Characterization of a Novel Type II Restriction-Modification System, Sth368I, Encoded by the Integrative Element ICESt1 of Streptococcus thermophilus CNRZ368

A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to ST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5′-GATC-3′. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5′-GATC-3′ was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5′-GATC-3′. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5′-GATC-3′ and is related to the Sau3AI and LlaKR2I restriction systems.     

Crystal structure and function of C-terminal Sau3AI domain

Sau3AI is a type II restriction enzyme that recognizes the 5'-GATC-3' sequence in double-strand DNA and cleaves at 5' to the G residue. The C-terminal domain of Sau3AI (Sau3AI-C), which contains amino acids from 233 to 489, was crystallized and its structure was solved by using the Multi-wavelength Anomalous Diffraction method. The Sau3AI-C structure at 1.9 A resolution is similar to the structure of MutH, a DNA mismatch repair protein that shares high sequence similarity with the N-terminal Sau3AI domain. The functional analysis shows that Sau3AI-C can bind DNA with one recognition sequence but has no cleavage activity. These results indicate that Sau3AI is a pseudo-dimer belonging to the type IIe restriction enzymes and the Sau3AI-C is the allosteric effector domain that assists DNA binding and cleavage.     

Characterization of a novel integrative element, ICESt1, in the lactic acid bacterium Streptococcus thermophilus

The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3' end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1 could be or could be derived from an integrative conjugative element.     

Crystallization and preliminary X-ray analysis of Sau3AI/E64A mutant protein

Sau3AI is a type II restriction endonuclease that recognizes the palindromic sequence 5'-GATC-3' and cleaves 5' to G residue on each strand. The E64A mutant full length protein was cloned and expressed in Escherichia coli. The purified (His) (6)-tagged protein has monomer and dimer fraction and was crystallized by the hanging-drop vapor-diffusion technique. The dimer protein crystals can diffract to 3.0A. resolution and the monomer protein crystals can diffract to better than 2.8A. resolution. One completed dataset has been collected and it shows that the monomer orthorhombic Sau3AI/E64A crystal is in space group C2221 with unit cell parameters (69.44, 197.60, 191.46, 90, 90, 90) and contains two molecules in one asymmetric unit.     

Distribution of the SsuDAT1I restriction-modification system among different serotypes of Streptococcus suis

The SsuDAT1I restriction-modification (R-M) system, which contains two methyltransferases and two restriction endonucleases with recognition sequence 5'-GATC-3', was first found in a field isolate of Streptococcus suis serotype 2. Isoschizomers of the R-M system were found in the same locus between purH and purD in a field isolate of serotype 1/2 and the reference strains of serotypes 3, 7, 23, and 26 among 29 strains of different serotypes examined in this study. The R-M gene sequences in serotypes 1/2, 3, 7, and 23 were very similar to those of SsuDAT1I, whereas those in serotype 26 were less similar. These results indicate intraspecies recombination among them and genetic divergence through their evolution.     

Transposition of pGh9:ISS1 is random and efficient in Streptococcus thermophilus CNRZ368

Streptococcus thermophilus bacteria are used as a starter in the fermentation of yogurts and many cheeses. To construct mutants of S. thermophilus CNRZ368, the use of the plasmid pGh9:ISS1 was considered. This plasmid is known to be a good tool for insertional mutagenesis in gram-positive bacteria, owing to its ability to integrate in the genome by a mechanism of replicative transposition. However, the presence of three endogenous ISS1 copies in the genome of S. thermophilus CNRZ368 and the possible occurrence of homologous recombination could reduce the efficiency of pGh9:ISS1 as a tool for generating mutants. To address this question, the ability of pGh9:ISS1 to transpose randomly in the genome of strain CNRZ368 was investigated. The results of our experiments indicated that: (i) the frequency of transposition of ISS1 was high, approximately 2 x 10(-2), in S. thermophilus CNRZ368; (ii) the integration of multiple tandem copies of the plasmid was frequent; (iii) homologous recombination events between ISS1 were not predominant; and (iv) plasmid pGh9:ISS1 transposed randomly around the S. thermophilus CNRZ368 chromosome. In addition, we describe the strategy used to localize the pGh9:ISS1 insertion locus on the physical map of strain CNRZ368 and the method used to clone the regions flanking this insertion site, especially when multiple copies of the plasmid were integrated in tandem.     

cse, a Chimeric and variable gene, encodes an extracellular protein involved in cellular segregation in Streptococcus thermophilus

