Lethal perturbation of an Escherichia coli regulatory network is triggered by a restriction-modification system's regulator and can be mitigated by excision of the cryptic prophage Rac.

Bacterial gene regulatory networks orchestrate responses to environmental challenges. Horizontal gene transfer can bring in genes with regulatory potential, such as new transcription factors (TFs), and this can disrupt existing networks. Serious regulatory perturbations may even result in cell death. Here, we show the impact on Escherichia coli of importing a promiscuous TF that has adventitious transcriptional effects within the cryptic Rac prophage. A cascade of regulatory network perturbations occurred on a global level. The TF, a C regulatory protein, normally controls a Type II restriction-modification system, but in E. coli K-12 interferes with expression of the RacR repressor gene, resulting in de-repression of the normally-silent Rac ydaT gene. YdaT is a prophage-encoded TF with pleiotropic effects on E. coli physiology. In turn, YdaT alters expression of a variety of bacterial regulons normally controlled by the RcsA TF, resulting in deficient lipopolysaccharide biosynthesis and cell division. At the same time, insufficient RacR repressor results in Rac DNA excision, halting Rac gene expression due to loss of the replication-defective Rac prophage. Overall, Rac induction appears to counteract the lethal toxicity of YdaT. We show here that E. coli rewires its regulatory network, so as to minimize the adverse regulatory effects of the imported C TF. This complex set of interactions may reflect the ability of bacteria to protect themselves by having robust mechanisms to maintain their regulatory networks, and/or suggest that regulatory C proteins from mobile operons are under selection to manipulate their host's regulatory networks for their own benefit.

Molecular basis for lethal cross-talk between two unrelated bacterial transcription factors - the regulatory protein of a restriction-modification system and the repressor of a defective prophage.

Bacterial gene expression depends on the efficient functioning of global transcriptional networks, however their interconnectivity and orchestration rely mainly on the action of individual DNA binding proteins called transcription factors (TFs). TFs interact not only with their specific target sites, but also with secondary (off-target) sites, and vary in their promiscuity. It is not clear yet what mechanisms govern the interactions with secondary sites, and how such rewiring affects the overall regulatory network, but this could clearly constrain horizontal gene transfer. Here, we show the molecular mechanism of one such off-target interaction between two unrelated TFs in Escherichia coli: the C regulatory protein of a Type II restriction-modification system, and the RacR repressor of a defective prophage. We reveal that the C protein interferes with RacR repressor expression, resulting in derepression of the toxic YdaT protein. These results also provide novel insights into regulation of the racR-ydaST operon. We mapped the C regulator interaction to a specific off-target site, and also visualized C protein dynamics, revealing intriguing differences in single molecule dynamics in different genetic contexts. Our results demonstrate an apparent example of horizontal gene transfer leading to adventitious TF cross-talk with negative effects on the recipient's viability. More broadly, this study represents an experimentally-accessible model of a regulatory constraint on horizontal gene transfer.

Regulator-dependent temporal dynamics of a restriction-modification system's gene expression upon entering new host cells: single-cell and population studies.

Restriction-modification (R-M) systems represent a first line of defense against invasive DNAs, such as bacteriophage DNAs, and are widespread among bacteria and archaea. By acquiring a Type II R-M system via horizontal gene transfer, the new hosts generally become more resistant to phage infection, through the action of a restriction endonuclease (REase), which cleaves DNA at or near specific sequences. A modification methyltransferase (MTase) serves to protect the host genome against its cognate REase activity. The production of R-M system components upon entering a new host cell must be finely tuned to confer protective methylation before the REase acts, to avoid host genome damage. Some type II R-M systems rely on a third component, the controller (C) protein, which is a transcription factor that regulates the production of REase and/or MTase. Previous studies have suggested C protein effects on the dynamics of expression of an R-M system during its establishment in a new host cell. Here, we directly examine these effects. By fluorescently labelling REase and MTase, we demonstrate that lack of a C protein reduces the delay of REase production, to the point of being simultaneous with, or even preceding, production of the MTase. Single molecule tracking suggests that a REase and a MTase employ different strategies for their target search within host cells, with the MTase spending much more time diffusing in proximity to the nucleoid than does the REase. This difference may partially ameliorate the toxic effects of premature REase expression.

