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Keywords:

  • Type III restriction and modification system;
  • Endonuclease;
  • ENase;
  • Methyltransferase;
  • Mtase;
  • BceS1;
  • Bacillus cereus

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The nucleotide sequence of an 11-kb chromosomal BglII fragment from Bacillus cereus American Type Culture Collection (ATCC) 10987 strain revealed two closely adjacent open reading frames organized in an operon, of which the deduced amino acids showed identity to the type III restriction and modification (R/M) subunits described in Gram-negative bacteria. An enhanced transcription level was revealed when the culture was grown in the presence of foreign DNA. A cell-free extract from this culture restricted pUC19, whereas from a plain medium the restriction was very weak. The in vitro methylation protected pUC 19 from restriction. The R/M system was designated BceS1 as this endonuclease required ATP and Mg2+ as cofactors like other type III endonucleases. BceS1 is the first chromosomal type III R/M system characterized in a Gram-positive bacterium.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The sequencing of genome fragments of Bacillus cereus American Type Culture Collection (ATCC) 10987 and B. cereus ATCC 14579 revealed genes with high identity to genes found in other bacteria [1,2], and a strategy for gene manipulation using a B. cereusEscherichia coli shuttle vector was established to study the function of these genes. All attempts to knock out genes in B. cereus ATCC 10987 using conjugation, transformation or electroporation were unsuccessful [3,4], whereas this was not a problem in the B. cereus ATCC 14579 strain. The deduced amino acid of an open reading frame (ORF) of a chromosomal fragment of B. cereus ATCC 10987 has shown identity to a segment of the type III EcoP1 restriction enzyme [5] and a very high identity to a segment of the Salmonella typhimurium type III StyLT1 restriction enzyme [6]. The activity of the potential type III restriction system in B. cereus ATCC 10987 might be the reason for the resistance against genetic manipulation in this strain.

Bacteria use restriction and modification R/M systems to protect themselves from attack of phages, incorporation of plasmids and to disintegrate foreign DNA [7–9]. DNA R/M systems are grouped in three classes, designated types I, II and III, based on their structure complexity and their need for cofactors. The type I and III endonucleases require both ATP and Mg2+ as their cofactors. The type I R/M system consists of three subunits, whereas the type III R/M system is composed of two subunits [7]. In the type III R/M system the Mod subunit is responsible for specific DNA recognition and methylation of the recognition site. The Res subunit cleaves the DNA approximately 24 to 27 bp downstream of the recognition site, but it has to be bound to the Mod subunit to do so [7]. The most characterized type III R/M systems belong to Gram-negative bacteria [10]. Many new putative type III R/M systems have been revealed in the sequencing of bacterial chromosomes and plasmids [9,11–13].

To gain more information on the potential type III R/M system and the gene organization in B. cereus ATCC 10987 we have determined the nucleotide sequence of an 11-kb fragment from a BglII library which contained the genes for the putative type III R/M enzymes. We here describe the effect on the transcription of these genes in the B. cereus strain when foreign DNA was added to the growth medium, and the capacity of a cell-free extract (CFE) from this culture to methylate and restrict foreign DNA.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacterial strains, plasmids, medium and growth conditions

B. cereus ATCC 10987 were obtained from the ATCC (Manassa, VA, USA); E. coli BK2118 [14] and E. coli XL1-Blue MRF′ (Epicurian Coli, Stratagene) were used as recombinant hosts; pUC19ampr was used as subcloning vector. The bacteria were grown in LB broth or on LB agar [15] at 37°C. LB agar containing ampicillin (50 μg ml−1) was used for selection of transformants. Shaker cultures (250 rpm) were used for RNA and CFE isolation of B. cereus ATCC 10987 grown in plain LB broth or grown with 0.02 μg ml−1 pUC19 DNA added to the medium just before onset of the exponential phase.

2.2Cloning, sequencing, RNA isolation and analysis of the type III R/M locus

A genomic BglII library of B. cereus ATCC 10987 [16] was screened using a probe that covered a fragment of the reported type III res gene [5]. Preparation, cloning and analysis of DNA was performed according to standard protocols [15].

