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1.  Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium. 
Journal of Bacteriology  1991;173(3):1321-1327.
The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.
PMCID: PMC207257  PMID: 1846861
2.  Characterization and expression of the Escherichia coli Mrr restriction system. 
Journal of Bacteriology  1991;173(16):5207-5219.
The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.
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PMCID: PMC208215  PMID: 1650347
3.  Cellular localization and dynamics of the Mrr type IV restriction endonuclease of Escherichia coli 
Nucleic Acids Research  2014;42(6):3908-3918.
In this study, we examined the intracellular whereabouts of Mrr, a cryptic type IV restriction endonuclease of Escherichia coli K12, in response to different conditions. In absence of stimuli triggering its activity, Mrr was found to be strongly associated with the nucleoid as a number of discrete foci, suggesting the presence of Mrr hotspots on the chromosome. Previously established elicitors of Mrr activity, such as exposure to high (hydrostatic) pressure (HP) or expression of the HhaII methyltransferase, both caused nucleoid condensation and an unexpected coalescence of Mrr foci. However, although the resulting Mrr/nucleoid complex was stable when triggered with HhaII, it tended to be only short-lived when elicited with HP. Moreover, HP-mediated activation of Mrr typically led to cellular blebbing, suggesting a link between chromosome and cellular integrity. Interestingly, Mrr variants could be isolated that were specifically compromised in either HhaII- or HP-dependent activation, underscoring a mechanistic difference in the way both triggers activate Mrr. In general, our results reveal that Mrr can take part in complex spatial distributions on the nucleoid and can be engaged in distinct modes of activity.
doi:10.1093/nar/gkt1370
PMCID: PMC3973329  PMID: 24423871
4.  Transfer of the genes for the StyLTI restriction-modification system of Salmonella typhimurium to strains lacking modification ability results in death of the recipient cells and degradation of their DNA. 
Journal of Bacteriology  1991;173(3):1328-1330.
The genes encoding the restriction-modification system StyLTI of Salmonella typhimurium were inserted in vivo into the conjugative plasmid pULB21. This allowed us to transfer the StyLTI genes at a very high frequency and to monitor the fate of recipient cells after mating. Transfer of the StyLTI restriction and modification genes into a modificationless recipient was lethal and resulted in degradation of the cell's DNA. This indicates that, in contrast to any other known restriction-modification systems, StyLTI cannot be established after horizontal transfer into a naive host.
PMCID: PMC207258  PMID: 1846862
5.  Phasevarions Mediate Random Switching of Gene Expression in Pathogenic Neisseria 
PLoS Pathogens  2009;5(4):e1000400.
Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes—modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5′-AGAAA-3′. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that phasevarions may be a common strategy used by host-adapted bacterial pathogens to randomly switch between “differentiated” cell types.
Author Summary
The pathogenic Neisseria are bacterial pathogens that cause meningitis and gonorrhoea. They have adapted to life exclusively in humans and have developed unique strategies to colonize the host and to evade the immune response. Central among these strategies are genetic switches that randomly turn genes on and off. In most cases, the genes controlled by these switches, contingency genes, are required for making bacterial surface structures. Recently we described a new class of contingency gene that methylates DNA. Rather than affecting the synthesis of a single surface structure, on/off switching of this DNA-methyltransferase gene leads to random switching of multiple genes. In this study, we have shown that this mechanism exists in all pathogenic Neisseria, and alters expression of multiple genes in all cases we examined. The two distinct populations of bacteria generated by this process had different behavior in model systems of colonization and infection. Understanding this process is key to understanding these human pathogens, and to developing strategies for treatment and prevention of the diseases they cause.
doi:10.1371/journal.ppat.1000400
PMCID: PMC2667262  PMID: 19390608
6.  DNA cleavage and methylation specificity of the single polypeptide restriction–modification enzyme LlaGI 
Nucleic Acids Research  2009;37(21):7206-7218.
LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5′-CrTCnAG-3′ being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).
doi:10.1093/nar/gkp790
PMCID: PMC2790903  PMID: 19808936
7.  Cloning of NruI and Sbo13I restriction and modification sstems in E. coli and amino acid sequence comparison of M.NruI and M.Sbo13I with other amino-methyltransferases 
BMC Research Notes  2010;3:139.
Background
NruI and Sbo13I are restriction enzyme isoschizomers with the same recognition sequence 5' TCG↓CGA 3' (cleavage as indicated↓). Here we report the cloning of NruI and Sbo13I restriction-modification (R-M) systems in E. coli. The NruI restriction endonuclease gene (nruIR) was cloned by PCR and inverse PCR using primers designed from the N-terminal amino acid sequence. The NruI methylase gene (nruIM) was derived by inverse PCR walking.
Results
The amino acid sequences of NruI endonuclease and methylase are very similar to the Sbo13I R-M system which has been cloned and expressed in E. coli by phage selection of a plasmid DNA library. Dot blot analysis using rabbit polyclonal antibodies to N6mA- or N4mC-modified DNA indicated that M.NruI is possibly a N6mA-type amino-methyltransferase that most likely modifies the external A in the 5' TCGCGA 3' sequence. M.Sbo13I, however, is implicated as a probable N4mC-type methylase since plasmid carrying sbo13IM gene is not restricted by Mrr endonuclease and Sbo13I digestion is not blocked by Dam methylation of the overlapping site. The amino acid sequence of M.NruI and M.Sbo13I did not show significant sequence similarity to many known amino-methyltransferases in the α, β, and γ groups, except to a few putative methylases in sequenced microbial genomes.
Conclusions
The order of the conserved amino acid motifs (blocks) in M.NruI/M.Sbo13I is similar to the γ. group amino-methyltranferases, but with two distinct features: In motif IV, the sequence is DPPY instead of NPPY; there are two additional conserved motifs, IVa and Xa as extension of motifs IV and X, in this family of enzymes. We propose that M.NruI and M.Sbo13I form a subgroup in the γ group of amino-methyltransferases.
doi:10.1186/1756-0500-3-139
PMCID: PMC2890505  PMID: 20497557
8.  The Transcription Factor Mrr1p Controls Expression of the MDR1 Efflux Pump and Mediates Multidrug Resistance in Candida albicans 
PLoS Pathogens  2007;3(11):e164.
Constitutive overexpression of the MDR1 (multidrug resistance) gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to fluconazole and other toxic compounds in clinical Candida albicans strains, but the mechanism of MDR1 upregulation has not been resolved. By genome-wide gene expression analysis we have identified a zinc cluster transcription factor, designated as MRR1 (multidrug resistance regulator), that was coordinately upregulated with MDR1 in drug-resistant, clinical C. albicans isolates. Inactivation of MRR1 in two such drug-resistant isolates abolished both MDR1 expression and multidrug resistance. Sequence analysis of the MRR1 alleles of two matched drug-sensitive and drug-resistant C. albicans isolate pairs showed that the resistant isolates had become homozygous for MRR1 alleles that contained single nucleotide substitutions, resulting in a P683S exchange in one isolate and a G997V substitution in the other isolate. Introduction of these mutated alleles into a drug-susceptible C. albicans strain resulted in constitutive MDR1 overexpression and multidrug resistance. By comparing the transcriptional profiles of drug-resistant C. albicans isolates and mrr1Δ mutants derived from them and of C. albicans strains carrying wild-type and mutated MRR1 alleles, we defined the target genes that are controlled by Mrr1p. Many of the Mrr1p target genes encode oxidoreductases, whose upregulation in fluconazole-resistant isolates may help to prevent cell damage resulting from the generation of toxic molecules in the presence of fluconazole and thereby contribute to drug resistance. The identification of MRR1 as the central regulator of the MDR1 efflux pump and the elucidation of the mutations that have occurred in fluconazole-resistant, clinical C. albicans isolates and result in constitutive activity of this trancription factor provide detailed insights into the molecular basis of multidrug resistance in this important human fungal pathogen.
