The EcoDXX1 restriction and modification system: Cloning the genes and homology to type I restriction and modification systems

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and Ecob. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.     

EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli

The genes (hsd A) encoding EcoA, a restriction and modification system first identified in Escherichia coli 15T-, behave in genetic crosses as alleles of the genes (hsd K) encoding the archetypal type I restriction and modification system of E. coli K12. Nevertheless, molecular experiments have failed to detect relatedness between the A and K systems. We have cloned the hsd A genes and have identified, on the basis of DNA homology, related genes (hsd E) conferring a new specificity to a natural isolate of E. coli. We show that the overall organization of the genes encoding EcoA and EcoE closely parallels that for EcoK. Each enzyme is encoded by three genes, of which only one, hsdS, confers the specificity of DNA interaction. The three genes are in the same order as those encoding EcoK, i.e. hsdR, hsdM and hsdS and, similarly, they include a promoter between hsdR and hsdM from which the M and S genes can be transcribed. The evidence indicates that EcoA and EcoE are type I restriction and modification enzymes, but they appear to identify an alternative family to EcoK. For both families, the hsdR polypeptide is by far the largest, but the sizes of the other two polypeptides are reversed, with the smallest polypeptide of EcoK being the product of hsd S, and the smallest for the EcoA family being the product of hsdM. Physiologically, the A restriction and modification system differs from that of K and its relatives, in that A-specific methylation of unmodified DNA is particularly effective.     

Analysis of type I restriction modification systems in the Neisseriaceae: genetic organization and properties of the gene products

The hsd locus (host specificity of DNA) was identified in the Neisseria gonorrhoeae genome. The DNA fragment encoding this locus produced an active restriction and modification (R/M) system when cloned into Escherichia coli. This R/M system was designated NgoAV. The cloned genomic fragment (7800 bp) has the potential to encode seven open reading frames (ORFs). Several of these ORFs had significant homology with other proteins found in the databases: ORF1, the hsdM, a methylase subunit (HsdM); ORF2, a homologue of dinD; ORF3, a homologue of hsdS; ORF4, a homologue of hsdS; and ORF5, an endonuclease subunit hsdR. The endonuclease and methylase subunits possessed strongest protein sequence homology to the EcoR124II R/M system, indicating that NgoAV belongs to the type IC R/M family. Deletion analysis showed that only ORF3 imparted the sequence specificity of the RM.NgoAV system, which recognizes an interrupted palindrome sequence (GCAN(8-)TGC). The genetic structure of ORF3 (208 amino acids) is almost identical to the structure of the 5' truncated hsdS genes of EcoDXXI or EcoR124II R/M systems obtained by in vitro manipulation. Genomic sequence analysis allowed us to identify hsd loci with a very high homology to RM.NgoAV in two strains of Neisseria meningitidis. However, significant differences in the organization and structure of the hsdS genes in both these systems suggests that, if functional, they would possess recognition sites that differ from the gonococcus and from themselves.     

Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes

EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.     

Sequence diversity among related genes for recognition of specific targets in DNA molecules

Escherichia coli strains K12 and B, and a new strain designated D, each encode a characteristic restriction and modification enzyme. These enzymes (EcoK, EcoB and presumably EcoD) comprise three subunits of which one, that encoded by the so-called specificity gene (hsdS), is responsible for recognition of the DNA sequence specific to that system. The other two subunits, encoded by hsdR and hsdM, are interchangeable between systems, and the available molecular evidence suggests that the hsdR and hsdM genes are highly conserved. The DNA sequence of a segment of the hsd region that includes the hsdS gene has been determined for each of the three strains. The hsdS gene varies in length from 1335 to 1425 base-pairs and the only regions showing obvious homology, one of about 100 base-pairs and a second of about 250 base-pairs, are highly conserved. The remainder of each hsd S gene shares little, or no, homology with either of the other related specificity genes. Thus, the specificity subunits, though components of a family of closely related enzymes with very similar functions, have remarkably dissimilar primary structure.     