Low-level expression of the Type II restriction-modification system confers potent bacteriophage resistance in Escherichia coli.

Restriction-modification systems (R-M) are one of the antiviral defense tools used by bacteria, and those of the Type II family are composed of a restriction endonuclease (REase) and a DNA methyltransferase (MTase). Most entering DNA molecules are usually cleaved by the REase before they can be methylated by MTase, although the observed level of fragmented DNA may vary significantly. Using a model EcoRI R-M system, we report that the balance between DNA methylation and cleavage may be severely affected by transcriptional signals coming from outside the R-M operon. By modulating the activity of the promoter, we obtained a broad range of restriction phenotypes for the EcoRI R-M system that differed by up to 4 orders of magnitude in our biological assays. Surprisingly, we found that high expression levels of the R-M proteins were associated with reduced restriction of invading bacteriophage DNA. Our results suggested that the regulatory balance of cleavage and methylation was highly sensitive to fluctuations in transcriptional signals both up- and downstream of the R-M operon. Our data provided further insights into Type II R-M system maintenance and the potential conflict within the host bacterium.

Transcriptome analyses of cells carrying the Type II Csp231I restriction-modification system reveal cross-talk between two unrelated transcription factors: C protein and the Rac prophage repressor.

Restriction-modification (R-M) systems represent an effective mechanism of defence against invading bacteriophages, and are widely spread among bacteria and archaea. In acquiring a Type II R-M system via horizontal gene transfer, the new hosts become more resistant to phage infection, through the action of a restriction endonuclease (REase), which recognizes and cleaves specific target DNAs. To protect the host cell's DNA, there is also a methyltransferase (MTase), which prevents DNA cleavage by the cognate REase. In some R-M systems, the host also accepts a cis-acting transcription factor (C protein), which regulates the counteracting activities of REase and MTase to avoid host self-restriction. Our study characterized the unexpected phenotype of Escherichia coli cells, which manifested as extensive cell filamentation triggered by acquiring the Csp231I R-M system from Citrobacter sp. Surprisingly, we found that the cell morphology defect was solely dependent on the C regulator. Our transcriptome analysis supported by in vivo and in vitro assays showed that C protein directly silenced the expression of the RacR repressor to affect the Rac prophage-related genes. The rac locus ydaST genes, when derepressed, exerted a toxicity indicated by cell filamentation through an unknown mechanism. These results provide an apparent example of transcription factor cross-talk, which can have significant consequences for the host, and may represent a constraint on lateral gene transfer.

Natural tuning of restriction endonuclease synthesis by cluster of rare arginine codons.

Restriction-modification (R-M) systems are highly widespread among bacteria and archaea, and they appear to play a pivotal role in modulating horizontal gene transfer, as well as in protecting the host organism against viruses and other invasive DNA particles. Type II R-M systems specify two independent enzymes: a restriction endonuclease (REase) and protective DNA methyltransferase (MTase). If the cell is to survive, the counteracting activities as toxin and antitoxin, must be finely balanced in vivo. The molecular basis of this regulatory process remains unclear and current searches for regulatory elements in R-M modules are focused mainly at the transcription step. In this report, we show new aspects of REase control that are linked to translation. We used the EcoVIII R-M system as a model. Both, the REase and MTase genes for this R-M system contain an unusually high number of rare arginine codons (AGA and AGG) when compared to the rest of the E. coli K-12 genome. Clusters of these codons near the N-terminus of the REase greatly affect the translational efficiency. Changing these to higher frequency codons for E. coli (CGC) improves the REase synthesis, making the R-M system more potent to defend its host against bacteriophages. However, this improved efficiency in synthesis reduces host fitness due to increased autorestriction. We hypothesize that expression of the endonuclease gene can be modulated depending on the host genetic context and we propose a novel post-transcriptional mode of R-M system regulation that alleviates the potential lethal action of the restriction enzyme.

