Restriction-modification (R-M) systems as defense mechanisms. R-M systems recognize the methylation status of incoming foreign DNA, e.g., phage genomes. Methylated sequences are recognized as self, while recognition sequences on the incoming DNA lacking methylation are recognized as nonself and are cleaved by the restriction endonuclease (REase). The methylation status at the genomic recognition sites is maintained by the cognate methyltransferase (MTase) of the R-M system. 

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Context 1

... REases” is not known. R-M systems are an extremely diverse group of enzymes and are ubiquitous among prokaryotes. To date, nearly 4,000 enzymes are known, with about 300 different specificities (21). The sequencing of more than 2,450 bacterial and archaeal genomes has only reaffirmed their vast diversity in the prokaryotic kingdom (21). In contrast to REases, MTases show highly conserved features, a surprising finding initially. The diversity and prevalence of R-M systems indicate their success in the bacterial world as a defense mechanism. To a large extent, the distribution of MTases among sequenced genomes seems to re- flect the distribution of R-M systems. It has been observed that ϳ 90% of the genomes contain at least one R-M system and that ϳ 80% contain multiple R-M systems ( /rebase/rebase.html). Interestingly, a positive correlation can be observed with respect to the number of R-M genes and the size of the genome (see the supplemental material). A general trend is an increase in the number of R-M systems with an increase in genome size (Fig. 2; see also Fig. S1 in the supplemental material). For example, organisms with a genome size of 2 to 3 Mbp have a median number of 3 R-M systems per genome, those with a genome size of 3 to 4 Mbp have 4 R-M systems per genome, and those with a genome size of 4 to 5 Mbp have 5 R-M systems per genome. However, an anomalous decrease in the 1- to 1.5-Mbp genome size class can be seen in the distribution of R-M systems because of many Brucella species harboring single R-M systems per chromosome (Fig. 2A). In contrast, the presence of multiple R-M systems among Helicobacter and Campylobacter species brings an anomalous increase in the 1.5- to 2-Mbp genome size class (Fig. 2A). A linear correlation can be observed when the above-mentioned bacterial species are omitted from the 1- to 1.49-Mbp and the 1.5- to 1.99-Mbp genome size classes (Fig. 2B). The significance of the presence of multiple R-M systems per organism observed for many bacterial species is discussed below in this review (see “Immigration Control, Maintenance of Species Identity, and Control of Speciation”). A further anomaly was observed for certain organisms wherein the correlation of the number of R-M systems to the genome size is not apparent. For instance, genomes of Buchnera , Borrelia , Chlamydia , Chlamydophila , Coxiella , Rickettsia , and Synechococcus vary in size (ranging from 1 to 2.5 Mb), and they do not appear to encode R-M systems. Notably, some of these organisms are obligate intracellular pathogens or endosymbiotic and therefore occupy the intracellular niche of infected cells. Hence, they may seldom encounter bacteriophages, obviating the need for R-M systems. Alternatively, a low frequency of horizontal gene transfer (HGT) in such species living in closed environments could account for the observed small number or total absence of the systems (see below). Another peculiarity is seen with respect to the occurrence of REases recognizing long or short palindromic DNA sequences. Some of the sequenced genomes belonging to the genera Bacillus , Nocardia , Pseudomonas , and Streptomyces have a larger propor- tion of R-M systems that recognize longer palindromic DNA sequences. Many of these genomes have a relatively large genome of Ͼ 5 Mbp. As a larger genome would have more 4-bp and 6-bp recognition sites than 8-bp sites, the utilization of an R-M system that recognizes the latter sites might prevent accidental double- stranded DNA (dsDNA) breaks inflicted by REases. For example, the probable occurrence of a particular 4-bp sequence in a 5-Mbp genome would be 19,531 times, while an 8-bp recognition sequence would be represented only 76 times, assuming equal base composition and an even 4-base distribution. Continuous selection against REases recognizing smaller target sequences could have resulted in the enrichment of enzymes recognizing longer sequences in the larger genomes. The preference for enzymes recognizing longer recognition sites in larger genomes appears to be an outcome of minimizing accidental double-strand breaks on the host DNA. However, the GC contents of the organism and the recognition site also play an important role. For example, an 8-base GC-rich recognition sequence (such as GGCCGGCC) would occur with a normal frequency in a highly GC-rich genome (e.g., Frankia species [ ϳ 73%]). The probable occurrence of a 4- or 6-base GC-rich sequence in the same genome would be greater than that of the 8-base sequence, and thus, in such a scenario, the organism may employ other mechanisms to protect the genome from accidental double-strand breaks. Restriction was first observed in the 1950s, when phage ␭ (prop- agated in Escherichia coli B) was found to grow poorly on E. coli K-12 (22, 23). Restriction is achieved by the cleavage of the phage DNA (foreign), which is unmethylated, while the genome of the host (self) remains protected due to methylation by the cognate MTase (Fig. 1). Because of their ability to recognize self versus nonself, a property observed for the immune systems of higher organisms, R-M systems are considered to function as primitive immune systems (24, 25). Various studies have demonstrated a 10- to 10 8 -fold protection of the host cell from phages by different R-M systems (reviewed in reference 26). Their role in curtailing the spread of phage is also evident from the fact that a number of phages have evolved to evade restriction, viz ., modification (methylation, glucosylation, and other modified nucleotides) of the phage DNA (1). These modifications of the phage genome directly affect DNA cleavage by REases and thus ensure the evasion of restriction. In turn, bacteria are known to express modification- specific endonucleases to restrict these adapted phages, resulting in a “coevolutionary arms race” (1, 27). The “cellular defense” function of R-M systems does not comprehensively provide an explanation for the following. (i) It does not provide an explanation for the high specificity in sequence recognition (28). A highly sequence-specific REase or a “rare cut- ter” would be less efficient in targeting an incoming DNA. Hence, it is not clear whether selection pressure on bacteria due to phages would be sufficient to maintain the high sequence specificity of the R-M systems (28). (ii) It does not provide an explanation for the presence of multiple R-M systems per organism in many bacterial species. While the antirestriction strategies evolved by phages may lead to the generation of multiple specificities, it is unclear why only certain organisms (e.g., naturally competent bacteria) have an abundance of R-M systems. For example, Neisseria gonorrhoeae contains 16 different biochemically identified systems (29). Moreover, some organisms, such as Helicobacter pylori , N. gonorrhoeae , Haemophilus influenzae , and Streptococcus pneumoniae , have an abundance of R-M systems (Fig. 3). (iii) It does not provide an explanation for the poor sequence homology of REases. While MTases share considerable homology and could be identified by primary sequence analysis, REases have very low levels of sequence similarity among themselves. A faster evolution of REases, if oc- curring, could be one way to account the low level of similarity among themselves. Alternatively, the evolution of REases could have taken place multiple times from different catalytic/structural folds. Although there is no sufficient evidence for the independent origins of REases and cognate MTases, such a scenario, rather than a coevolutionary strategy, would explain their diversification. In addition to the R-M systems, other gene loci are also involved in limiting the entry of invasive DNA elements. Short stretches of direct repeats interrupted by unique sequences, termed “clustered regularly interspaced short palindromic repeats” (CRISPRs), are found in many eubacteria and archaea (30). The nonrepetitive sequences of the CRISPR loci exhibit homology to previously en- countered phage genomes (31). These loci were proposed to serve as memory for the bacteria with respect to earlier phage encounters (32). Recent evidence suggests that CRISPRs along with their associated genes ( cas genes) are involved in adaptive immunity against phages (33). It appears that R-M systems and CRISPRs are the strategies employed by bacteria to serve as innate and adaptive immune systems, respectively, to evade invading genomes. It would be interesting to study the functional cooperation, if any, between the CRISPR and the R-M systems. Another cellular machine which functions similarly to REases in limiting invasive genome elements is RecBCD. RecBCD functions both in restricting foreign genomes and in host DNA repair by recombination (34). The DNA repair function on the phage genome or the restriction function on host DNA could potentially be lethal to the host. RecBCD distinguishes the host genome from the phage DNA by means of a cis element, the Chi sequence, which is absent in phages but present at high frequencies in bacterial genomes (35). RecBCD is a bipolar helicase with nuclease activity that hydrolyzes DNA from a double-strand end (36). When RecBCD reaches a Chi sequence, the hydrolysis of DNA is arrested, and recombination is initiated (37) (Fig. 4). The Chi sequences differ among bacteria and serve as a bar code (38). The recognition of Chi sequences by the RecBCD enzyme is now understood at an atomic resolution (39). The RecBCD enzyme degrades phage DNA after restriction breakage but repairs chromosomal DNA after restriction (40). Alternative roles of RecBCD systems have been proposed by Kobayashi et al. (40; reviewed in reference 41). Similar to RecBCD, the RecFOR pathway has also been shown to repair lethal double-strand breaks on the chromosomes generated by REase and degrade restricted nonself DNA (40, 42). In contrast to the defense systems discussed above, abortive infection of phage (termed ...

