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The Salmonella research community has used strains and bacteriophages over decades, exchanging useful new isolates among laboratories for the study of cell surface antigens, metabolic pathways and restriction-modification (RM) studies. Here we present the sequences of two laboratory Salmonella strains (STK005, an isolate of LB5000; and its descendant ER3625). In the ancestry of LB5000, segments of ∼15 and ∼42 kb were introduced from Salmonella enterica sv Abony 803 into S. enterica sv Typhimurium LT2, forming strain SD14; this strain is thus a hybrid of S. enterica isolates. Strains in the SD14 lineage were used to define flagellar antigens from the 1950s to the 1970s, and to define three RM systems from the 1960s to the 1980s. LB5000 was also used as a host in phage typing systems used by epidemiologists. In the age of cheaper and easier sequencing, this resource will provide access to the sequence that underlies the extensive literature.

The genome of Salmonella enterica serovar Typhimurium LT7 comprises a chromosome and two plasmids. One plasmid is very close to pSLT of Salmonella Typhimurium LT2; the second harbors a shufflon region. Prophage content is distinct: LT7 lacks Fels-1, while Gifsy-1 and Fels-2 show island-like divergence and likely programmed inversion, respectively.

We report a new subgroup of Type III Restriction-Modification systems that use m4C methylation for host protection. Recognition specificities for six such systems, each recognizing a novel motif, have been determined using single molecule real-time DNA sequencing. In contrast to all previously characterized Type III systems which modify adenine to m6A, protective methylation of the host genome in these new systems is achieved by the N4-methylation of a cytosine base in one strand of an asymmetric 4 to 6 base pair recognition motif. Type III systems are heterotrimeric enzyme complexes containing a single copy of an ATP-dependent restriction endonuclease-helicase (Res) and a dimeric DNA methyltransferase (Mod). The Type III Mods are beta-class amino-methyltransferases, examples of which form either N6-methyl adenine or N4-methyl cytosine in Type II RM systems. The Type III m4C Mod and Res proteins are diverged, suggesting ancient origin or that m4C modification has arisen from m6A MTases multiple times in diverged lineages. Two of the systems, from thermophilic organisms, required expression of both Mod and Res to efficiently methylate an E. coli host, unlike previous findings that Mod alone is proficient at modification, suggesting that the division of labor between protective methylation and restriction activities is atypical in these systems. Two of the characterized systems, and many homologous putative systems, appear to include a third protein; a conserved putative helicase/ATPase subunit of unknown function and located 5’ of the mod gene. The function of this additional ATPase is not yet known, but close homologs co-localize with the typical Mod and Res genes in hundreds of putative Type III systems. Our findings demonstrate a rich diversity within Type III RM systems.

Restriction enzymes that combine DNA methylation and cleavage activities into a single polypeptide or protein assemblage and that modify just one DNA strand for host protection are capable of more efficient adaptation towards novel target sites. However, they must solve the problem of discrimination between newly replicated and unmodified host sites (needing methylation) and invasive foreign site (needing to lead to cleavage). One solution to this problem might be that the activity that occurs at any given site is dictated by the oligomeric state of the bound enzyme. Methylation requires just a single bound site and is relatively slow, while cleavage requires that multiple unmethylated target sites (often found in incoming, foreign DNA) be brought together into an enzyme-DNA complex to license rapid cleavage. To validate and visualize the basis for such a mechanism, we have determined the catalytic behavior of a bifunctional Type IIL restriction-modification (‘RM’) enzyme (DrdV) and determined its high-resolution structure at several different stages of assembly and coordination with multiple bound DNA targets using CryoEM. The structures demonstrate a mechanism of cleavage by which an initial dimer is formed between two DNA-bound enzyme molecules, positioning the single endonuclease domain from each enzyme against the other’s DNA and requiring further oligomerization through differing protein-protein contacts of additional DNA-bound enzyme molecules to enable cleavage. The analysis explains how endonuclease activity is licensed by the presence of multiple target-containing DNA duplexes and provides a clear view of the assembly through 3D space of a DNA-bound RM enzyme ‘synapse’ that leads to rapid cleavage of foreign DNA.

In this announcement, we present the complete annotated genome sequence of an Escherichia coli MC4100 mutant strain, BE104. This strain has several methionine sulfoxide reductase gene deletions, making it ideal for studying enzymes that alter the redox state of methionine.

Deinococcus wulumuqiensis 479 (formerly known as Deinococcus radiodurans 479) is the original source strain for the restriction enzyme DrdI. Its complete sequence and full methylome were determined using Pacific Biosciences single-molecule real-time (SMRT) sequencing.

Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage λ infection, induction of λ prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.