Sequencing of the recombinant plasmid DNA was performed on both strands using a fluorescence-based automatic DNA sequencer (ALFwin version 1.00) (Pharmacia Biotech) at the DNA Sequencing Laboratory, Biotechnology Centre of Oslo, Oslo, Norway. Following the sequence determination, PCR primers were designed to extend into unknown regions. The sequence analyses were performed with the GCG Sequence Analysis Software Program [17]. Translated ORFs were used for BLAST and gapped BLAST searches [18,19].

Total mRNA was isolated from 1-ml culture samples from the cultures in mid-exponential phase using RNeasy kit (Qiagen) as described by the manufacturer. Northern blotting using MagnaCharge nylon transfer membranes (MSI, Westboro, MA, USA) and hybridization was performed essentially as in the standard protocols [15], using probes from the mod and res genes respectively. RNA was visualized by phosphorimaging (Storm 840, Molecular Dynamics, USA).

2.3Preparation of crude enzyme extracts, and in vitro methylation and restriction endonuclease assay

CFEs were isolated as described by Su et al. [9], from the cultures in the late-exponential phase of B. cereus ATCC 10987.

Methylation of pUC19 was performed according to Donahue et al. [20] using 0.6 μg plasmid DNA, 200 mM S-adenosyl-methionine (SAM, New England Biolabs) and 10 μl CFE in a reaction volume of 50 μl. Mock-treated plasmid DNA was used as a control.

Restriction endonuclease assay was performed according to Su [9] using a reaction mixture (20 μl) containing 0.5 μg of pUC DNA, 10 μl of CFE (in extraction buffer with 20 mM Mg2+) and a buffer containing 10 mM Tris–HCl pH 8; 10 mM MgCl2; 0.1 mM EDTA; 0.1 mM DTT; 50 μg ml−1 BSA; 10 mM KCl; 1 mM ATP with and without 5 μM SAM. Incubation temperature was 37°C, and the reactions were stopped by heating the mixtures to 65°C for 20 min. Each mixture was applied to an 0.8% agarose gel for electrophoresis.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Nucleotide sequence and the organization of the mod/res region

Six ORFs were identified (Fig. 1), all exhibited the same 5′–3′ order. The deduced amino acids of five of the ORFs showed similarity to submitted entries in the protein sequence database. ORF1 showed 35% identity to the central part of the McrB-related protein in Methanobacterium thermoautotrophicum[21]. The McrB enzyme belongs to a small family of methylation-dependent restriction enzymes first described in E. coli K-12 [10,22]. ORF2 showed 20% identity to a segment of the C-terminal part of a transcriptional activator in Plasmodium falciparum that belongs to the SNF2 family of proteins [23]. ORF3 (mod) and ORF4 (res) showed a high overall identity to the type III StyLT1 enzymes in S. typhimurium[6]. ORF6 showed 23% identity to a segment of the C-terminal part of a SNF2 protein in B. cereus[4], and to a segment in the N-terminal part of the SNF2 in P. falciparum[23].

image

Figure 1. Genetic organization of the 11-kb chromosomal BglII fragment containing the type III modification and restriction enzyme. The amino acid size of the ORFs are indicated. The sequence of the 10 915-bp BglII fragment has been deposited in the EMBL database under accession number AJ007510.

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The two adjacent ORFs (mod and res) in B. cereus showed an overall identity to other type III R/M enzymes (Table 1), and were separated by a few bp, each ORF was preceded by a putative ribosomal binding site. This organization is seen in most of the reported type III R/M systems [6,9,13,24]. Several type III R/M systems are reported in the chromosomes of the two strains of Helicobacter pylori[11,12], and some of the enzymes show similarity to the system in B. cereus. The Mod enzyme in the LlaFI system was the homolog to the B. cereus Mod enzyme (Table 1), but the corresponding Res enzyme showed no similarity to the B. cereus Res enzyme when the GAP data program was used [17]. The LlaFI Mod enzyme was shown to be more related to the HinfIII Mod enzyme than to the E. coli and S. typhimurium enzymes [9]. All the homolog type III methyltransferases (Mtases) showed the highly conserved motif IV and I present in all adenine Mtases [25].