Context 2

... (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuni- form distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population. O ne diversity of the is attributes the ability for of bacteria success to in recognize microbial and evolution distinguish and incoming foreign DNA from self DNA. The organisms have evolved strategies to limit constant exposure to extraneous foreign DNA elements. Mechanisms involving restriction-modification (R-M) systems directly target invading DNA elements. To begin with, this review covers the various aspects of R-M systems that target invading DNA elements and counterstrategies employed by the invading genomes to evade restriction. From analyses of these defense and counterdefense measures, it is apparent that the cellular defense function does not comprehensively provide an explanation for (i) the uneven distribution of R-M systems in the bacterial kingdom, (ii) the high level of specificity in sequence recognition, and (iii) the independent evolution of restriction endonucleases (REases) with respect to methyltransferases (MTases). The present review deals with new developments that provide insights into the roles of R-M systems in other aspects of cellular function. The review is not intended to cover the vast literature on structure-func- tion studies, modes of recognition, catalytic motifs, or mechanisms of catalysis by these enzymes. Instead, the major emphasis is to under- stand the reasons for their diversity and to discuss additional biological roles. Restriction-modification (R-M) systems are important components of prokaryotic defense mechanisms against invading genomes. They occur in a wide variety of unicellular organisms, including eubacteria and archaea (1, 2), and comprise two con- trasting enzymatic activities: a restriction endonuclease (REase) and a methyltransferase (MTase). The REase recognizes and cleaves foreign DNA sequences at specific sites, while MTase activity ensures discrimination between self and nonself DNA, by transferring methyl groups to the same specific DNA sequence within the host’s genome (Fig. 1). Functionally, REases cleave en- donucleolytically at phosphodiester bonds, generating 5 = or 3 = overhangs or blunt ends. MTases transfer the methyl group from S -adenosyl methionine to the C-5 carbon or the N 4 amino group of cytosine or to the N 6 amino group of adenine (3). R-M systems are classified mainly into four different types based on their subunit composition, sequence recognition, cleavage position, cofactor requirements, and substrate specificity (4). Type I enzymes consist of a hetero-oligomeric protein complex encompassing both restriction and modification activities. These enzymes bind to a bipartite DNA sequence and cleave from ϳ 100 bp to tens of thousands of base pairs away from the target (5). Typical examples are EcoKI and EcoR124I (5, 6). In contrast, most type II systems contain separate REase and MTase enzymes. Generally, type II REases are homodimeric or homotetrameric and cleave DNA within or near their target site. These enzymes are highly diverse and are known to utilize at least five types of folds: PD-(D/E)XK, PLD, HNH, GIY-YIG, and halfpipe, e.g., R.EcoRI, R.BfiI, R.KpnI, R.Eco29kI, and R.PabI enzymes, respectively (2, 7–10). Type II enzymes are the most widely studied and are also extensively utilized nucleases in genetic engineering. Type III enzymes are heterotrimers (M 2 R 1 ) (11) or heterotetramers (M 2 R 2 ) (12) containing restriction-, methylation-, and DNA-dependent NTPase activities. As a consequence, they compete within themselves for modification or restriction in the same catalytic cycle (13). These enzymes recognize short asymmetric sequences of 5 to 6 bp, translocate along DNA, and cleave the 3 = side of the target site at a distance of ϳ 25 bp (1, 5). Restriction is elicited only when two recognition sequences are in an inverse orientation with respect to each other. Typical examples are EcoP1I and EcoP15I (5, 14). In contrast to the above-described three groups, the type IV systems cleave only DNA substrates containing methylated, hydroxymethylated, or glucosyl-hydroxymethylated bases at specific sequences (4). For example, EcoKMcrBC, a well-studied type IV enzyme, targets A/G m C (methylated cytosine, either m 4 C or m 5 C) separated by ϳ 40 to 3,000 bases (15). The recently discovered type IV enzyme GmrSD specifically digests DNAs containing sugar- modified hydroxymethylated cytosine (16). However, the sequence specificity of the enzyme is not well studied. In addition to the above-described four groups, a number of genomes are also known to encode MTases that are not associated with REases and are thus termed “orphan/solitary MTases.” Examples of this group of enzymes are the N 6 -adenine MTases Dam and CcrM (cell cycle-regulated MTase) and the C-5– cytosine MTase Dcm (17– 19). Interestingly, unlike the vast majority of REases, which are accompanied by MTases to protect the genomic DNA from self- digestion, some of the rare-cutting REases, viz ., R.PacI and R.P- meI, seem to be solitary enzymes with no cognate MTase (http: //rebase.neb.com/rebase/rebase.html) (20). It appears that genome protection in these organisms is dependent on the underrepresentation of the recognition sequences in the genome (20). However, the biological significance of “solitary REases” is not known. R-M systems are an extremely diverse group of enzymes and are ubiquitous among prokaryotes. To date, nearly 4,000 enzymes are known, with about 300 different specificities (21). The sequencing of more than 2,450 bacterial and archaeal genomes has only reaffirmed their vast diversity in the prokaryotic kingdom (21). In contrast to REases, MTases show highly conserved features, a surprising finding initially. The diversity and prevalence of R-M systems indicate their success in the bacterial world as a defense mechanism. To a large extent, the distribution of MTases among sequenced genomes seems to re- flect the distribution of R-M systems. It has been observed that ϳ 90% of the genomes contain at least one R-M system and that ϳ 80% contain multiple R-M systems ( /rebase/rebase.html). Interestingly, a positive correlation can be observed with respect to the number of R-M genes and the size of the genome (see the supplemental material). A general trend is an increase in the number of R-M systems with an increase in genome size (Fig. 2; see also Fig. S1 in the supplemental material). For example, organisms with a genome size of 2 to 3 Mbp have a median number of 3 R-M systems per genome, those with a genome size of 3 to 4 Mbp have 4 R-M systems per genome, and those with a genome size of 4 to 5 Mbp have 5 R-M systems per genome. However, an anomalous decrease in the 1- to 1.5-Mbp genome size class can be seen in the distribution of R-M systems because of many Brucella species harboring single R-M systems per chromosome (Fig. 2A). In contrast, the presence of multiple R-M systems among Helicobacter and Campylobacter species brings an anomalous increase in the 1.5- to 2-Mbp ...