Restriction and modification of SP10 phage by BsuM of Bacillus subtilis Marburg

FEMS Microbiology Letters 244 (2005) 335–339 www.fems-microbiology.org

Restriction and modification of SP10 phage by BsuM of Bacillus subtilis Marburg Satoshi Matsuoka, Kei Asai, Yoshito Sadaie

*

Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Saitama 338-8570, Japan Received 6 January 2005; received in revised form 2 February 2005; accepted 3 February 2005 First published online 14 February 2005 Edited by A. Klier

Abstract Bacillus subtilis Marburg has only one intrinsic restriction and modification system BsuM that recognizes the CTCGAG (XhoI site) sequence. It consists of two operons, BsuMM operon for two cytosine DNA methyltransferases, and BsuMR operon for a restriction nuclease and two associated proteins of unknown function. In this communication, we analyzed the BsuM system by utilizing phage SP10 that possesses more than twenty BsuM target sequences on the phage genome. SP10 phages grown in the restriction and modification-deficient strain could not make plaques on the restriction-proficient BsuMR+ indicator strain. An enforced expression of the wild type BsuMM operon in the BsuMR+ indicator strain, however, allowed more than thousand times more plaques. DNA extracted from SP10 phages, thus, propagated became more but not completely refractory to XhoI digestion in vitro. Thus, the SP10 phage genome DNA is able to be nearly full-methylated but some BsuM sites are considered to be unmethylated.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Bacillus subtilis; SP10; BsuM

1. Introduction Bacillus subtilis Marburg 168 strain possesses an inherent type II restriction and modification system BsuM that recognizes the CTCGAG sequence (XhoI site) [1–7]. The BsuM system consists of two separate operons responsible for modification (BsuMM) and restriction (BsuMR) in the 13 kb-long prophage 3 region of the chromosome between groEL and gutR [8,9]. They are the BsuMM (ydiO–ydiP) operon with the predicted products YdiO and YdiP for cytosine DNA methyltransferases, and the BsuMR (ydiR–ydiS–ydjA) operon *

Corresponding author. Tel.: +81 48 858 3399; fax: +81 48 858 3384. E-mail address: [email protected] (Y. Sadaie).

with the predicted product YdiS for a restriction nuclease [6,8]. While YdiR has no ortholog, YdjA has orthologs in Bacillus cereus and Helicobacter pylori, Lactococcal plasmid pNP40, Streptococcus pyogenes [10–13]. Both ydiS and ydjA genes are retained in these bacteria. Restriction of the /105 phage genome was weak [1] probably because of few restriction sites on the phage genome, as efficient restriction required the presence of multiple target sites for XhoI (BsuM) on a plasmid pHV105 DNA [4]. SP10 phage genome possesses more than twenty XhoI sites and is considered to be useful for the study of BsuM activity [7]. SP10 phage infects and multiplies in the nonA–nonB strain [2]. Mutant nonA allele is found to be a cured state of SPb, and the nonB allele is a nonsense mutation in ydiR, the first gene in the

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2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.02.006

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S. Matsuoka et al. / FEMS Microbiology Letters 244 (2005) 335–339

BsuMR operon, therefore SP10 infection requires the cured state of SPb and a defective BsuMR [7]. Furthermore, the BsuMM (ydiO–YdiP) operon locates downstream to the groESL operon. Deleting the transcription termination signal sequence of the groESL operon resulted in an enhanced read-through from the groESL operon to the ydiOP operon. That leads to an enhanced transcription of the ydiOP operon and protection of XhoI sites of the plasmid pHV1401 from BsuMR digestion [6]. This communication describes the use of SP10 phage for a further analysis of the BsuM system of B. subtilis.

2. Materials and methods 2.1. Bacterial strains, phages and plasmids used Bacterial strains and plasmids used in this study are described in Table 1. Phage SP10 [14] was propagated through B. subtilis Marburg strain.

