8
nucleotide sequence GCCGGC . In this report, we describe the appearance of a silent RM system after phage infection in S. aureofuciens mutant strains, as well as the characterization of the induced restriction endonuclease. This endonuclease recognized the DNA nucleotide sequence %C/TCGAG-3’ and it was designated a SauLPI (isoschizomer XlzoI).
LKB. I .2 X IO cm). The endonuclease activity was eluted with 160 ml of a linear gradient of KC1 (O-O.6 M) in buffer A. Active fractions were pooled and applied to a phosphocellulose column (Whatman Pl 1. I .2 X IO cm). Fractions containing restriction activity were dialyzed against storage buffer (IO mM KH,P0,/K2HP0,. pH=7.0; 0.1 mM EDTA; I mM NaN,; 7 mM 2-mercaptoethanol, 50% glycerine) and stored at -20°C.
2. Material and methods 2.3. Determination 2.1. Bacrerial srrains. phages. conditions
S. aureqfuciens strains BMK, NMU and B96 were TC-producing, phage sensitive strains . Phage resistant mutants of individual strains. obtained through this study. were designated BMK-PR. NMU-PR and B96-PR. S. uureofaciens 3239 was a TC-nonproducing, phage resistant strain expressing the RM system Suu32391 (isoschizomer XlujI) [lo]. All strains used were from the collection of microorganisms at the Institute of Molecular Biology (Bratislava). pl/6 and B I were Iytic host-specific actinophages used in this study [I I]. Streptomycete strains were grown in GPY medium [IO] at 30°C for 8 h. Actinophages p I /6 and B 1 were propagated according to the methods described by Hopwood et al. [ 121. Phage infections were done by surface plating technique [ 121. 2.2. Restriction
growth und it+ction
60 g (wet weight) of cells were resuspended in 100 ml of buffer A (IO mM K,HP0,/KH2P0,, 1 mM EDTA. 7 mM 2-mercaptoethanol; pH = 7.0) and disintegrated by sonication (Soniprep 150 homogenizer; 10 bursts for 30 s at 0°C). Bacterial debris was removed by centrifugation for 1 h at 100000 X g. Streptomycin sulfate (final concentration 1.2%) was then added to the supernatant and the nucleic acid precipitate was removed by centrifugation. The resulting supernatant was brought to 70% saturation with (NH,J2S0,. The precipitated proteins were collected by centrifugation and dissolved in buffer A, dialyzed and subsequently applied to a heparin-Sepharose column (Pharmacia
The recognition sequence of SauLPII restriction endonuclease was identified by digestion of pBluescriptIIKS+ and ADNA. Their restriction patterns were compared with those obtained with known restriction endonucleases. The position of the cleavage site within the recognition sequence was determined by the examination of a primed synthesis reaction using pBluescriptIIKS’ as a substrate [ 131. Sequencing reactions were performed as described by Sanger et al. [ 141. The fifth reaction containing no dideoxy nucleotides was extended through the SauLPII site. The double stranded DNA recovered from the fifth reaction was used as a substrate for the isolated endonuclease. The resulting cleavage products were separated on a sequencing gel alongside the sequencing reaction. 2.4. DNA preparation
Isolation of total DNA from S. aureofaciens was carried out according to Hopwood et al. [ 121. Restriction endonucleases were isolated as described previously  and used under optimal conditions.
