Restriction-modification Genes Research Articles

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems.

The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.

Conjugation-Based Genome Engineering in Deinococcus radiodurans.

Deinococcus radiodurans has become an attractive microbial platform for the study of extremophile biology and industrial bioproduction. To improve the genomic manipulation and tractability of this species, the development of tools for whole genome engineering and design is necessary. Here, we report the development of a simple and robust conjugation-based DNA transfer method from E. coli to D. radiodurans, allowing for the introduction of stable, replicating plasmids expressing antibiotic resistance markers. Using this method with nonreplicating plasmids, we developed a protocol for creating sequential gene deletions in D. radiodurans by targeting restriction-modification genes. Importantly, we demonstrated a conjugation-based method for cloning the large (178 kb), high G+C content MP1 megaplasmid from D. radiodurans in E. coli. The conjugation-based tools described here will facilitate the development of D. radiodurans strains with synthetic genomes for biological studies and industrial applications.

Comparative genomic analysis of Staphylococcus aureus isolates associated with either bovine intramammary infections or human infections demonstrates the importance of restriction-modification systems in host adaptation.

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. The versatile and adaptative evolutionary strategies of this bacterium have challenged mastitis control and prevention globally, and the high incidence of S. aureus mastitis increases concerns about antimicrobial resistance (AMR) and zoonosis. This study aims to describe the evolutionary relationship between bovine intramammary infection (IMI)-associated S. aureus and human pathogenic S. aureus and further elucidate the specific genetic composition that leads to the emergence of successful bovine IMI-associated S. aureus lineages. We performed a phylogenomic analysis of 187 S . aureus isolates that originated from either dairy cattle or humans. Our results revealed that bovine IMI-associated S. aureus isolates showed distinct clades compared to human-originated S. aureus isolates. From a pan-genome analysis, 2070 core genes were identified. Host-specific genes and clonal complex (CC)-specific genes were also identified in bovine S. aureus isolates, mostly located in mobile genetic elements (MGEs). Additionally, the genome sequences of three apparent human-adapted isolates (two from CC97 and one from CC8), isolated from bovine mastitis samples, may provide an snapshot of the genomic characteristics in early host spillover events. Virulence and AMR genes were not conserved among bovine IMI-associated S. aureus isolates. Restriction-modification (R-M) genes in bovine IMI-associated S. aureus demonstrated that the Type I R-M system was lineage-specific and Type II R-M system was sequence type (ST)-specific. The distribution of exclusive, virulence, and AMR genes were closely correlated with the presence of R-M systems in S. aureus , suggesting that R-M systems may contribute to shaping clonal diversification by providing a genetic barrier to the horizontal gene transfer (HGT). Our findings indicate that the CC or ST lineage-specific R-M systems may limit genetic exchange between bovine-adapted S. aureus isolates from different lineages.

Genetic Markers of Genome Rearrangements in Helicobacter pylori.

Helicobacter pylori exhibits a diverse genomic structure with high mutation and recombination rates. Various genetic elements function as drivers of this genomic diversity including genome rearrangements. Identifying the association of these elements with rearrangements can pave the way to understand its genome evolution. We analyzed the order of orthologous genes among 72 publicly available complete genomes to identify large genome rearrangements, and rearrangement breakpoints were compared with the positions of insertion sequences, genomic islands, and restriction modification genes. Comparison of the shared inversions revealed the conserved genomic elements across strains from different geographical locations. Some were region-specific and others were global, indicating that highly shared rearrangements and their markers were more ancestral than strain—or region—specific ones. The locations of genomic islands were an important factor for the occurrence of the rearrangements. Comparative genomics helps to evaluate the conservation of various elements contributing to the diversity across genomes.

Correlating prophage presence in Helicobacter pylori with restriction-modification systems

The human pathogen Helicobacter pylori colonises approximately half of the global population and infectioncan lead to a range of gastric diseases. The temperate bacteriophages in H. pylorihave been poorly characterised, most likely due to a very high number and strain-to-strain variability of restriction modification (RM) systems, which can be easily more than 20 in any strain. This work aims to study the prevalence of bacteriophages and RM systems in over 460 strains of H. pyloriisolated from 184 gastric samples from asymptomatic subjects. H. pyloriprophages were identified using the PHASTER tool. The gold standard RM systems were downloaded from Rebase and the RM genes were identified in the 460 genomes using Blast+. The analysis for phage genomes showed 57 intact bacteriophages, ranging from 12 to 30 Kb in length, in 25 subjects (14 %). Approximately half of the bacteriophages were shown to have integrated in the Lipid A biosynthesis gene lpxD. Using 107 RM system genes, we built a presence/absence matrix of the genes in all our H. pylori genomes, which was ordered according to a maximum likelihood gene presence/absence tree generated by FastTree. This clustering revealed the presence of multiple pairs of strains, one strain carrying an intact prophage whereas the other is lacking a prophage but containing the same RM systems, potentially allowing for a prophage donor and recipient pair. The availability of this panel of donor and recipient strain pairs is an ideal starting point to study the molecular biology of bacteriophages in H. pylori.