Author Summary
The Candida albicans MDR1 (multidrug resistance) gene encodes a multidrug efflux pump of the major facilitator superfamily that is constitutively overexpressed in many fluconazole-resistant strains. Although MDR1 overexpression is a major cause of resistance to this widely used antifungal agent and other metabolic inhibitors, so far the molecular basis of MDR1 upregulation in resistant strains has remained elusive. By comparing the transcription profiles of MDR1 overexpressing, clinical C. albicans isolates and matched, drug-susceptible isolates from the same patients, we identified a transcription factor, termed multidrug resistance regulator 1 (MRR1), which was upregulated in all resistant isolates and turned out to be a central regulator of MDR1 expression. Resistant isolates contained point mutations in MRR1, which rendered the transcription factor constitutively active. Introduction of these mutated alleles into a susceptible strain caused MDR1 overexpression und multidrug resistance. Inactivation of MRR1 in clinical isolates abolished MDR1 expression and affected fluconazole resistance even more strongly than deletion of the MDR1 efflux pump itself, indicating that additional Mrr1p target genes, which were identified by genome-wide gene expression analysis, contribute to fluconazole resistance. These findings provide detailed insights into the molecular basis of multidrug resistance in one of the most important human fungal pathogens.
doi:10.1371/journal.ppat.0030164
PMCID: PMC2048531  PMID: 17983269
9.  Opioid μ-Receptors in Medullary Raphe Region Affect the Hypoxic Ventilation in Anesthetized Rats 
Opioids can attenuate the peripheral chemoreceptor-mediated hypoxic ventilatory response (HVR) by acting on central μ-type opioid receptors. Since the medullary raphe region (MRRs) expresses abundant μ-receptors and participates in modulating HVR, we tested the role of μ-receptors within the caudal, medial, and rostral MRR (cMRR, mMRR, and rMRR) in modulating the HVR. We recorded cardiorespiratory activities and their responses to isocapnic hypoxia in anesthetized rats before and after local microinjection of DAMGO into the MRR, and intravenous administration of DAMGO (100 μg/kg) alone or coupled with a previous local injection of CTAP. Microinjecting DAMGO into the cMRR or mMRR but not the rMRR significantly attenuated the HVR. However, systemic DAMGO-induced HVR attenuation was not significantly affected by pretreating the cMRR and mMRR with CTAP. Our data suggest that cMRR and mMRR μ-receptors are capable of depressing the HVR, while their contribution to the attenuated HVR by systemic DAMGO is limited.
doi:10.1016/j.resp.2009.07.015
PMCID: PMC3438222  PMID: 19632358
brainstem; carotid body; breathing
10.  Regulation of Efflux Pump Expression and Drug Resistance by the Transcription Factors Mrr1, Upc2, and Cap1 in Candida albicans▿† 
Constitutive overexpression of the Mdr1 efflux pump is an important mechanism of acquired drug resistance in the yeast Candida albicans. The zinc cluster transcription factor Mrr1 is a central regulator of MDR1 expression, but other transcription factors have also been implicated in MDR1 regulation. To better understand how MDR1-mediated drug resistance is achieved in this fungal pathogen, we studied the interdependence of Mrr1 and two other MDR1 regulators, Upc2 and Cap1, in the control of MDR1 expression. A mutated, constitutively active Mrr1 could upregulate MDR1 and confer drug resistance in the absence of Upc2 or Cap1. On the other hand, Upc2 containing a gain-of-function mutation only slightly activated the MDR1 promoter, and this activation depended on the presence of a functional MRR1 gene. In contrast, a C-terminally truncated, activated form of Cap1 could upregulate MDR1 in a partially Mrr1-independent fashion. The induction of MDR1 expression by toxic chemicals occurred independently of Upc2 but required the presence of Mrr1 and also partially depended on Cap1. Transcriptional profiling and in vivo DNA binding studies showed that a constitutively active Mrr1 binds to and upregulates most of its direct target genes in the presence or absence of Cap1. Therefore, Mrr1 and Cap1 cooperate in the environmental induction of MDR1 expression in wild-type C. albicans, but gain-of-function mutations in either of the two transcription factors can independently mediate efflux pump overexpression and drug resistance.