Distribution and diversity of hsd genes in Escherichia coli and other enteric bacteria.

We screened Salmonella typhimurium, Citrobacter freundii, Klebsiella pneumoniae, Shigella boydii, and many isolates of Escherichia coli for DNA sequences homologous to those encoding each of two unrelated type I restriction and modification systems (EcoK and EcoA). Both K- and A-related hsd genes were identified, but never both in the same strain. S. typhimurium encodes three restriction and modification systems, but its DNA hybridized only to the K-specific probe which we know to identify the StySB system. No homology to either probe was detected in the majority of E. coli strains, but in C. freundii, we identified homology to the A-specific probe. We cloned this region of the C. freundii genome and showed that it encoded a functional, A-related restriction system whose specificity differs from those of known type I enzymes. Sequences immediately flanking the hsd K genes of E. coli K-12 and the hsd A genes of E. coli 15T- were shown to be homologous, indicating similar or even identical positions in their respective chromosomes. E. coli C has no known restriction system, and the organization of its chromosome is consistent with deletion of the three hsd genes and their neighbor, mcrB.     

The DNA sequence recognized by the EcoDXX1 restriction endonuclease

EcoDXX1 is a type-I restriction enzyme coded for by the plasmid pDXX1. Like other type-I restriction endonucleases, the enzyme catalyses the modification of susceptible DNA. We have determined the DNA sequence recognised by EcoDXX1 to be: 5'TCANNNNNNNATTC-3' 3'-AGTNNNNNNNTAAG-5' where N can be any nucleotide. This sequence has an overall structure very similar to previously determined type-I sequences.     

The EcoR124 and EcoR124/3 restriction and modification systems: Cloning the genes

The Escherichia coli plasmid R124 codes for a type I restriction and modification system EcoR124 and carries genetic information, most probably in the form of a "silent copy," for the expression of a different R-M specificity R124/3. Characteristic DNA rearrangements have been shown to accompany the switch in specificity from R124 to R124/3 and vice versa. We have cloned a 14.2-kb HindIII fragment from R124 and shown that it contains the hsdR, hsdM, and hsdS genes which code for the EcoR124 R-M system. An equivalent fragment from the plasmid R124/3 following the switch in R-M specificity has also been cloned and shown to contain the genes coding for the EcoR124/3 R-M system. These fragments, however, lack a component present on the wild-type plasmid essential for the switch in specificity. Restriction fragment maps and preliminary heteroduplex analysis indicate the near identity of the genes that encode the two different DNA recognition specificities. Transposon mutagenesis was used to locate the positions of the hsdR, hsdM, and hsdS genes on the cloned fragments in conjunction with complementation tests for gene function. Indirect evidence indicates that hsdR is expressed from its own promoter and that hsdM and hsdS are expressed from a single promoter, unidirectionally.     

Weakening of type I restriction in E. coli: the effect of dam mutation

The host-controlled EcoK-restriction of unmodified phages lambda.0 and T7ocr. is 100-fold alleviated in dam- mutants of E. coli. In addition the EcoK modification activity is considerably decreased in dam- strains. The I and III types restriction (EcoB, EcoD, EcoK and EcoP1) were relieved in dam- mutants, but no alleviation of EcoRI restriction occurred in dam- strains. We interpret the alleviation of the I type restriction in dam- mutants as consequence of induction of the function, which interferes with the I type restriction systems.     

