Evidence of participation of McrB(S) in McrBC restriction in Escherichia coli K-12.

The McrBC restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N4-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 51-kDa protein (McrB(L)) and a 33-kDa protein (McrB(S)). The smaller McrB polypeptide is produced from an in-frame, internal translational start site in the mcrB gene. The McrB(S) sequence is identical to that of McrB(L) except that it lacks 161 amino acids present at the N-terminal end of the latter protein. It has been suggested that McrB(L) is the DNA binding restriction subunit. The function of McrB(S) is unknown, although there has been speculation that it plays some role in the modulation of McrBC restriction. Studies of the function of McrB(S) have been challenging since it is produced in frame with McrB(L). In this study, we tested the effects of underproduction (via antisense RNA) and overproduction (via gene dosage) of mcrBC gene products on restriction levels of the mcrBC+ strain JM107. Among the parameters monitored was the induction of SOS responses, which indicate of DNA damage. Evidence from this study suggests that McrB(S) is necessary for stabilization of the McrBC restriction complex in vivo.     

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modified DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB(+)-dependent restriction can be divided into two sets: some modified sequences containing 5-methylcytosine are restricted by McrB+ cells even when McrC-, but most such sequences are restricted in vivo only by McrB+ McrC+ cells. The sequences restricted only by McrB+C+ include T-even bacteriophage containing 5-hydroxymethylcytosine (restriction of this phage is the RglB+ phenotype), some sequences containing N4-methylcytosine, and some sequences containing 5-methylcytosine. The sequence codes for two polypeptides of 54 (McrB) and 42 (McrC) kilodaltons, whereas in vitro translation yields four products, of approximately 29 and approximately 49 (McrB) and of approximately 38 and approximately 40 (McrC) kilodaltons. The McrB polypeptide sequence contains a potential GTP-binding motif, so this protein presumably binds the nucleotide cofactor. The deduced McrC polypeptide is somewhat basic and may bind to DNA, consistent with its genetic activity as a modulator of the specificity of McrB. At the nucleotide sequence level, the G+C content of mcrBC is very low for E. coli, suggesting that the genes may have been acquired recently during the evolution of the species.     

Purification and N-terminal amino acid sequences of two polypeptides encoded by the mcrB gene from Escherichia coli K-12.

This report provides a purification method for the two proteins, 51 kDa and 33 kDa, both encoded by the same mcrB gene of the McrBC restriction system in Escherichia coli K-12. The two proteins were produced in large quantity using a T7 expression system and copurified to near homogeneity by DEAE-Sepharose and Affi-Gel blue column chromatography. The N-terminal amino acid sequences of these purified McrB proteins were the same as those predicted from the mcrB DNA sequence by Ross et al. [J. Bacteriol. 171 (1989b) 1974-1981]. The 33-kDa protein totally overlaps the C-terminal part of the 51-kDa protein.     

Characterization of the mcrBC region of Escherichia coli K-12 wild-type and mutant strains.

We have carried out an analysis of the Escherichia coli K-12 mcrBC locus in order to (1) elucidate its genetic organization, (2) to identify the proteins encoded by this region, and (3) to characterize their involvement in the restriction of DNA containing methylated cytosine residues. In vitro expression of recombinant plasmids carrying all or portions of the mcrBC region revealed that the mcrB and mcrC genes are organized as an operon. The mcrBC operon specifies five proteins, as evident from parallel in vitro and in in vivo expression studies. Three proteins of 53, 35 and 34 kDa originate from mcrB expression, while two proteins of 37 and 16 kDa arise from mcrC expression. Products of both the mcrB and mcrC genes are required to restrict the methylated substrate DNA used in this study. We also determined the nature of mutant mcrBC loci in comparison to the E. coli K-12 wild-type mcrBC locus. A major goal of these studies was to clarify the nature of the mcrB-1 mutation, which is carried by some strains employed in previous analyses of the E. coli K-12 McrBC system. Based on our analyses the mutant strains investigated could be divided into different complementation groups. The mcrB-1 mutation is a nonsense or frameshift mutation located within mcrB. It causes premature termination of mcrB gene product synthesis and reduces the level of mcrC gene expression. This finding helps to understand an existing conflict in the literature. We also describe temperature-sensitive McrA activity in some of the strains analysed and its relationship to the previously defined differences in the tolerance levels of E. coli K-12 mcrBC mutants to cytosine methylation.     

Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12

Summary The McrB restriction system in Escherichia coli K12 causes sequence-specific recognition and inactivation of DNA containing 5-methylcytosine residues. We have previously located the mcrB gene near hsdS at 99 min on the E. coli chromosome and demonstrated that is encodes a 51 kDa polypeptide required for restriction of M.AluI methylated (A-G-5mC-T) DNA. We show here, by analysis of maxicell protein synthesis of various cloned fragments from the mcrB region, that a second protein of approximately 39 kDa is also required for McrB-directed restriction. The new gene, designated mcrC, is adjacent to mcrB and located distally to hsdS. The McrB phenotype has been correlated previously with restriction of 5-hydroxy-methylcytosine (HMC)-containing T-even phage DNA that lacks the normal glucose modification of HMC, formally designated RglB (for restriction of glucoseless phage). This report reveals a difference between the previously correlated McrB and RglB restriction systems: while both require the mcrB gene product only the McrB system requires the newly identified mcrC-encoded 39-kDa polypeptide.     

Localization of a genetic region involved in McrB restriction by Escherichia coli K-12.

A 5,500-base-pair BglII-EcoRI fragment proximal to the hsd genes of Escherichia coli K-12 has been cloned in the plasmid vector pUC9. The resultant hybrid plasmid was shown to complement the mcrB mutation of E. coli K802. The presence of the hybrid plasmid in strain K802 caused an 18.3-fold drop in transformation efficiency with AluI-methylated pACYC184 relative to unmethylated pACYC184. These results indicate that the cloned DNA is involved in the McrB system restriction of 5-methylcytosine DNA.     

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains.

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.     

Genetic and Physical Mapping of the Mcra (Rgla) and Mcrb (Rglb) Loci of Escherichia Coli K-12

We have genetically analyzed, cloned and physically mapped the modified cytosine-specific restriction determinants mcrA (rglA) and mcrB (rglB) of Escherichia coli K-12. The independently discovered Rgl and Mcr restriction systems are shown to be identical by three criteria: 1) mutants with the RglA- or RglB- phenotypes display the corresponding McrA- or McrB- phenotypes, and vice versa; 2) the gene(s) for RglA and McrA reside together at one locus, while gene(s) for RglB and McrB are coincident at a different locus; and 3) RglA+ and RglB+ recombinant clones complement for the corresponding Mcr-deficient lesions. The mcrA (rglA) gene(s) is on the excisable element e14, just clockwise of purB at 25 min. The mcrB (rglB) gene(s), at 99 min, is in a cluster of restriction functions that includes hsd and mrr, determinants of host-specific restriction (EcoK) and methyladenine-specific restriction respectively. Gene order is mcrB-hsdS-hsdM-hsdR-mrr-serB. Possible models for the acqusition of these restriction determinants by enteric bacteria are discussed.     

Methyl-cytosine specific restriction in Escherichia coli K-12

Experiments on transformation of Escherichia coli K-12 cells by plasmids carrying RM systems with different recognition sites containing 5-methylcytosine have shown that the gene mcrB determines the function of restriction. The data obtained made it possible to believe that E. coli possesses no restriction system recognizing specifically cytosine methylated in position 4.     

The McrBC restriction endonuclease assembles into a ring structure in the presence of G nucleotides

McrBC from Escherichia coli K-12 is a restriction enzyme that belongs to the family of AAA(+) proteins and cuts DNA containing modified cytosines. Two proteins are expressed from the mcrB gene: a full-length version, McrB(L), and a short version, McrB(S). McrB(L) binds specifically to the methylated recognition site and is, therefore, the DNA-binding moiety of the McrBC endonuclease. McrB(S) is devoid of DNA-binding activity. We observed that the quaternary structure of the endonuclease depends on binding of the cofactors. In gel filtration experiments, McrB(L) and McrB(S) form high molecular weight oligomers in the presence of Mg(2+) and GTP, GDP or GTP-gamma-S. Oligomerization did not require the presence of DNA and was independent of GTP hydrolysis. Electron micrographs of negatively stained McrB(L) and McrB(S) revealed ring-shaped particles with a central channel. Mass analysis by scanning transmission electron microscopy indicates that McrB(L) and McrB(S) form single heptameric rings as well as tetradecamers. In the presence of McrC, a subunit that is essential for DNA cleavage, the tetradecameric species was the major form of the endonuclease.     

Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene.

Using the vector pGEM-4-blue, a 4,251-base-pair DNA fragment containing the gene for the surface (S)-layer protein of Bacillus sphaericus 2362 was cloned into Escherichia coli. Determination of the nucleotide sequence indicated an open reading frame (ORF) coding for a protein of 1,176 amino acids with a molecular size of 125 kilodaltons (kDa). A protein of this size which reacted with antibody to the 122-kDa S-layer protein of B. sphaericus was detected in cells of E. coli containing the recombinant plasmid. Analysis of the deduced amino acid sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The first amino acid of the N-terminal sequence of the 122-kDa S-layer protein followed the predicted cleavage site of the leader peptide in the 125-kDa protein. A sequence characteristic of promoters expressed during vegetative growth was found within a 177-base-pair region upstream from the ORF coding for the 125-kDa protein. This putative promoter may account for the expression of this gene during the vegetative growth of B. sphaericus and E. coli. The gene for the 125-kDa protein was followed by an inverted repeat characteristic of terminators. Downstream from this gene (11.2 kilobases) was an ORF coding for a putative 80-kDa protein having a high sequence similarity to the 125-kDa protein. Evidence was presented indicating that this gene is cryptic.     

Organization and function of the mcrBC genes of Escherichia coli K-12

Many natural DNA sequences are restricted in Escherichia coli K-12, not only by the classic Type I restriction system EcoK, but also by one of three modification-specific restriction systems found in K-12. The McrBC system is the best studied of these. We infer from the base composition of the mcrBC genes that they were imported from an evolutionarily distant source. The genes are located in a hypervariable cluster of restriction genes that may play a significant role in generation of species identity in enteric bacteria. Restriction activity requires the products of two genes for activity both in vivo and in vitro. The mcrB gene elaborates two protein products, only one of which is required for activity in vitro, but both of which contain a conserved amino acid sequence motif identified as a possible GTP-binding site. The mcrC gene product contains a leucine heptad repeat that could play a role in protein-protein interactions. McrBC activity in vivo and in vitro depends on the presence of modified cytosine in a specific sequence context; three different modifications are recognized. The in vitro activity of this novel multi-subunit restriction enzyme displays an absolute requirement for GTP as a cofactor.     

A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli.

The 27-kilodalton (kDa) mosquitocidal protein gene from Bacillus thuringiensis subsp. israelensis has been cloned as a 10-kilobase (kb) HindIII fragment from plasmid DNA; efficient expression in Escherichia coli KM1 depends on a region of DNA located approximately 4 kb upstream (K. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987). We have cloned the upstream DNA region and show that it contains a complete open reading frame (ORF) encoding a protein with a molecular mass of 19,584 Da. Sequencing of adjacent stretches of DNA revealed two partial ORFs: one has 55.2% identity in an overlap of 319 amino acids to the putative transposase of IS231 of B. thuringiensis subsp. thuringiensis, and the other, a 78-codon partial ORF, may be the carboxyl terminus of the 67-kDa protein previously observed in maxicells of strain KM1. A 0.8-kb fragment containing only the 20-kDa protein gene greatly enhanced the expression of the 27-kDa protein in E. coli. The introduction of nonsense codons into the 20-kDa protein gene ORF abolished this effect, indicating that the gene product, not the mRNA or DNA, is required for the enhancement. The effect of the 20-kDa protein gene on various fusions of lacZ to the 27-kDa protein gene suggests that the 20-kDa protein acts after the initiation of translation of the 27-kDa protein gene.