The isolation of a Streptococcus thermophilus CNRZ368 mutant displaying a long-chain phenotype allowed us to identify the cse gene (for cellular segregation). The N terminus of Cse exhibits high similarity to Streptococcus agalactiae surface immunogenic protein (SIP), while its C terminus exhibits high similarity to S. thermophilus PcsB. In CNRZ368, deletion of the entire cse open reading frame leads to drastic lengthening of cell chains and altered colony morphology. Complementation of the Deltacse mutation with a wild-type allele restored both wild-type phenotypes. The central part of Cse is a repeat-rich region with low sequence complexity. Comparison of cse from CNRZ368 and LMG18311 strains reveals high variability of this repeat-rich region. To assess the impact of this central region variability, the central region of LMG18311 cse was exchanged with that of CNRZ368 cse. This replacement did not affect chain length, showing that divergence of the central part does not modify cell segregation activity of Cse. The structure of the cse locus suggests that the chimeric organization of cse results from insertion of a duplicated sequence deriving from the pcsB 3' end into an ancestral sip gene. Thus, the cse locus illustrates the module-shuffling mechanism of bacterial gene evolution.     

Synthesis of decadeoxyribonucleotides containing 5-modified uracils and their interactions with restriction endonucleases Bgl II, Sau 3AI and Mbo I (nucleosides and nucleotides 82)

Decadeoxyribonucleotides containing uracil, 5-bromouracil, 5-cyanouracil and 5-ethyluracil in recognition sequences of restriction endonucleases Bgl II, Sau 3AI, Mbo I were synthesized. Decanucleotides containing 5-bromouracil in place of thymine had essentially the same susceptibility to all the restriction endonucleases. Uracil-containing decanucleotides were however very resistant to attack. Decanucleotides containing 5-cyanouracil in the recognition sequence were strongly resistant to hydrolysis by Sau 3AI, but were hydrolysed by Bgl II and Mbo I as well as the parent decanucleotide. Decanucleotides containing 5-ethyluracil were strongly resistant to hydrolysis by Sau 3AI, but were partially resistant to hydrolysis by Bgl II and Mbo I.     

The ICESt1 element of Streptococcus thermophilus belongs to a large family of integrative and conjugative elements that exchange modules and change their specificity of integration

The 34,734-bp element ICESt1 from Streptococcus thermophilus CNRZ368 is site-specifically integrated into the 3(') end of the gene fda. ICESt1 encodes integrative functions and putative transfer functions. Six proteins of the putative conjugative system of ICESt1 are related to those encoded by the conjugative transposon Tn916 from Enterococcus faecalis. A comparison of these proteins with those encoded by the complete or partial genome sequences of various low G+C bacteria including Bacillus subtilis, Clostridium difficile, E. faecalis, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus mutans revealed the presence of numerous putative site-specific integrative conjugative elements and/or conjugative transposons within these genomes. Sequence comparisons revealed that these elements possess a modular structure and that exchanges of unrelated or distantly related modules and genes have occurred between these elements, and also plasmids and prophages. These exchanges have probably led to modifications in the site specificity of integration of these elements. Therefore, a distinction between low specificity integrative conjugative elements (i.e., conjugative transposons) and site-specific integrative conjugative elements does not appear to be relevant. We propose to call all the conjugative elements that excise by site-specific recombination and integrate by recombination between a specific site of a circular intermediate and another site, "Integrative and Conjugative Elements" (ICEs), irrespective of the integration specificity.     

Are horizontal transfers involved in the evolution of the Streptococcus thermophilus exopolysaccharide synthesis loci?

A 32.5kb variable locus of the Streptococcus thermophilus CNRZ368 chromosome, the eps locus, contains 25 ORF and seven insertion sequences (IS). The putative products of 17 ORF are related to proteins involved in the synthesis of polysaccharides in various bacteria. The two distal regions and a small central region of the eps locus are constant and present in all or almost all of the S. thermophilus strains tested. The other regions are variable and present in only some S. thermophilus strains tested, particularly in the closely related strains CNRZ368 and A054. A 13.6kb variable region of the eps locus of S. thermophilus CNRZ368 contains two ORF that are almost identical to epsL and orfY of the eps locus of Lactococcus lactis NIZOB40 and seven IS belonging to four different families, ISS1, IS981, IS1193 and IS1194. Five of these sequences were probably acquired by horizontal transfer from L. lactis (Bourgoin, F., et al., 1996. Gene 178, 15-23). Three probes of this 13.6kb region hybridized with the DNA of several L. lactis strains tested. A specific probe for another sequence within the S. thermophilus eps locus, epsF, hybridized with the DNA of one of the L. lactis strains tested. Sequence comparisons also suggest that five ORF of the eps locus have a mosaic structure and probably result from recombinations between sequences that are 10 to 50% divergent. The chimeric structure of the eps locus suggests a very complex evolution. This evolution probably involves both the acquisition of the 13.6kb region from L. lactis by horizontal transfer and exchanges within the S. thermophilus species.     