Bacillus subtilis (Ehrenburg) Cohn ATCC 49760, deposited as Bacillus globigii , is the source strain for the restriction enzymes BglI and BglII. Its complete sequence and full methylome were determined using single-molecule real-time (SMRT) sequencing.

The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5’-TCCRAC-3’; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5’-TCCRAC-3’). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5’-TCCRAC-3’). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others., The structure of the bifunctional Type IIL restriction-and-modification enzyme MmeI provides a basis for understanding how such enzymes recognize their substrates and a framework for manipulating their specificities., Author Summary Type II restriction endonucleases (REases) are the bedrock of modern biotechnology. Type II REases were essential for the recombinant DNA revolution and the development of gene technology. However, despite the discovery of more than 4,000 REases, the DNA recognition specificities are limited to only ~365. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes provides a framework for understanding and engineering new specificities. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation. The results establish a platform for rationally engineering further derivatives from MmeI and its homologs that will possess new, intentionally chosen, specificities.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems., Author Summary DNA methylation is a chemical modification of DNA present in many prokaryotic genomes. The best-known role of DNA methylation is as a component of restriction-modification systems. In these systems, restriction enzymes target foreign DNA for cleavage, while DNA methylation protects the host genome from destruction. Studies in a handful of organisms show that DNA methylation may also act independently of restriction systems and function in genome regulation. However, a lack of technologies has limited the study of DNA methylation to a small number of organisms, and the broader patterns and functions of DNA methylation remain unknown. Here we use SMRT-sequencing to determine the genome wide DNA methylation patterns of more than 200 diverse bacteria and archaea. We show that DNA methylation is pervasive and present in more than 90% of studied organisms. Analysis of this data enabled annotation of the specific DNA binding sites of more than 600 restriction systems, revealing their extraordinary diversity. Strikingly, we observed widespread DNA methylation in the absence of restriction systems. Analyses of these patterns reveal that they are conserved through evolution, and likely function in genome regulation. Thus DNA methylation may play a far wider function in prokaryotic genome biology than was previously supposed.

The genomes of two Bacillus cereus strains (ATCC 10987 and ATCC 14579) have been sequenced. Here, we report the specificities of type II/III restriction (R) and modification (M) enzymes. Found in the ATCC 10987 strain, BceSI is a restriction endonuclease (REase) with the recognition and cut site CGAAG 24-25/27-28. BceSII is an isoschizomer of AvaII (G/GWCC). BceSIII cleaves at ACGGC 12/14. The BceSIII C terminus resembles the catalytic domains of AlwI, MlyI, and Nt.BstNBI. BceSIV is composed of two subunits and cleaves on both sides of GCWGC. BceSIV activity is strongly stimulated by the addition of cofactor ATP or GTP. The large subunit (R1) of BceSIV contains conserved motifs of NTPases and DNA helicases. The R1 subunit has no endonuclease activity by itself; it strongly stimulates REase activity when in complex with the R2 subunit. BceSIV was demonstrated to hydrolyze GTP and ATP in vitro. BceSIV is similar to CglI (GCSGC), and homologs of R1 are found in 11 sequenced bacterial genomes, where they are paired with specificity subunits. In addition, homologs of the BceSIV R1-R2 fusion are found in many sequenced microbial genomes. An orphan methylase, M.BceSV, was found to modify GCNGC, GGCC, CCGG, GGNNCC, and GCGC sites. A ParB-methylase fusion protein appears to nick DNA nonspecifically. The ATCC 14579 genome encodes an active enzyme Bce14579I (GCWGC). BceSIV and Bce14579I belong to the phospholipase D (PLD) family of endonucleases that are widely distributed among Bacteria and Archaea. A survey of type II and III restriction-modification (R-M) system genes is presented from sequenced B. cereus, Bacillus anthracis, and Bacillus thuringiensis strains.

The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight-base-pair target recognition sequence 5'-TTAATTAA-3' has been determined to 1.9 A resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a betabetaalpha-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA base pairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in noncanonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures.

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.

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N(6)-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N(6)-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY M6A: G-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC M6A: CC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC M6A: GC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY M6A: G-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC M6A: GG-3' sites, to 100% at 5'-ACGT M6A: GG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.

How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (downward arrow CCNGG), PspGI (downward arrow CCWGG), Eco-RII (downward arrow CCWGG), NgoMIV (G downward arrow CCGGC), and Cfr10I (R downward arrow CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of approximately 70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (downward arrow GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of approximately 70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.

iceA1 in Helicobacter pylori is a homolog of nlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity.