Table 1 Bacterial strains and plasmids used Strain or plasmid Bacillus subtilis 168 Marburg [6] HLL3g [7] BSU1 [6] BSU2 [6] BSU3 [6] BSU4 [6] BSU5 [6] BSU9 [6] BSU51 BSU52

IPTG inducible promoter was introduced in the promoter region of ydiR–ydiS–ydjA operon by integration of pMUTIN NC plasmid [15,16] carrying N terminal region of the ydiR gene with SD sequence, which was synthesized by PCR with primer pairs, 5 0 -AAGAAGCTTGCTTGTGTGATTTTATGGGG and 5 0 -CGCGGATCCATTTATGTTTTATCTTA. 2.3. Cytological observation Phase contrast microscopy and DAPI stained fluorescence microscopy were performed by the methods described elsewhere [17]. Cells grown in 2 ml LB broth were collected by centrifugation and suspended in 200 ll fresh LB broth. 1 ll DAPI (50%, Sigma) was added and the sample was kept on ice for 15 min. Cells were fixed on object slide coated with a layer of 1% agarose. Fluorescence images were viewed with UV-1A filter (excitation 365/10 emission 400, Nikon).

3. Results and discussion

Genotype and/or relevant phenotypea

3.1. Restriction and modification of phage SP10 in vivo

BSU54b BSU55b BSU56b BSU57b BSU58b BSU59c BSU60d

trpC2 nonA thr met lys leu his trp ade trpC2 ydiO::pet24b (km) ydiS::pMUTIN2 (em) trpC2 ydiP::pet24b (km) ydiS::pMUTIN2 (em) trpC2 ydiR::pMUTIN2 (em) trpC2 ydiS::pMUTIN2 (em) trpC2 ydjA::pMUTIN2 (em) trpC2 Dter(groESL) trpC2 ydiR::pMUTIN NC(Pspac-ydiR em) trpC2 ydiO::pet24b (km) ydiR::pMUTIN NC (Pspac-ydiR em) ydiO::pet24b neo (km) ydiS::pMUTIN2 (em) ydiP::pet24b neo (km) ydiS::pMUTIN2 (em) ydiR::pMUTIN2 (em) ydiS::pMUTIN2 (em) ydjA::pMUTIN2 (em) Dter(groESL) Dter(groESL) ydiS::pMUTIN2 (em)

Escherichia coli C600 [6]

thi-1 thr-1 leuB6 lacY1 tonA21 supE44

Plasmids pMUTIN2 [15]

em ap

a

2.2. Construction of IPTG inducible BsuMR (ydiR–ydiS– ydjA) operon on the chromosome

km, em, and ap: kanamycin, erythromycin, and ampicillin genes, respectively. b These strains were km and/or em transformants of HLL3g with DNA from BSU1, BSU2, BSU3, BSU4, or BSU5 DNAs, and carried the same genetic markers as those of HLL3g. c Lys+ Dter(groESL) transformants of HLL3g with DNA from BSU9. d Eryr transformant of BSU59 with DNA from BSU4. BSU59 and BSU60 carried the same genetic markers as those of HLL3g except for lys.

SP10 can infect and multiply in B. subtilis cells, which are SPb free and possess a defective BsuMR restriction system [7]. To see the effect of the modification or restriction activity of the BsuM system on SP10 phage multiplication, all the indicator strains were cured of SPb. As shown in Table 2, SP10 grown in restriction–modification defective strain BSU54 (ydiO–ydiS) (m
r
) could not grow in the wild type cell (PFU less than 5/ml) but could grow in the cell of restriction-defective strains, BSU56 (ydiR), BSU57 (ydiS), and BSU58 (ydjA) (PFU, ca. 109). These three disruptants possess wild type modification genes ydiO and ydiP. Disruption of ydiO or ydiP requires disruption of one of the following genes: ydiR, ydiS, or ydjA. Modification-deficient strains BSU54 (ydiO–ydiS) and BSU55(ydiP–ydiS), which are restriction-defective, also support the growth of above SP10 phage (PFU, ca. 109). Even in the presence of the wild type restriction system, the strain BSU59 (Dter(groESL)), carrying a deletion in the transcription termination signal sequence of the groESL operon, allowed plaque formation of SP10 phages. The number of plaque forming unit increased more than ten thousand times. This is probably because of enhancement in methylation activity through enhanced read-through from groESL operon to BsuMM operon [6].