doi:10.1128/AAC.01343-10
PMCID: PMC3088179  PMID: 21402859
11.  Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains. 
Journal of Bacteriology  1991;173(15):4707-4716.
Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E. coli genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.
PMCID: PMC208148  PMID: 1649819
12.  Site-specific methylases induce the SOS DNA repair response in Escherichia coli. 
Journal of Bacteriology  1987;169(7):3243-3250.
Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GAmeATTC] or induced SOS to a lesser extent (dam [GmeATC]). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [meCCGG], HaeIII [GGmeCC], BamHI [GGATmeCC], HhaI [GmeCGC], BsuRI [GGmeCC], and M.Spr) also induced SOS, whereas one indigenous to E. coli (EcoRII [CmeCA/TGG]) did not. SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986).
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PMCID: PMC212376  PMID: 3036779
13.  Functional Dissection of a Candida albicans Zinc Cluster Transcription Factor, the Multidrug Resistance Regulator Mrr1 ▿ 
Eukaryotic Cell  2011;10(8):1110-1121.
The overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the widely used antimycotic agent fluconazole and other toxic compounds in the pathogenic yeast Candida albicans. The zinc cluster transcription factor Mrr1 controls MDR1 expression in response to inducing chemicals, and gain-of-function mutations in MRR1 are responsible for the constitutive MDR1 upregulation in fluconazole-resistant C. albicans strains. To understand how Mrr1 activity is regulated, we identified functional domains of this transcription factor. A hybrid protein consisting of the N-terminal 106 amino acids of Mrr1 and the transcriptional activation domain of Gal4 from Saccharomyces cerevisiae constitutively induced MDR1 expression, demonstrating that the DNA binding domain is sufficient to target Mrr1 to the MDR1 promoter. Using a series of C-terminal truncations and systematic internal deletions, we could show that Mrr1 contains multiple activation and inhibitory domains. One activation domain (AD1) is located in the C terminus of Mrr1. When fused to the tetracycline repressor TetR, this distal activation domain induced gene expression from a TetR-dependent promoter. The deletion of an inhibitory region (ID1) located near the distal activation domain resulted in constitutive activity of Mrr1. The additional removal of AD1 abolished the constitutive activity, but the truncated Mrr1 still could activate the MDR1 promoter in response to the inducer benomyl. These results demonstrate that the activity of Mrr1 is regulated in multiple ways and provide insights into the function of an important mediator of drug resistance in C. albicans.
doi:10.1128/EC.05100-11
PMCID: PMC3165443  PMID: 21685320
14.  An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI 
Nucleic Acids Research  2009;37(21):7231-7238.
Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction–modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.
doi:10.1093/nar/gkp795
PMCID: PMC2790908  PMID: 19793866
15.  The other face of restriction: modification-dependent enzymes 
Nucleic Acids Research  2013;42(1):56-69.