The type IC hsd loci of the enterobacteria are flanked by DNA with high homology to the phage P1 genome: implications for the evolution and spread of DNA restriction systems

Eco R124I, Eco DXXI and Eco prrI are the known members of the type IC family of DNA restriction and modification systems. The first three are carried on large, conjugative plasmids, while Eco prrI is chromosomally encoded. The enzymes are coded by three genes, hsdR , hsdM and hsdS . Analysis of the DNA sequences upstream and downstream of the type IC hsd loci shows that all are highly homologous to each other and also to sequences present in the bacteriophage P1 genome. The upstream sequences include functional phd and doc genes, which encode an addiction system that stabilizes the P1 prophage state, and extend to and beyond pac , the site at which phage DNA packaging begins. Downstream of the hsd loci, P1 DNA sequences begin at exactly the same place for all of the systems. For Eco DXXI and Eco prrI the P1 homology extends for thousands of base pairs while for Eco R124I an IS 1 insertion and an associated deletion have removed most of the P1-homologous sequences. The significance of these results for the evolution of DNA restriction and modification systems is discussed.     

Alleviation of type I restriction in the presence of plasmids of group inc I. General characteristics and molecular cloning of the ard gene

The capability of a number of plasmids of incN and incI groups to alleviate an action of type I EcoK, EcoB, EcoD, and EcoA restriction endonucleases on the unmodified DNA was revealed. The efficiency of EcoK action on lambda 0 DNA is alleviated about 10 divided by 100 fold in E. coli K12 AB 1157 bacteria containing the plasmid of incN group (pKM101, N3, pJA4733) or incI group (R144, R648; R621a; ColIb-P9). We have cloned ard gene of ColIb-P9 plasmid (SalI-C fragment) in pBR322 multicopying vector. A hybrid clone abolishing the EcoK restriction has been received. Ard gene activity is independent of the recA, recBc, recF, lexA, umuC, lon bacterial genes activity. Ard gene's product does not inhibit the EcoK restriction endonuclease action as well as ocr protein (phage T7) and does not increase the process of methylation of DNA as well as ral protein of phage lambda.     

Inhibition of the type I restriction-modification enzymes EcoB and EcoK by the gene 0.3 protein of bacteriophage T7

The gene 0.3 protein of bacteriophage T7 is a potent inhibitor of the restriction-modification enzymes EcoB and EcoK, both in vivo and in vitro. We have analyzed the ability of purified 0.3 protein to inhibit different steps in the reactions of EcoB and EcoK with DNA. Most of our experiments were done with EcoK, but selected tests with EcoB indicate that the two enzymes are affected by 0.3 protein in the same way. Purified 0.3 protein binds tightly to free enzyme, apparently to one of the small subunits, and prevents it from binding to DNA. If EcoK is allowed to form specific recognition complexes with unmodified DNA before 0.3 protein is added, relatively low levels of 0.3 protein prevent the nuclease activity that would otherwise appear upon addition of ATP, but considerably higher levels are needed to prevent formation of filter-binding complexes or ATPase activity. This, together with other results, suggests that the binding site for 0.3 protein is protected in recognition complexes and in the early stages of the ATP-stimulated reactions, but that it becomes accessible again before cleavage of the DNA, perhaps after the translocation step. If added after the nuclease reaction is substantially over, 0.3 protein has little effect on ATPase activity, and indeed, the subunit having the binding site for 0.3 protein apparently dissociates from the enzyme-DNA complex. The methylase activity of EcoK on hemi-methylated recognition sites is strongly inhibited by 0.3 protein added at any stage of the reaction.     

Weakening of type I restriction in E. coli: the effect of 2-aminopurine and 5-bromouracil

2-aminopurine (2-AP) and 5-bromouracil, strong mutagens of base analog type, were found to induce efficiently the alleviation of I type restriction in Escherichia coli. 2-AP induced restriction alleviation occurs in recA, lexA and mut- mutants, but no additional relief of restriction is registered in dam-bacteria in the presence of sublethal 2-AP concentrations. 2-AP specifically alleviates I type restriction in Escherichia coli (EcoA, EcoB, EcoD, and EcoK) and does not affect restriction systems of II (EcoRI) and III (EcoP1) types. We suggest that 2-AP-induced mismatches and other replication errors may be signals inducing restriction alleviation in Escherichia coli.     