Table 1.  Comparison of BceS1, the type III R/M system in B. cereus ATCC 10987 and type III R/M enzymes in other species
Bacterial strainType III R/M systemIdentity (%)
  Mod enzymesRes enzymes
  1. aNo similarity to BceS1 or the other type III restriction enzymes

S. typhimurium LT7StyLT154.3 (651 aa)61.6 (984 aa)
E. coli, bacteriophage P1EcoP1I38.7 (646 aa)33.1 (970 aa)
E. coli, plasmid p15BEcoP15I35.1 (645 aa) 
H. pylori (J99)HPmod2/HPres234.9 (444 aa)30.8 (970 aa)
H. pylori (26695)HP0593/HP059234.6 (599 aa)31.6 (1002 aa)
H. pylori (26695)a/HP152131.4 (968 aa) 
Pasteurella haemolytica A1PHmod/PHres32.5 (707 aa)29.6 (880 aa)
Lactococcus lactis subsp. cremoris, UC503LlaFI/a30.3 (681 aa) 
H. pylori (J99)HPmod1/HPres1a26.4 (621 aa) 

The type III restriction endonuclease (ENase) like the type I ENases contains sequence motifs characteristic of superfamily-II helicases, which may be involved in DNA unwinding at the cleavage site [26]. The seven conserved helicase motifs were observed in all the type III Res sequences (Table 2) and it can be seen that the motifs indeed are very conserved. The reported type III LlaFI Res subunit in L. lactis[9] showed only weak similarity to these motifs. These results might indicate that there are various subgroups of the type III R/M system, the EcoP1, StyLT1, B. cereus system and the system in Pasteurella haemolytica[13], representing one subgroup and the system in HinfIII, LlaF1 and some of the Res enzymes in H. pylori representing another subgroup. The DEAD box [26] in the helicase motifs is different in the two subgroups.

Table 2.  Sequence alignment of the seven conserved helicase motifs, found type III restriction enzymes
Restriction enzymesHelicase motifs
 IIAIIIIIIVVVI
  1. The DEAD box is shown in bold in motif II.

BceS1IDVKMETGTGKTYVYTIFKFVIVVPSPAIKEGVIIDEPHRIRFGATFLSLFFIDTRRFLISKWTLREGWDNPNVFVISKIQEVGRGLRL
StyLT1IDVKMETGTGKTYVYTLFKFVLVVPTPAIKEGVIIDEPHRVRFGATFLSLFFIDTRRFLISKWTLREGWDNPNVFVISKIQEVGRAVRL
EcoP1IDVSMETGTGKTYTYTINKFIIIVPTLSIKAGIIIDEPHKIRYGATFLTLFFIDPRRFIISKWTLREGWDNPNVFQISKLQEVGRGLRL
HP0592VDVLMETGTGKTFTYLQNKFIIFVPRKAILESSIIDEPHLFRFGATFLSLFFIPPLRFIISVWALQEGWDNPNIFTLSRHQQVGRGLRI
HP1521CDILMETGTGKTFCFLLSKFIVLTPSNAIKLGVIMDEPHRLRFGATFLCMVFIS.LRFIFSQWALQEGWDNPNVMTITKLQQIGRGLRL
HPres2CDILMETGTGKTFCFLLSKFIVLAPSNAIKLGVIMDEPHRLRFGATFLCMVFIN.LRFIISQWALQEGWDNPNVMTITKLQQIGRGLRL
PHresFTVEMETGTGKTYVYLWQKFVIVVPSVAIREGVIIDEPQNLRYSATHLSLFFIDPLRFIISHSALKEGWDNPNVFQIKKRQQIGRGLRL

3.2Expression of the B. cereus R/M system

Despite the mentioned similarities of the type III R/M genes, the regulation of the expression of the enzymatic activities seem to differ. In the E. coli the EcoP1I and EcoP15I genes are freely transferable from cell to cell by phage infection, conjugation and transformation, whereas horizontal transfer of the StyLT1 genes resulted in cell death of the recipients caused by extensive DNA breakdown [6]. The same problem was reported in P. haemolytica[13], and the presence of the type III R/M system in L. lactis rendered the strain resistant to bacteriophage attack.