The 1952 observation of host-induced non-hereditary variation in bacteriophages by Salvador Luria and Mary Human led to the discovery in the 1960s of modifying enzymes that glucosylate hydroxymethylcytosine in T-even phages and of genes encoding corresponding host activities that restrict non-glucosylated phage DNA: rglA and rglB (restricts glucoseless phage). In the 1980’s, appreciation of the biological scope of these activities was dramatically expanded with the demonstration that plant and animal DNA was also sensitive to restriction in cloning experiments. The rgl genes were renamed mcrA and mcrBC (modified cytosine restriction). The new class of modification-dependent restriction enzymes was named Type IV, as distinct from the familiar modification-blocked Types I–III. A third Escherichia coli enzyme, mrr (modified DNA rejection and restriction) recognizes both methylcytosine and methyladenine. In recent years, the universe of modification-dependent enzymes has expanded greatly. Technical advances allow use of Type IV enzymes to study epigenetic mechanisms in mammals and plants. Type IV enzymes recognize modified DNA with low sequence selectivity and have emerged many times independently during evolution. Here, we review biochemical and structural data on these proteins, the resurgent interest in Type IV enzymes as tools for epigenetic research and the evolutionary pressures on these systems.
doi:10.1093/nar/gkt747
PMCID: PMC3874153  PMID: 23990325
16.  Response to UV damage by four Escherichia coli K-12 restriction systems. 
Journal of Bacteriology  1994;176(19):5888-5896.
To understand the role of restriction in regulating gene flow in bacterial populations, we would like to understand the regulation of restriction enzyme activity. Several antirestriction (restriction alleviation) systems are known that reduce the activity of type I restriction enzymes like EcoKI in vivo. Most of these do not act on type II or type III enzymes, but little information is available for the unclassified modification-dependent systems, of which there are three in E. coli K-12. Of particular interest are two physiological controls on type I enzymes: EcoKI restriction is reduced 2 to 3 orders of magnitude following DNA damage, and a similar effect is seen constitutively in Dam- cells. We used the behavior of EcoKI as a control for testing the response to UV treatment of the three endogenous modification-dependent restriction systems of K-12, McrA, McrBC, and Mrr. Two of these were also tested for response to Dam status. We find that all four resident restriction systems show reduced activity following UV treatment, but not in a unified fashion; each response was genetically and physiologically distinct. Possible mechanisms are discussed.
PMCID: PMC196804  PMID: 7928948
17.  Transcriptional Phase Variation of a Type III Restriction-Modification System in Helicobacter pylori 
Journal of Bacteriology  2002;184(23):6615-6623.
Phase variation is important in bacterial pathogenesis, since it generates antigenic variation for the evasion of immune responses and provides a strategy for quick adaptation to environmental changes. In this study, a Helicobacter pylori clone, designated MOD525, was identified that displayed phase-variable lacZ expression. The clone contained a transcriptional lacZ fusion in a putative type III DNA methyltransferase gene (mod, a homolog of the gene JHP1296 of strain J99), organized in an operon-like structure with a putative type III restriction endonuclease gene (res, a homolog of the gene JHP1297), located directly upstream of it. This putative type III restriction-modification system was common in H. pylori, as it was present in 15 out of 16 clinical isolates. Phase variation of the mod gene occurred at the transcriptional level both in clone MOD525 and in the parental H. pylori strain 1061. Further analysis showed that the res gene also displayed transcriptional phase variation and that it was cotranscribed with the mod gene. A homopolymeric cytosine tract (C tract) was present in the 5′ coding region of the res gene. Length variation of this C tract caused the res open reading frame (ORF) to shift in and out of frame, switching the res gene on and off at the translational level. Surprisingly, the presence of an intact res ORF was positively correlated with active transcription of the downstream mod gene. Moreover, the C tract was required for the occurrence of transcriptional phase variation. Our finding that translation and transcription are linked during phase variation through slipped-strand mispairing is new for H. pylori.
doi:10.1128/JB.184.23.6615-6623.2002
PMCID: PMC135423  PMID: 12426350
18.  A new method for the rapid identification of genes encoding restriction and modification enzymes. 
Nucleic Acids Research  1991;19(8):1831-1835.