Effects of rodA and pbp2b disruption on cell morphology and oxidative stress response of Streptococcus thermophilus CNRZ368

Insertional mutagenesis was used to isolate clones from Streptococcus thermophilus CNRZ368 that were modified in their abilities to tolerate oxidative stress. During this process, two menadione-sensitive clones (6G4 and 18C3) were found to display abnormal cell morphologies and distorted chain topologies and were further studied. Molecular characterization of both 6G4 and 18C3 mutants indicated that they were disrupted in open reading frames homologous to rodA and pbp2b, respectively. Both genes encoded proteins in Escherichia coli that were described as being implicated in peptidoglycan synthesis during the process of cell elongation and to function in determining the rod shape of the cell. This work reports a possible connection between peptidoglycan biosynthesis and oxidative stress defense in S. thermophilus CNRZ368.     

Restriction endonucleases in Clostridium pasteurianum ATCC 6013 and C. thermohydrosulfuricum DSM 568

A small collection of clostridia was surveyed for type II restriction endonucleases. Enzymes were detected in two organisms. Clostridium pasteurianum ATCC 6013 contains an isoschizomer of ThaI (FnuDII) [5'-CGCG-3'] and preliminary evidence suggests that cleavage generates blunt-ended fragments. Clostridium thermohydrosulfuricum DSM 568 contains an isoschizomer of MboI (Sau3A) [5'-GATC-3'] that is inactive on dam methylated substrates. The DNA of this latter organism shows dam methylation.     

In silico restriction landmark genome scanning analysis of Xanthomonas oryzae pathovar oryzae MAFF 311018

We have developed a restriction landmark genome scanning (RLGS) system in silico, involving two-dimensional electrophoretic analysis of DNA by computer simulation that is based on the availability of whole-genome sequences for specific organisms. We applied the technique to the analysis of the Xanthomonas oryzae pathovar oryzae (Xoo) MAFF 311018, which causes bacterial blight in rice. The coverage that was found to be achievable using RLGS in silico, as a percentage of the genomic regions that could be detected, ranged from 44.5% to 72.7% per image. However, this reached a value of 96.7% using four images that were obtained with different combinations of landmark restriction enzymes. Interestingly, the signal intensity of some of the specific spots obtained was significantly lower than that of other surrounding spots when MboI, which cleaves unmethylated 5'-GATC-3' sites, was used. DNA gel blot analysis with both DNA adenine methylase (Dam)-sensitive and -insensitive isoschizomers (MboI and Sau3AI) revealed that Dam-mediated DNA adenine methylation had indeed occurred at these particular sites. These results suggest that a significant portion of the 5'-GATC-3' sites within the Xoo genome is stably methylated by Dam.     

Purification of the novel endonuclease, Hpy188I, and cloning of its restriction-modification genes reveal evidence of its horizontal transfer to the Helicobacter pylori genome

We have isolated a novel restriction endonuclease, Hpy188I, from Helicobacter pylori strain J188. Hpy188I recognizes the unique sequence, TCNGA, and cleaves the DNA between nucleotides N and G in its recognition sequence to generate a one-base 3' overhang. Cloning and sequence analysis of the Hpy188I modification gene in strain J188 reveal that hpy188IM has a 1299-base pair (bp) open reading frame (ORF) encoding a 432-amino acid product. The predicted protein sequence of M.Hpy188I contains conserved motifs typical of aminomethyltransferases, and Western blotting indicates that it is an N-6 adenine methyltransferase. Downstream of hpy188IM is a 513-bp ORF encoding a 170-amino acid product, that has a 41-bp overlap with hpy188IM. The predicted protein sequence from this ORF matches the amino acid sequence obtained from purified Hpy188I, indicating that it encodes the endonuclease. The Hpy188I R-M genes are not present in either strain of H. pylori that has been completely sequenced but are found in two of 11 H. pylori strains tested. The significantly lower G + C content of the Hpy188I R-M genes implies that they have been introduced relatively recently during the evolution of the H. pylori genome.     