Northern blots using probes from mod and res genes indicated that the transcription was enhanced when foreign DNA was added to the plain LB medium and a transcript of approximately 6.5 kb was seen (data not shown). This could be the transcript of a dicistronic message. In E. coli the existence of independent transcription units for the res and mod genes of bacteriophage P1 has been reported [27]. Our finding might indicate that DNA breakdown could be the reason for the unsuccessful attempt to knock out genes in this particular B. cereus strain.

The enhanced mRNA level of the B. cereus R/M genes in cells growing in medium with foreign DNA added, might indicate that a signal transduction had taken place. Work is in progress to study this preliminary finding further.

3.3Characterization of the restriction and methylation activity of B. cereus Res enzyme

CFE of cultures of the B. cereus strain grown in media with and without DNA added (CFEa and CFEb respectively) were tested for methylation and restriction activity. The CFEa restriction digests of pUC19 obtained in the presence and absence of cofactors are shown in Fig. 2a. Adding the cofactors enhanced the restriction of the circular pUC19 DNA showing a clear band at the size of the linear pUC19.

image

Figure 2. Agarose gel electrophoresis of pUC 19 DNA restricted with a CFE from B. cereus ATCC 10987 grown in medium with and without foreign DNA added, CFEa and CFEb respectively. Molecular size markers are shown in kb on the left border. a: Effects of Mg2+ and ATP on the endonuclease activity in CFEa, 1 h incubation time. b: Effect of SAM on the endonuclease activity in CFEa, 1 h (lane 1 and 3) and 2 h (lane 2 and 4) incubation time. c: Effect of in vitro methylation on pUC19 in lane 1, and a mock-treated pUC19 in lane 2, 2 h incubation time. d: Effect of SAM on the endonuclease activity in CFEb, 1 h (lane 1 and 5) and 2 h (lane 2 and 6) incubation time.

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Based on these results and the gene homology to other type III R/M systems, the B. cereus ATCC 10987 R/M system was designated BceS1 in accordance with the nomenclature proposed by Smith and Nathans [28].

Comparing the restriction pattern when using the CFEa and CFEb, it is clearly seen that the restriction enzyme activity in the CFEb is very low, only partly linearizing the plasmid (Fig. 2d), whereas with the CFEa several restriction fragments of pUC19 is seen (Fig. 2b). This confirms that the BceS1 system was inducible as was seen in the RNA blot. The digestion of pUC19 DNA indicates that the plasmid has at least three recognition sites for the BceSI restriction enzyme. Adding SAM to the restriction assay did not seem to enhance restriction. Restriction of the CFEa-methylated and mock-treated pUC19 showed that the in vitro methylation of the plasmid protected it against further restriction by the CFEa (Fig. 2c). This result implies that a similar procedure could be used to make knock outs of these genes in B. cereus ATCC 10987.

All this taken together may signify that the BceS1 belongs to the type III R/M system.

BceS1 is the first chromosomal type III R/M system reported in a Gram-positive bacterium. The high identity to the chromosomal StyLT1 in S. typhimurium suggests a horizontal gene transfer between the Gram-negative and Gram-positive bacterium. Our results also indicate that a complete cistron including the regulatory gene was transferred, since we have demonstrated an active type III R/M system. The genes have not been identified in any of 32 other B. cereus/thuringiensis strains.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The authors are thankful to Henning A. Johansen and Liv A. Bj?rnland at the DNA sequencing laboratory and to Ewa Jaroszewicz for skilful technical assistance and to Toril Lindbäck for providing the library for B. cereus ATCC 10987 genomic DNA.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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