We have constructed derivatives of Escherichia coli that can be used for the rapid identification of recombinant plasmids encoding DNA restriction enzymes and methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding DNA methyltransferases. The strains of E. coli that we have constructed are temperature-sensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure that is based on the induction at the restrictive temperature of the lacZ gene. Transformants encoding DNA methyltransferase genes are detected on LB agar plates supplemented with X-gal as blue colonies. Using this method, we have cloned a variety of DNA methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema, Pseudomonas, Xanthomonas and Saccharopolyspora.
PMCID: PMC328112  PMID: 1851562
19.  Origin of the Diversity in DNA Recognition Domains in Phasevarion Associated modA Genes of Pathogenic Neisseria and Haemophilus influenzae 
PLoS ONE  2012;7(3):e32337.
Phase variable restriction-modification (R-M) systems have been identified in a range of pathogenic bacteria. In some it has been demonstrated that the random switching of the mod (DNA methyltransferase) gene mediates the coordinated expression of multiple genes and constitutes a phasevarion (phase variable regulon). ModA of Neisseria and Haemophilus influenzae contain a highly variable, DNA recognition domain (DRD) that defines the target sequence that is modified by methylation and is used to define modA alleles. 18 distinct modA alleles have been identified in H. influenzae and the pathogenic Neisseria. To determine the origin of DRD variability, the 18 modA DRDs were used to search the available databases for similar sequences. Significant matches were identified between several modA alleles and mod gene from distinct bacterial species, indicating one source of the DRD variability was via horizontal gene transfer. Comparison of DRD sequences revealed significant mosaicism, indicating exchange between the Neisseria and H. influenzae modA alleles. Regions of high inter- and intra-allele similarity indicate that some modA alleles had undergone recombination more frequently than others, generating further diversity. Furthermore, the DRD from some modA alleles, such as modA12, have been transferred en bloc to replace the DRD from different modA alleles.
doi:10.1371/journal.pone.0032337
PMCID: PMC3311624  PMID: 22457715
20.  A unique family of Mrr-like modification-dependent restriction endonucleases 
Nucleic Acids Research  2010;38(16):5527-5534.
Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli’s Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes mCNNR (R = G/A) sites and cleaves DNA at fixed distances (N12/N16) away from the modified cytosine at the 3′ side (or N9/N13 from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example ∼32 bp around symmetrically methylated CG sites and ∼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.
doi:10.1093/nar/gkq327
PMCID: PMC2938202  PMID: 20444879
21.  Phasevarion Mediated Epigenetic Gene Regulation in Helicobacter pylori 
PLoS ONE  2011;6(12):e27569.
Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis.
doi:10.1371/journal.pone.0027569
PMCID: PMC3230613  PMID: 22162751
22.  DNA cleavage by Type ISP Restriction–Modification enzymes is initially targeted to the 3′-5′ strand 
Nucleic Acids Research  2012;41(2):1081-1090.
The mechanism by which a double-stranded DNA break is produced following collision of two translocating Type I Restriction–Modification enzymes is not fully understood. Here, we demonstrate that the related Type ISP Restriction–Modification enzymes LlaGI and LlaBIII can cooperate to cleave DNA following convergent translocation and collision. When one of these enzymes is a mutant protein that lacks endonuclease activity, DNA cleavage of the 3′-5′ strand relative to the wild-type enzyme still occurs, with the same kinetics and at the same collision loci as for a reaction between two wild-type enzymes. The DNA nicking activity of the wild-type enzyme is still activated by a protein variant entirely lacking the Mrr nuclease domain and by a helicase mutant that cannot translocate. However, the helicase mutant cannot cleave the DNA despite the presence of an intact nuclease domain. Cleavage by the wild-type enzyme is not activated by unrelated protein roadblocks. We suggest that the nuclease activity of the Type ISP enzymes is activated following collision with another Type ISP enzyme and requires adenosine triphosphate binding/hydrolysis but, surprisingly, does not require interaction between the nuclease domains. Following the initial rapid endonuclease activity, additional DNA cleavage events then occur more slowly, leading to further processing of the initial double-stranded DNA break.
doi:10.1093/nar/gks1210
PMCID: PMC3553963  PMID: 23221632
23.  A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300 
Nucleic Acids Research  2011;39(13):5597-5610.