Isospecific adenine DNA methyltransferases show distinct preferences towards DNA substrates.

Here, we report results on systematic analysis of DNA substrate preferences of three N6-adenine ß-class DNA methyltransferases that are part of the type II restriction-modification systems. The studied enzymes were: M.EcoVIII, M.HindIII and M.LlaCI, which although found in phylogenetically distant bacteria (γ-proteobacteria and low-GC Gram-positive bacteria), recognize the same palindromic specific sequence 5'-AAGCTT-3' and catalyze formation of N6-methyladenine at the first A-residue. As expected overall the enzymes share the most analyzed features, but they show also some distinct differences in substrate recognition. Therefore DNA methylation reactions were carried out not only under standard, but also under relaxed conditions using DMSO or glycerol. We found that all of these enzymes preferred DNA containing a hemimethylated target site, but differ in modification of ssDNA, especially more pronounced for M.EcoVIII under relaxed conditions. In these conditions they also have shown varied preferences toward secondary sites, which differ by one nucleotide from specific sequence. They preferred sequences with substitutions at the 1st (A1 → G/C) and at the 2nd position (A2 → C), while sites with substitutions at the 3rd position (G3 → A/C) were modified less efficiently. Kinetic parameters of the methylation reaction carried out by M.EcoVIII were determined. Methylation efficiency (kcat/Km) of secondary sites was 4.5-10 times lower when compared to the unmethylated specific sequences, whilst efficiency observed for the hemimethylated substrate was almost 4.5 times greater. We also observed a distinct effect of analyzed enzymes on unspecific interaction with DNA phosphate backbone. We concluded that for all three enzymes the most critical is the phosphodiester bond between G3-C4 nucleotides at the center of the target site.

Natural C-independent expression of restriction endonuclease in a C protein-associated restriction-modification system.

Restriction-modification (R-M) systems are highly prevalent among bacteria and archaea, and appear to play crucial roles in modulating horizontal gene transfer and protection against phage. There is much to learn about these diverse enzymes systems, especially their regulation. Type II R-M systems specify two independent enzymes: a restriction endonuclease (REase) and protective DNA methyltransferase (MTase). Their activities need to be finely balanced in vivo Some R-M systems rely on specialized transcription factors called C (controller) proteins. These proteins play a vital role in the temporal regulation of R-M gene expression, and function to indirectly modulate the horizontal transfer of their genes across the species. We report novel regulation of a C-responsive R-M system that involves a C protein of a poorly-studied structural class - C.Csp231I. Here, the C and REase genes share a bicistronic transcript, and some of the transcriptional auto-control features seen in other C-regulated R-M systems are conserved. However, separate tandem promoters drive most transcription of the REase gene, a distinctive property not seen in other tested C-linked R-M systems. Further, C protein only partially controls REase expression, yet plays a role in system stability and propagation. Consequently, high REase activity was observed after deletion of the entire C gene, and cells bearing the ΔC R-M system were outcompeted in mixed culture assays by those with the WT R-M system. Overall, our data reveal unexpected regulatory variation among R-M systems.

Relaxed specificity of prokaryotic DNA methyltransferases results in DNA site-specific modification of RNA/DNA heteroduplexes.

RNA/DNA hybrid duplexes regularly occur in nature, for example in transcriptional R loops. Their susceptibility to modification by DNA-specific or RNA-specific enzymes is, thus, a biologically relevant question, which, in addition, has possible biotechnological implications. In this study, we investigated the activity of four isospecific DNA methyltransferases (M.EcoVIII, M.LlaCI, M.HindIII, M.BstZ1II) toward an RNA/DNA duplex carrying one 5'-AAGCUU-3'/3'-TTCGAA-5' target sequence. The analyzed enzymes belong to the ß-group of adenine N6-methyltransferases and recognize the palindromic DNA sequence 5'-AAGCTT-3'/3'-TTCGAA-5'. Under standard conditions, none of these isospecific enzymes could detectibly methylate the RNA/DNA duplex. However, the addition of agents that generally relax specificity, such as dimethyl sulfoxide (DMSO) and glycerol, resulted in substantial methylation of the RNA/DNA duplex by M.EcoVIII and M.LlaCI. Only the DNA strand of the RNA/DNA duplex was methylated. The same was not observed for M.HindIII or M.BstZ1II. This is, to our knowledge, the first report that demonstrates such activity by prokaryotic DNA methyltransferases. Possible applications of these findings in a laboratory practice are also discussed.