S. Matsuoka et al. / FEMS Microbiology Letters 244 (2005) 335–339

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Table 2 Restriction and modification of SP10 phage Number of plaques · 109/ml

Indicator

Phages grown in BSU54 (M
R
) +

+

HLL3g (M R ) BSU54 (M
R
) BSU55 (M
R
) BSU56 (M+R
) BSU57 (M+R
) BSU58 (M+R
) BSU59 (M+R+)
Heat BSU59 (M+R+) + Heat BSU60 (M+R
)


9

<5 · 10 1.88 3.56 2.32 2.45 2.04 1.70 · 10
4 1.00 · 10
3 4.36

BSU57 (M+R
)
9

<5 · 10 1.76 3.11 2.39 1.95 2.11 1.60 · 10
1 1.34 3.83

BSU59
Heat (M+R+)
9

<5 · 10 0.50 0.40 0.41 0.48 0.49 0.52 0.60 0.69

BSU59 + Heat (M+R+) <5 · 10 9.69 9.26 7.78 8.73 9.40 6.92 7.30 10.7


9

BSU60 (M+R
) <5 · 10
9 2.33 4.24 3.13 2.80 2.62 0.27 0.90 5.62

SP10 phage propagated in the wild type HLL3g, BSU54, BSU57, BSU59, or BSU60 strain was grown on the indicator strains HLL3g (wild), BSU54, BSU55, BSU56, BSU57, BSU58, BSU59, or BSU60. All the strains are cured of SPb. Heat treatment was at 48 C for 30 min.

Heat induction of the groESL operon in the BSU59 (Dter(groESL)) strain further increased the plaqueforming unit more than five times. However, elevated expression of BsuMM (ydiO–ydiP) operon was not fully sufficient for SP10 phage development, as disruption of restriction activity (ydiS) in addition to enhanced wild type BsuMM (ydiO–ydiP) transcription (Dter(groESL)) in BSU60 (ydiS Dter(groESL)) strain further enhanced plaque forming unit more than a thousand times. SP10 phage grown in the cell of modificationproficient strains such as BSU57 (ydiS), BSU59 (Dter(groESL)), BSU60 (ydiSDter(groESL)) showed more than a thousand times higher number of plaque forming unit in the BSU59 (Dter(groESL)) strain compared to those grown in modification-deficient BSU54 (ydiO ydiS) strain. Therefore, plaque-forming ability of SP10 phage seemed to depend on the degree of methylation. However, even fully methylated SP10 phage grown in heat-treated mutant cells of BSU59 (Dter (groESL)) with elevated methylation activity by enhanced read-through transcription from the groESL operon could not grow in the cells of wild type strain. Our results also indicate that ydiR, ydiS and ydjA are all required for restriction.

mined accurately but was more than 20. SP10 phage DNA from phages grown in heat-treated BSU59 (Dter(groESL)) strain (lane 12) was less refractory to XhoI digestion compared to untreated BSU59 (Dter (groESL)) strain. It is probable that overexpression of ydiOP operon protected but did not methylate some XhoI sites or modifying activity of BsuMM methylases was partially lost by heat. In vivo results also support above possibility. The number of plaque forming unit of SP10 phage, which

3.2. Restriction and modification of phage SP10 in vitro To elucidate the degree of modification and susceptibility to restriction, DNA extracted from SP10 phages grown in the cells of modification-defective or modification-proficient strain was subjected to XhoI digestion. As shown in Fig. 1, phage DNA extracted from SP10 phages grown in modification proficient strain became more refractory to XhoI digestion (lane 6) compared to the unmethylated DNA (lane 3) from SP10 phage grown in the cell of the BsuM deficient strain. It is notable that DNA from phages grown in the BSU59 (Dter (groESL)) strain (lane 9) was much more refractory to XhoI digestion. The number of XhoI sites was not deter-

Fig. 1. Digestion of SP10 phage DNA with XhoI. DNA extracted from SP10 phages developed in BSU54 (M
R
) (lanes 1, 2, 3), BSU57 (M+R
) (lanes 4, 5, 6), BSU59 (Dter(groESL) M+R+) (lanes 7, 8, 9), and heat (48 C, 30 min) treated BSU59 (Dter(groESL) M+R+) (lanes 10, 11, 12) was not digested (lanes 1, 4, 7, 10), or digested with NruI (lanes 2, 5, 8, 11) or XhoI (lanes 3, 6, 9, 12), and subjected to electrophoresis. MW lane shows molecular weight marker (EcoT14I digested k phage DNA). 3 lg Phage DNA was digested with 6U restriction enzyme at 37 C for 60 min.