The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca(2+) is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca(2+), possibly a (MboII)(2)-(DNA)(2) complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme)(2)-(DNA)(2) complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.

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

Restriction endonucleases (REases) with 8-base specificity are rare specimens in nature. NotI from Nocardia otitidis-caviarum (recognition sequence 5'-GCGGCCGC-3') has been cloned, thus allowing for mutagenesis and screening for enzymes with altered 8-base recognition and cleavage activity. Variants possessing altered specificity have been isolated by the application of two genetic methods. In step 1, variant E156K was isolated by its ability to induce DNA-damage in an indicator strain expressing M.EagI (to protect 5'-NCGGCCGN-3' sites). In step 2, the E156K allele was mutagenized with the objective of increasing enzyme activity towards the alternative substrate site: 5'-GCTGCCGC-3'. In this procedure, clones of interest were selected by their ability to eliminate a conditionally toxic substrate vector and induce the SOS response. Thus, specific DNA cleavage was linked to cell survival. The secondary substitutions M91V, F157C and V348M were each found to have a positive effect on specific activity when paired with E156K. For example, variant M91V/E156K cleaves 5'-GCTGCCGC-3' with a specific activity of 8.2 x 10(4) U/mg, a 32-fold increase over variant E156K. A comprehensive analysis indicates that the cleavage specificity of M91V/E156K is relaxed to a small set of 8 bp substrates while retaining activity towards the NotI sequence.

The Nla III restriction enzyme isolated from Neisseria lactamica recognizes the sequence 5′-CATG-3′, cleaving after the G to generate a four base 3′ overhang. The Nla III methylase and a portion of the Nla III endonuclease gene were cloned into E. coli by the methylase selection method, and the remaining portion of the Nla III endonuclease gene was cloned by inverse PCR. The nucleotide sequence of the endonuclease gene and the methylase gene were determined. The Nla III endonuclease gene is 693 bp, encoding a protein with predicted molecular weight of 26 487. The Nla III methylase gene was identical with that previously reported [Labbe, D., Joltke, H.J. and Lau, P.C. (1990) Cloning and characterization of two tandemly arranged DNA methyltransferse genes of Neisseria lactamica : an adenine-specific M.NlaIII and a cytosine-type methylase. Mol. Gen. Genet. 224, 101–110]. The endonuclease and methylase genes overlap by four bases and are transcribed in the same orientation. The endonuclease gene was cloned into an improved T7 vector, and a high level of Nla III endonuclease expression was achieved in E. coli

BcgI and BcgI-like restriction endonucleases have a very distinct characteristic which causes them to differ from the other classified restriction enzymes; they all cleave double-stranded DNA specifically on both sides of the recognition sequence to excise a short DNA fragment including the recognition sites. Here we report a new BcgI-like restriction endonuclease, BaeI, isolated from Bacillus sphaericus. Like BcgI, BaeI also cleaves double-stranded DNA on both strands upstream and downstream of its recognition sequence (10/15)ACNNNNGTAYC(12/7). There are two dominant polypeptides in the final preparation of BaeI with molecular masses of approximately 80 and 55 kDa. Both are slightly larger than the two BcgI subunits. BaeI requires both Mg2+ and AdoMet to cleave DNA. Accompanying bilateral cleavage activity, the heteromeric BaeI also has an N6-adenine methyltransferase activity which modifies the symmetrically located adenines within its recognition sequence.

A site-specific endonuclease was isolated from a eukaryotic Chlorella-like green alga infected with the dsDNA-containing virus NYs-1. The enzyme recognizes the sequence 5'-CC-3' and cleaves 5' to the first C. It cleaves 5'-CmC-3' sequences but not 5'-mCC-3' sequences. The enzyme creates breaks in dsDNA whenever two 5'-CC-3' sequences on opposite strands are close enough for the two strands to separate; when the 5'-CC-3' sequences on opposite strands are further apart only a portion of the strands separate. Consequently, NYs-1 endonuclease does not produce a completely stable DNA digestion pattern. The enzyme probably does not cleave ssDNA and definitely does not cleave ssRNA or dsRNA.

R.MwoI, a type-II restriction enzyme with the new specificity 5'-GCN7GC-3', was found in extracts of the thermophilic archaebacterium, Methanobacterium wolfei. R.MwoI cleaves duplex DNA producing fragments with 3-nt, 3'-terminal extensions, thus: GCN5/N2GC. The genes coding for the MwoI restriction and modification enzymes were cloned into Escherichia coli on the plasmid vector pBR322. The clones synthesize a low level of R.MwoI endonuclease. The plasmids display incomplete MwoI-specific modification, suggesting that the clones synthesize a low level of the M.MwoI methyltransferase, too.