To be or not to be: regulation of restriction-modification systems and other toxin-antitoxin systems.

One of the simplest classes of genes involved in programmed death is that containing the toxin-antitoxin (TA) systems of prokaryotes. These systems are composed of an intracellular toxin and an antitoxin that neutralizes its effect. These systems, now classified into five types, were initially discovered because some of them allow the stable maintenance of mobile genetic elements in a microbial population through postsegregational killing or the death of cells that have lost these systems. Here, we demonstrate parallels between some TA systems and restriction-modification systems (RM systems). RM systems are composed of a restriction enzyme (toxin) and a modification enzyme (antitoxin) and limit the genetic flux between lineages with different epigenetic identities, as defined by sequence-specific DNA methylation. The similarities between these systems include their postsegregational killing and their effects on global gene expression. Both require the finely regulated expression of a toxin and antitoxin. The antitoxin (modification enzyme) or linked protein may act as a transcriptional regulator. A regulatory antisense RNA recently identified in an RM system can be compared with those RNAs in TA systems. This review is intended to generalize the concept of TA systems in studies of stress responses, programmed death, genetic conflict and epigenetics.

Antisense RNA associated with biological regulation of a restriction-modification system.

Restriction-modification systems consist of a modification enzyme that methylates a specific DNA sequence and a restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI system, where the restriction gene is located upstream of the modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88 nt) antisense RNA (Rna0) transcribed from a reverse promoter (P(REV0)) at the 3' end of the restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the P(M1,M2) promoter. P(M1,M2) promoter-initiated transcription, in turn, appeared to be inhibited by P(REV0). Mutational inactivation of P(REV0) increased expression of the restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in P(REV0) increased restriction of incoming DNA. Second, the presence of the antisense RNA gene (ecoRIA) in trans alleviated cell killing after loss of the EcoRI plasmid (post-segregational killing). Taken together, these results strongly suggested the involvement of an antisense RNA in the biological regulation of this restriction-modification system.

Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system.

Most type II restriction-modification (R-M) systems produce separate endonuclease (REase) and methyltransferase (MTase) proteins. After R-M genes enter a new cell, MTase activity must appear before REase or the host chromosome will be cleaved. Temporal control of these genes thus has life-or-death consequences. PvuII and some other R-M systems delay endonuclease expression by cotranscribing the REase gene with the upstream gene for an autogenous activator/repressor (C protein). C.PvuII was previously shown to have low levels early, but positive feedback later boosts transcription of the C and REase genes. The MTase is expressed without delay, and protects the host DNA. C.PvuII binds to two sites upstream of its gene: O(L), associated with activation, and O(R), associated with repression. Even when symmetry elements of each operator are made identical, C.PvuII binds preferentially to O(L). In this study, the intra-operator spacers are shown to modulate relative C.PvuII affinity. In light of a recently reported C.Esp1396I-DNA co-crystal structure, in vitro and in vivo effects of altering O(L) and O(R) spacers were determined. The results suggest that the GACTnnnAGTC consensus is the primary determinant of C.PvuII binding affinity, with intra-operator spacers playing a fine-tuning role that affects mobility of this R-M system.

Real-time kinetics of restriction-modification gene expression after entry into a new host cell.