Cellular localization of Type I restriction-modification enzymes is family dependent

Cellular localization of Type I restriction-modification enzymes EcoKI, EcoAI, and EcoR124I-the most frequently studied representatives of IA, IB, and IC families-was analyzed by immunoblotting of subcellular fractions isolated from Escherichia coli strains harboring the corresponding hsd genes. EcoR124I shows characteristics similar to those of EcoKI. The complex enzymes are associated with the cytoplasmic membrane via DNA interaction as documented by the release of the Hsd subunits from the membrane into the soluble fraction following benzonase treatment. HsdR subunits of the membrane-bound enzymes EcoKI and EcoR124I are accessible, though to a different extent, at the external surface of cytoplasmic membrane as shown by trypsinization of intact spheroplasts. EcoAI strongly differs from EcoKI and EcoR124I, since neither benzonase nor trypsin affects its association with the cytoplasmic membrane. Possible reasons for such a different organization are discussed in relation of the control of the restriction-modification activities in vivo.     

DNA recognition by a new family of type I restriction enzymes: a unique relationship between two different DNA specificities.

The DNA sequences recognized by the allelic type I restriction enzymes EcoR124 and EcoR124/3 were determined. EcoR124 recognizes 5'-GAA(N6)RTCG-3' and EcoR124/3 recognizes 5'-GAA(N7)RTCG-3'. These are typical of sequences recognized by type I recognition enzymes in that they consist of two specific domains separated by a non-specific spacer sequence. For these two enzymes, the specific sequences are identical but the length of the non-specific spacer is different. The specific domains of EcoR124/3 are thus 3.4 A further apart than those of EcoR124 and rotated with respect to each other through a further 36 degrees.     

Restriction alleviation by bacteriophages lambda and lambda reverse.

Deletion analysis indicated that the phage lambda restriction alleviation gene(s) ral resides between the cIII and N genes. The Ral+ phenotype was expressed only when lambda ral+ carried a modification such that it was resistant to restriction by the host specificity system. Under these conditions, Ral function protected superinfecting unmodified phages from restriction by EcoK or EcoB but not from restriction by EcoP1. Ral-protected phage DNA was not concomitantly K and B modified, but rather received only the modification specified by the system of the restricting host. Possible mechanisms for Ral action are discussed. Of the other lambdoid phages tested, the hybrid phage lambda rev had Ral activity, whereas phi 80vir and one lambda-P22 hybrid did not. The restriction alleviation activity of lambda rev called Lar, may be the same as the activity expressed in sbcA- strains of Escherichia coli, but it was functionally separable from exonuclease VIII activity (the product of the recE gene), which is also expressed in sbcA- strains.     

Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis

An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R-M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R-M enzymes previously found only in enteric bacteria.     

Molecular analysis of the cryptic and functional phn operons for phosphonate use in Escherichia coli K-12.

We cloned the cryptic phn operon of a K-12 strain, phn(EcoK), and analyzed the nucleotide sequence of the phn region (11,672 bp). An mRNA start site upstream of the phnC gene was identified by S1 nuclease mapping. The pho regulon activator PhoB protects a pho box region near the mRNA start in DNase I footprinting and methylation protection experiments. The sequence of the cryptic phn(EcoK) operon was very similar to that of the functional phn operon of an Escherichia coli B strain, phn(EcoB) (C.-M. Chen, Q.-Z. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh, J. Biol. Chem. 265:4461-4471, 1990). The phnE(EcoK) gene has an 8-bp insertion, absent from the phnE(EcoB) gene, which causes a frameshift mutation. The spontaneous activation of the cryptic phn(EcoK) operon is accompanied by loss of this additional 8-bp insertion. Studies of the structure, regulation, and function of the phn region suggest that the phosphate starvation-inducible phn operon consists of 14 cistrons from phnC to phnP.