3. Results and discussion Actinophages PI/~ and B 1 showed an extremely narrow host range. They can only infect TC-producing strains of S. aureofaciens Ill]. In typical infection experiments phage resistant mutants of all strains analyzed arose with a frequency of about 1Oph. Analysis of their restriction activity revealed that all of these mutant strains expressed a new, previously unidentified restriction endonuclease. It was shown that in all of them the second restriction enzyme is
A. [email protected]
et al./ FEMS Microbiology Letters 138 (1996) 123-127
present and was able to cleave actinophage DNAs (data not shown). In the B96-PR strain of 5. aureofaciens the restriction endonuclease was further purified and characterized. This restrictase was named SauLPI according to the nomenclature used in our previous work . Restriction endonuclease SauLPI was purified from cell extracts of S. aureofuciens B96-PR without contaminating nucleases. Recovery of SuuLPII was rather low, about 1000 U per g of cells. The purified restriction endonuclease was tested under a variety of reaction conditions and it showed maximal activity at 37°C within a pH range of 7.5-8.0. SuuLPII was rather insensitive to salt concentration; no changes in activity were observed within a salt concentration range of O-250 mM NaCl or KCl. The purified SuuLPII was used to digest various DNA substrates. The digestion profiles of and ADNA with SuuLPII indipBluescriptIIKS+ cated that this restriction endonuclease could be an isoschizomer of XhoI , having the recognition sequence S-CTCGAG-3’. A digest of Su~lLP11 and X/z01 on ADNA confirmed that these enzymes are isoschizomers (Fig. 1). Sequence analysis of a SuuLPII cleavage site within the polylinker region of pBluescriptIIKS+ plasmid indicated that restriction endonuclease
cleaved DNA after the first C in the sequence 5’C/TCGAG-3’ (Fig. 2, lane I). After the reaction with Klenow fragment, the migration distance of cleaved products was changed (Fig. 2, lane II). Therefore SuuLPII recognized and cleaved the following sequence: 5’-C/TCGAG-3’ 3’-GAGCT/C-5’ Modification activities in S. uureofuciens strains were analyzed using digestion of chromosomal DNAs (modified in viva) by restriction endonucleases (Table 1). The digestion of chromosomal DNAs isolated from both parental and resistant strains with SuuLPII indicated that parental strains were deficient in SuuLPII cognate methylation. The DNAs from parental strains were digested, while those from phage resistant strains were not cleaved by SuuLPII. Thus both genes coding for the SuuLPII RM system were activated after actinophage infection only. Once activated, the second RM system was stably expressed in new strains without any selection pressure provided by actinophage action. There has to be some regulatory facto&) keeping these RM system genes repressed or regulated in some way. This suggestion can be supported by the detection and characterization of the RM system Sun32391 (another XhoI isoschizomer) in the TC-
XhoI Hind111 Hind111
J3coRI + NluI
Fig. I. Gel electrophoresis
of the ADNA digest.
A. God&y et 01./ fi~EMS Micrrhiolqq
Letters 138 (I 9961 123-127
II G G
G G -* A G C SauLPI T C -C A G C T G C C A .T 5’
Fig. 2. Determination of the SauLPI cleavage position within the recognition sequence: ssDNA of pBluescriptIIKS+ plasmid and the universal -40 sequencing primer were used for standard dideoxy DNA sequencing reactions (lanes G, A. T and C). II and I denote extension product cleaved with SUULPII and treated with Klenow fragment (lane II). or untreated (lane I). Lane X: extension product cleaved with Xhol.
nonproducing S. aureqfaciens 3239 [lo]. This RM system was detected without actinophage action. The similarity of the optimal reaction conditions for SauLPII and Sau3239I [ 161 and the resistance of S. aureqfaciens 3239 chromosomal DNA against cleavage by SauLPII indicated the presence of a second common RM system in S. aureofaciens strains. We can speculate on the molecular basis of its regulation. Possibly, a mechanism similar to those observed in Streptococcus pneumoniae [ 171 is involved Table I Cleavage
DNA from different by endonuclease
B96/GCCGGC S. aureqfaciens B96-PR/GCCGGC, S. aureofaciens BMK/GCCGGC S. aureofrrciens BMK-PR/GCCGGC. S. uureofariens NMU/GCCGGC S. aureqfaciens NMU-PR/GCCGGC, S. uureofuciens 3239/CTCGAG -
CTCGAG CTCGAG CTCGAG
denotes DNA is resistant to cleavage.
SauLPII RM system. The cloning RM systems in S. aureofaciens needed to understand the appearRM system.
Acknowledgements This work was in part supported from research grant of Biotika Slovenskd L’upEa. We are indebted to K. Straussova for technical assistance.
in switching on the of genes encoding strains is therefore ante of this second
Cleavage by SauLPI GCC/GGC
_ _ _ _ _
+ denotes DNA is cleavable
+ _ + _ -
A. God&y et al. / FEMS Microbiology Letters 138 (1996) 123-127
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