A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. The protein was purified to over 90% purity by chromatography. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. Cleavage of 5mC-modified plasmids indicated that the sites S5mCNGS (S = C or G) are preferentially digested. The endonuclease activity requires the presence of adenosine triphosphate (ATP) or dATP whereas the non-hydrolyzable γ-S-ATP does not support activity. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. It is most active in Mg++ buffers. No companion methylase gene was found near the SauUSI restriction gene. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage λ infection when the endonuclease is expressed in E. coli.
doi:10.1093/nar/gkr098
PMCID: PMC3141236  PMID: 21421560
24.  The Type ISP Restriction–Modification enzymes LlaBIII and LlaGI use a translocation–collision mechanism to cleave non-specific DNA distant from their recognition sites 
Nucleic Acids Research  2012;41(2):1071-1080.
The Type ISP Restriction–Modification (RM) enzyme LlaBIII is encoded on plasmid pJW566 and can protect Lactococcus lactis strains against bacteriophage infections in milk fermentations. It is a single polypeptide RM enzyme comprising Mrr endonuclease, DNA helicase, adenine methyltransferase and target-recognition domains. LlaBIII shares >95% amino acid sequence homology across its first three protein domains with the Type ISP enzyme LlaGI. Here, we determine the recognition sequence of LlaBIII (5′-TnAGCC-3′, where the adenine complementary to the underlined base is methylated), and characterize its enzyme activities. LlaBIII shares key enzymatic features with LlaGI; namely, adenosine triphosphate-dependent DNA translocation (∼309 bp/s at 25°C) and a requirement for DNA cleavage of two recognition sites in an inverted head-to-head repeat. However, LlaBIII requires K+ ions to prevent non-specific DNA cleavage, conditions which affect the translocation and cleavage properties of LlaGI. By identifying the locations of the non-specific dsDNA breaks introduced by LlaGI or LlaBIII under different buffer conditions, we validate that the Type ISP RM enzymes use a common translocation–collision mechanism to trigger endonuclease activity. In their favoured in vitro buffer, both LlaGI and LlaBIII produce a normal distribution of random cleavage loci centred midway between the sites. In contrast, LlaGI in K+ ions produces a far more distributive cleavage profile.
doi:10.1093/nar/gks1209
PMCID: PMC3553950  PMID: 23222132
25.  Haemophilus influenzae phasevarions have evolved from type III DNA restriction systems into epigenetic regulators of gene expression 
Nucleic Acids Research  2007;35(15):5242-5252.
Phase variably expressed (randomly switching) methyltransferases associated with type III restriction-modification (R-M) systems have been identified in a variety of pathogenic bacteria. We have previously shown that a phase variable methyltransferase (Mod) associated with a type III R-M system in Haemophilus influenzae strain Rd coordinates the random switching of expression of multiple genes, and constitutes a phase variable regulon—‘phasevarion’. We have now identified the recognition site for the Mod methyltransferase in H. influenzae strain Rd as 5′-CGAAT-3′. This is the same recognition site as the previously described HinfIII system. A survey of 59 H. influenzae strains indicated significant sequence heterogeneity in the central, variable region of the mod gene associated with target site recognition. Intra- and inter-strain transformation experiments using Mod methylated or non-methylated plasmids, and a methylation site assay demonstrated that the sequence heterogeneity seen in the region encoding target site specificity does correlate to distinct target sites. Mutations were identified within the res gene in several strains surveyed indicating that Res is not functional. These data suggest that evolution of this type III R-M system into an epigenetic mechanism for controlling gene expression has, in some strains, resulted in loss of the DNA restriction function.
doi:10.1093/nar/gkm571
PMCID: PMC1976455  PMID: 17675301

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