Elisabeth A. Raleigh

McrBC : a multisubunit GTP-dependent restriction endonuclease

McrBC-mediated restriction of modified DNA has been studied extensively by genetic methods, but little is known of its molecular action. We have used overproducing plasmid constructs to facilitate purification of the McrBL and McrC proteins, and report preliminary characterization of the activity of the complex. Both proteins are required for cleavage of appropriately modified DNA in vitro, in a reaction absolutely dependent on GTP. ATP inhibits the reaction. The sequence and modification requirements for cleavage of the substrate reflect those seen in vivo. The position of cleavage was examined at the nucleotide level, revealing that cleavage occurs at multiple positions in a small region. Based upon these observations, and upon cleavage of model oligonucleotide substrates, it is proposed that the recognition site for this enzyme consists of the motif RmC(N40-80)RmC, with cleavage occurring at multiple positions on both strands, between the modified C residues. In subunit composition, cofactor requirement, and relation between cleavage and recognition site, McrBC does not fit into any of the classes (types I to IV) of restriction enzyme so far described.

Highlights of the DNA cutters: a short history of the restriction enzymes

In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.

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.

Discovery and distribution of super‐integrons among Pseudomonads

Until recently, integrons (systems for acquisition and expression of new genetic materials) have been associated generally with antibiotic resistance gene cassettes. The discovery of ‘super‐integrons’ in Vibrionaceae suggests a greater impact of this gene acquisition mechanism on bacterial genome evolution than initially believed. Super‐integrons may contain more than 100 gene cassettes and may encode other determinants, including biochemical functions or virulence factors. Here, we report the genetic organization of a super‐integron from Pseudomonas alcaligenes ATCC 55044. This is the first evidence of a super‐integron in a non‐pathogenic bacterium, one which is widely distributed in a great number of ecological niches such as soil and aquatic habitats. Here, the sequence composition, open reading frame (ORF) content and organization of In55044 are described and found to have features intermediate between the multidrug‐resistant integrons and the Vibrio cholerae super‐integron. Similar structures are inferred to be present in several Pseudomonas species, based on polymerase chain reaction (PCR) experiments.

Type I restriction enzymes and their relatives

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes.

RimO, a MiaB-like enzyme, methylthiolates the universally conserved Asp88 residue of ribosomal protein S12 in Escherichia coli

Ribosomal protein S12 undergoes a unique posttranslational modification, methylthiolation of residue D88, in Escherichia coli and several other bacteria. Using mass spectrometry, we have identified the enzyme responsible for this modification in E. coli, the yliG gene product. This enzyme, which we propose be called RimO, is a radical-S-adenosylmethionine protein that bears strong sequence similarity to MiaB, which methylthiolates tRNA. We show that RimO and MiaB represent two of four subgroups of a larger, ancient family of likely methylthiotransferases, the other two of which are typified by Bacillus subtilis YqeV and Methanococcus jannaschii Mj0867, and we predict that RimO is unique among these subgroups in its modification of protein as opposed to tRNA. Despite this, RimO has not significantly diverged from the other three subgroups at the sequence level even within the C-terminal TRAM domain, which in the methyltransferase RumA is known to bind the RNA substrate and which we presume to be responsible for substrate binding and recognition in all four subgroups of methylthiotransferases. To our knowledge, RimO and MiaB represent the most extreme known case of resemblance between enzymes modifying protein and nucleic acid. The initial results presented here constitute a bioinformatics-driven prediction with preliminary experimental validation that should serve as the starting point for several interesting lines of further inquiry.

The other face of restriction: modification-dependent enzymes

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.

Selected Topics from Classical Bacterial Genetics

Current cloning technology exploits many facts learned from classical bacterial genetics. This unit covers those that are critical to understanding the techniques described in this book. Topics include antibiotics, the LAC operon, the F factor, nonsense suppressors, genetic markers, genotype and phenotype, DNA restriction, modification and methylation and recombination.

Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis

Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (–SCH3). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N6-isopentenyladenosine (i6A) to 2-methylthio-N6-isopentenyladenosine (ms2i6A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms2i6A formation (MiaB activity), and yqeV is required for modification of N6-threonylcarbamoyladenosine (t6A) to 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNA. The enzyme responsible for the latter activity belongs to a third MTTase subgroup, no member of which has previously been characterized. We performed domain-swapping experiments between YmcB and YqeV to narrow down the protein domain(s) responsible for distinguishing i6A from t6A and found that the C-terminal TRAM domain, putatively involved with RNA binding, is likely not involved with this discrimination. Finally, we performed a computational analysis to identify candidate residues outside the TRAM domain that may be involved with substrate recognition. These residues represent interesting targets for further analysis.

Evolution of Bacterial Phosphoglycerate Mutases: Non-Homologous Isofunctional Enzymes Undergoing Gene Losses, Gains and Lateral Transfers

Background The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms. Methods/Principal Findings To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect. Conclusions/Significance Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non-uniform PGM distribution we report across the bacterial domain.