Evolution and function of the neisserial dam-replacing gene

Phase variation through slippage-like mechanisms involving homopolymeric tracts depends in part on the absence of Dam-methylase in several pathogenic isolates of Neisseria meningitidis. In Dam-defective strains drg (dam-replacing gene), flanked by pseudo-transposable small repeated elements (SREs), replaced dam. We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5'-GmeATC-3'. drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE-providing promoter. This is associated with the presence of GATmeC, suggesting an alternative restriction-modification system (RM) specific for 5'-GATC-3', similar to Sau3AI-RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.     

A model of structure and action of Sau3AI restriction endonuclease that comprises two MutH-like endonuclease domains within a single polypeptide

Sau3AI is a type II endonuclease that cleaves GATC sequences, producing sticky ends with 4-nucleotide 5'-overhangs. Its activity is inhibited by cytosine C5-methylation within the target sequence. In the N-terminus, Sau3AI exhibits sequence similarity to the GATC-specific single-strand nicking endonuclease MutH implicated in mismatch repair (Ban and Yang, 1998). Sequence analysis of Sau3AI and its homologs reveals that Sau3AI possesses an additional MutH-like domain in the C-terminus. Structure prediction suggests that the C-terminal domain lacks the endonuclease active site but retains all putative DNA-binding elements. As an illustration of these findings, a model of quaternary structure of Sau3AI complexed with the target DNA is presented. These predictions have implications for evolution, structure and function of bacterial DNA repair enzymes and restriction endonucleases.     

Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae

The genetic basis of the unique restriction endonuclease DpnI, that cleaves only at a methylated sequence, 5'-GmeATC-3', and of the complementary endonuclease DpnII, which cleaves at the same sequence when it is not methylated, was investigated. Different strains of Streptococcus pneumoniae isolated from patients contained either DpnI (two isolates) or DpnII (six isolates). The latter strains also contained DNA methylated at the 5'-GATC-3' sequence. A restrictable bacteriophage, HB-3, was used to characterize the various strains and to select for transformants. One laboratory strain contained neither DpnI nor Dpn II. It was probably derived from a DpnI-containing strain, and its DNA was not methylated at 5'-GATC-3'. Cells of this strain were transformed to the DpnI restriction phenotype by DNA from a DpnI-containing strain and to the DpnII restriction phenotype by DNA from a DpnII-containing strain. Neither cross-transformation, that is, transformation to one phenotype by DNA from a strain of the other phenotype, nor spontaneous conversion was observed. Extracts of transformants to the new restriction phenotype were shown to contain the corresponding endonuclease.     

Site specific endonuclease from Fusobacterium nucleatum.

Four different isolates of Fusobacterium nucleatum (A,C,D and E) contain restriction endonucleases of differing specificity. Whilst many of the endonucleases are isochizomers of known enzymes, two novel activities are Fnu DII which recognizes and cleaves the sequence 5'-CGCT-3'/3'-GCGC-5' AND Fnu EI which recognizes and cleaves the sequence 5'-GATC-3'/3'-CTAG-5' irrespective of the extent of methylation of the adenine residues.     

The Sse9I restriction-modification system: organization of genes and comparative analysis of proteins structures

A nucleotide sequence was established for the full-length Sporosarcina species 9D operon coding for enzymes of type II restriction-modification system Sse9I. These enzymes recognize the tetranucleotide DNA sequence 5'-AATT-3'. The operon was shown to consist of three genes that are situated with the order: sse9IC-sse9IR-sse9IM and are transcribed in the same direction. These genes encode the control protein (C.Sse9I), restriction endonuclease (R.Sse9I) and DNA-methyltransferase (M.Sse9I), respectively. A specific DNA sequence (C-box) presumably recognized by C-protein was found immediately upstream of sse9IC gene. The comparative analysis of amino acid sequences of C.Sse9I and R.Sse9I with those of relative proteins has been done. It was found that R.Sse9I revealed the most homology with the segments of R.MunI (5'-CAATTG-3') and R.EcoRI (5'-GAATTC-3'), where amino acid residues, responsible for recogniton of AATT core sequence are located. The sse9IR gene was cloned into the temperature-inducible expression vector, and recombinant Sse9I restriction endonuclease preparation was isolated.     

Modification methylase M.Sau3239I from Streptomyces aureofaciens 3239

By chromatography on phosphocellulose and Heparin-Sepharose the modification methylase M.Sau3239I was detected and partly purified from cells of Streptomyces aureofaciens 3239. Methylation by this enzyme protects DNA from cleavage by the restriction endonuclease R.Sau3239I. The enzyme catalyzes methylation of adenine to N-6-methyladenine in the 5'-CTCGmAG-3' recognition sequence.