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was grown in heat treated BSU59 (Dter(groESL)), on the wild type strain was nil, but ca. 109 on the BSU59 (Dter(groESL)) strain. So some BsuM recognition sites of SP10 phage genome may not be methylated but protected in the BSU59 (Dter(groESL)) strain by BsuMM protein from BsuMR digestion. SP10 phage genome DNA (G + C, 42 mol%) contains incompletely characterized hypermodified thymine (YThy) residues in an amount of 5% in addition to 21% thymine residues [18]. The hypermodification Y is composed of L-glutamate and a basic component, and hypermodification-defective mutant SP10 phages are isolated [19]. YThy is partially responsible for the resistance of SP10 genome to cleavage and modification by BsuRI [19]. Therefore among ca. twenty BsuM target (5 0 CTCGAG 3 0 ) sequences, one or two sites may contain hypermodified thymine residues, which may make such BsuM sites refractory to BsuMM modification and susceptible to BsuMR restriction. In the above in vitro restriction experiments, we used exogenous XhoI restriction enzyme in stead of endogenous BsuMR enzyme complex. The latter may correctly recognize BsuMM-mediated modifiable BsuM sites. BsuM restriction and modification may recognize some DNA sequences around or near XhoI target sequence 5 0 CTCGAG 3 0 . Sequencing of SP10 phage genome will reveal such site-specificity.

In this study, enhanced expression of BsuMM depends on artificial read-through from groESL operon in the cell of BSU59 (Dter(groESL)) strain, however, even wild type strain shows read-through [6]; that may be the reason why heat treated B. subtilis cells become permissive to SP20 or SP10 phage [20], although heat treatment of our wild type B. subtilis Marburg strain did not show such enhancement of plaque formation (data not shown). 3.3. Restriction and modification of the B. subtilis chromosome Above results suggest that the SP10 genome is not fully methylated even in the presence of strong BsuMM activity or the BsuMM protein complex protects some CTCGAG sites from the attack by BsuMR. On the other hand, the wild type chromosomal DNA of B. subtilis carries more than 200 CTCGAG sites but is refractory to BsuMR digestion in the wild type BsuMM+ strain in vivo. It is subjected to degradation, observed by DAPI staining, when the BsuMR operon is placed under Pspac promoter and induced upon IPTG addition only in the modification deficient, but not in the modification proficient strain (Fig. 2). Viability was also lost in parallel with imbalance between modification and restriction [6].

Fig. 2. Cytological observation of restriction of B. subtilis chromosome by endogenous BsuMR. DAPI staining of the cells of BsuMM+ or BsuMM
strains carrying IPTG inducible BsuMR operon was carried out as described in Section 2. Upper panel, wild type strain 168 which did not accept IPTG. Middle panel, BsuMM+ strain BSU51 carrying IPTG inducible BsuMR operon which accepted 1 mM IPTG. Lower panel, BsuMM
strain BSU52 carrying IPTG inducible BsuMR operon which accepted 1 mM IPTG. Zero minutes indicate cells stained just after IPTG addition. Sixty minutes indicate cells stained 60 min after IPTG addition. Phase-contrast means images by phase-contrast microscopy. DAPI means cells stained with DAPI.

S. Matsuoka et al. / FEMS Microbiology Letters 244 (2005) 335–339

Modification activity of BsuM system of B. subtilis Marburg is sufficient for maintaining the integrity of the chromosome but not sufficient for the protection of invading SP10 phage DNA.

Acknowledgements We are grateful to R.H. Doi for critically reading the manuscript. This work was partially supported by a Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science, Sports and Culture of Japan.