Most type II restriction-modification (R-M) systems produce separate restriction endonuclease (REase) and methyltransferase (MTase) proteins. After R-M system genes enter a new cell, protective MTase must appear before REase to avoid host chromosome cleavage. The basis for this apparent temporal regulation is not well understood. PvuII and some other R-M systems appear to achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator/repressor (the 'C' protein C.PvuII). To test this model, bacteriophage M13 was used to introduce the PvuII genes into a bacterial population in a relatively synchronous manner. REase mRNA and activity appeared approximately 10 min after those of the MTase, but never rose if there was an inactivating pvuIIC mutation. Infection with recombinant M13pvuII phage had little effect on cell growth, relative to infection with parental M13. However, infection of cells pre-expressing C.PvuII led to cessation of growth. This study presents the first direct demonstration of delayed REase expression, relative to MTase, when type II R-M genes enter a new host cell. Surprisingly, though the C and REase genes are cotranscribed, the pvuIIC portion of the mRNA was more abundant than the pvuIIR portion after stable establishment of the R-M system.

Regulatory circuit based on autogenous activation-repression: roles of C-boxes and spacer sequences in control of the PvuII restriction-modification system.

Type II restriction-modification (R-M) systems comprise a restriction endonuclease (REase) and a protective methyltransferase (MTase). After R-M genes enter a new cell, MTase must appear before REase or the chromosome will be cleaved. PvuII and some other R-M systems achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator (the controlling or 'C' protein C.PvuII). This study reveals, through in vivo titration, that C.PvuII is not only an activator but also a repressor for its own gene. In other systems, this type of circuit can result in oscillatory behavior. Despite the use of identical, symmetrical C protein-binding sequences (C-boxes) in the left and right operators, C.PvuII showed higher in vitro affinity for O(L) than for O(R), implicating the spacer sequences in this difference. Mutational analysis associated the repression with O(R), which overlaps the promoter -35 hexamer but is otherwise dispensable for activation. A nonrepressing mutant exhibited poor establishment in new cells. Comparing promoter-operator regions from PvuII and 29 R-M systems controlled by C proteins revealed that the most-highly conserved sequence is the tetranucleotide spacer separating O(L) from O(R). Any changes in that spacer reduced the stability of C.PvuII-operator complexes and abolished activation.

A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid.

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.

Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction-modification systems.

The gene encoding the LlaCI methyltransferase (M.LlaCI) from Lactococcus lactis subsp. cremoris W15 was overexpressed in Escherichia coli. The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein of M(r) 31 300+/-1000 under denaturing conditions. The exact position of the start codon AUG was determined by protein microsequencing. This enzyme recognizes the specific palindromic sequence 5'-AAGCTT-3'. Purified M.LlaCI was characterized. Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer. It modifies the first adenine residue at the 5' end of the specific sequence to N(6)-methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII (Haemophilus influenzae Rd) and EcoVIII (Escherichia coli E1585-68) restriction-modification systems. This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved among N(6)-adenine beta-class methyltransferases. However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII. Restriction endonucleases require Mg(2+) for phosphodiester bond cleavage. Mg(2+) was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues. This observation suggests that sensitivity of the M.LlaCI to Mg(2+) may strengthen the restriction activity of the cognate endonuclease in the bacterial cell. Other biological implications of this finding are also discussed.

Genetic organization and molecular analysis of the EcoVIII restriction-modification system of Escherichia coli E1585-68 and its comparison with isospecific homologs.

The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome. The 921-bp EcoVIII endonuclease (R. EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an M(r) of 35,554. The convergently oriented EcoVIII methyltransferase (M. EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an M(r) of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5'-AAGCTT-3'. Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized. R. EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5' protruding ends. M. EcoVIII functions as a monomer and modifies the first adenine residue at the 5' end of the specific sequence to N(6)-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M. EcoVIII with M. HindIII and M. LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m(6) N-adenine beta-class methyltransferases. The deduced amino acid sequence of R. EcoVIII shows weak homology with its two isoschizomers, R. HindIII (26%) and R. LlaCI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R. EcoVIII (D(108)X(12)DXK(123)), as well as in the primary structures of R. LlaCI and R. HindIII. Polyclonal antibodies raised against R. EcoVIII did not react with R. HindIII, while anti-M. EcoVIII antibodies cross-reacted with M. LlaCI but not with M. HindIII. R. EcoVIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M. EcoVIII enzyme. The biological implications of this finding are discussed.