References [1] Shibata, T. and Ando, T. (1974) Host controlled modification and restriction in Bacillus subtilis. Mol. Gen. Genet. 131, 275–280. [2] Saito, H., Shibata, T. and Ando, T. (1977) Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Marburg. Mol. Gen. Genet. 170, 117–122. [3] Jentsch, S. (1983) Restriction and modification in Bacillus subtilis: sequence specificities of restriction/modification systems BsuM, BsuE, and BsuF. J. Bacteriol. 156, 800–808. [4] Bron, S., Janniere, L. and Ehrlich, S.D. (1988) Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation. Mol. Gen. Genet. 211, 186– 189. [5] Trautner, T.A. and Noyer-Weidner, M. (1993) Restriction/modification and methylation systems in Bacillus subtilis, related species, and their phages In: Bacillus Subtilis and Other Grampositive Bacteria (Sonenshein, A.L., Hoch, J.A. and Losick, R., Eds.), pp. 539–552. American Society for Microbiology. [6] Ohshima, H., Matsuoka, S., Asai, K. and Sadaie, Y. (2002) Molecular organization of intrinsic restriction and modification genes BsuM of Bacillus subtilis Marburg. J. Bacteriol. 184, 381– 389. [7] Matsuoka, S., Arai, T., Murayama, R., Kawamura, F., Asai, K. and Sadaie, Y. (2004) Identification of the nonA and nonB loci of Bacillus subtilis supporting the growth of SP10 phage. Genes Genet. Syst. 79, 311–317. [8] Kasahara, Y., Nakai, S., Ogasawara, N., Yata, K. and Sadaie, Y. (1997) Sequence analysis of the groESL–cotA region of the Bacillus subtilis genome containing the restriction/modification system genes. DNA Res. 4, 335–339.

339

[9] Kunst, F. and Ogasawara, N., et al. (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249–256. [10] Alm, R.A., Ling, L.-S.L., Moir, D.T., King, B.L., Brown, E.D., Doig, P.C., Smith, D.R., Noonan, B., Guild, B.C., deJonge, B.L., Carmel, G., Tummino, P.J., Caruso, A., Uria-Nickelsen, M., Mills, D.M., Ives, C., Gibson, R., Merberg, D., Mills, S.D., Jiang, Q., Taylor, D.E., Vovis, G.F. and Trust, T.J. (1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397, 176– 180. [11] Ivanova, N., Sorokin, A., Anderson, I., Galleron, N., Candelon, B., Kapatral, V., Bhattacharyya, A., Reznik, G., Mikhailova, N., Lapidus, A., Chu, L., Mazur, M., Goltsman, E., Larsen, N., DÕSouza, M., Walunas, T., Grechkin, Y., Pusch, G., aselkorn, R., Fonstein, M., Ehrlich, D.S.D., Overbeek, R. and Kyrpides, N. (2003) Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423, 87–91. [12] Santagati, M., Iannelli, F., Cascone, C., Campanile, F., Oggioni, M.R., Stefani, S. and Pozzi, G. (2003) The novel conjugative transposon Tn1207.3 carries the macrolide efflux gene met(A) in Streptococcus pyogenes Microb. Drug Resist. 9, 243–247. [13] OÕDriscoll, J., Glynn, F., Cahalane, O., OÕConnell-Motherway, M., Fitzgerald, G.F. and Van Sinderen, D. (2004) Lactococcal plasmid pNP40 encodes a novel, temperature-sensitive restrictionmodification system. Appl. Environ. Microbiol. 70, 5546–5556. [14] Inatsu, Y., Kimura, K and Itoh, Y. (2002) Characterization of Bacillus subtilis strains isolated from fermented soybean foods in Southeast Asia: comparison with B. subtilis (natto) starter strains. JARQ 36, 169–175. [15] Vagner, V., Dervyn, E. and Ehrlich, S.D. (1998) A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144, 3097–3104. [16] Morimoto, T., Loh, P.C., Hirai, T., Asai, K., Kobayashi, K., Moriya, S. and Ogasawara, N. (2002) Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis. Microbiology 148, 3539–3552. [17] Kawai, F., Shoda, M., Harashima, R., Sadaie, Y., Hara, H. and Matsumoto, K. (2004) Cardiolipin domains in Bacillus subtilis Marburg membranes. J. Bacteriol. 186, 1475–1483. [18] Wiatr, C.L. and Witmer, H.J. (1984) Selective protection of 5 0 GGCC-3 0 and 5 0 -GCNGC-3 0 sequences by the hypermodified oxopyrimidine in Bacillus subtilis bacteriophage SP10 DNA. J. Virol. 52, 47–54. [19] Witmer, H. (1981) Synthesis of deoxythymidylate and the unusual deoxynucleotide in mature DNA of Bacillus subtilis bacteriophage SP10 occurs by postreplicational modification of 5-hydroxymethyldeoxyuridylate. J. Virol. 39, 536–547. [20] Gwinn, D.D. and Lawton, W.D. (1968) Alteration of host specificity in Bacillus subtilis. Bacteriol. Rev. 32, 297–301.