Periplasmic expression of a restriction endonuclease in Escherichia coli and its effect on the antiviral activity of the host

One possible mechanism preventing phage infection of the bacterial cells is related to the presence of an effective restriction-modification system (R-M) which allows restriction of the invading DNA. However, there are some limitations to the absolute restriction of foreign DNA. Since there is a serious conflict between increase in the restriction-modification genes expression level and cell viability, we examined the antiviral effect of EcoRI restriction endonuclease after its translocation to the periplasmic space of the cell. We assumed that such reconstructed R-M system could be able to degrade foreign DNA at the stage of its passage through the cell envelope of Gram-negative bacteria, before its penetration into the bacterial cytoplasm. The Tat secretion pathway of Escherichia coli was used to export R.EcoRI fused to the TorA leader peptide across the cytoplasmic membrane. However, although we observed a huge accumulation of the TorAss-R.EcoRI pre-protein in the cytoplasm the Tat system did not provide an efficient transport across the cytoplasmic membrane. Moreover, our data strongly suggest that endonuclease cannot function under the conditions prevailing in periplasmic space, therefore, the transported endonuclease could not contribute to an increase in restriction properties of the host.

Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing.

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeon Haloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase genes in H. volcanii and sequenced the methylomes of the resulting deletion mutants via SMRT sequencing to characterize the genes responsible for DNA methylation. The results indicate that deletion of putative RM genes HVO_0794, HVO_A0006, and HVO_A0237 in a single strain abolished methylation of the sole cytosine motif in the genome (Cm4TAG). Amino acid alignments demonstrated that HVO_0794 shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for Cm4TAG methylation in H. volcanii. The CTAG motif has high density at only one of the origins of replication, and there is no relative increase in CTAG motif frequency in the genome of H. volcanii, indicating that CTAG methylation might not have effectively taken over the role of regulating DNA replication and mismatch repair in the organism as previously predicted. Deletion of the putative Type I RM operon rmeRMS (HVO_2269-2271) resulted in abolished methylation of the adenine motif in the genome (GCAm6BN6VTGC). Alignments of the MTase (HVO_2270) and site specificity subunit (HVO_2271) demonstrate homology with other characterized Type I MTases and site specificity subunits, indicating that the rmeRMS operon is responsible for adenine methylation in H. volcanii. Together with HVO_0794, these genes appear to be responsible for all detected methylation in H. volcanii, even though other putative MTases (HVO_C0040, HVO_A0079) share homology with characterized MTases in other organisms. We also report the construction of a multi-RM deletion mutant (ΔRM), with multiple RM genes deleted and with no methylation detected via SMRT sequencing, which we anticipate will be useful for future studies on DNA methylation in H. volcanii.

TfoI produced by Tepidimonas fonticaldi PL17, a moderate thermophilic bacterium, is an isoschizomer of MseI.

A moderately thermophilic Gram-negative bacterium isolated from the Polok hot spring, Sikkim, India, was identified as a strain (PL17) of Tepidimonas fonticaldi by 16S rDNA sequencing. T. fonticaldi PL17 produces a Type IIP restriction endonuclease; named TfoI. Restriction mapping, run-off sequencing of TfoI-digests of dsDNA fragments, and end compatibility of TfoI with NdeI confirmed that the enzyme recognizes and cleaves the sequence 5'-T^TAA-3', and is thus an isoschizomer of MseI. The TfoI restriction-modification genes in the T. fonticaldi PL17 genome were identified, and the annotated TfoI protein encodes a protein of 181 amino acid residues that shares 47.2% sequence identity with MseI. The native enzyme was purified using a four-column chromatography protocol, and its functional homogeneity was confirmed by standard quality control tests. The ESI-MS measured molecular weight of purified TfoI (20.696kDa) is in agreement with that of the calculated monomeric molecular weight of the predicted TfoI protein sequence (20.694kDa). TfoI exhibits optimal activity in the temperature range of 55-70 °C with Mg+2 or Co+2 as cofactor. Similar to its isoschizomers, TfoI can be used as the frequent cutter for genome analysis.

A Novel Approach to Helicobacter pylori Pan-Genome Analysis for Identification of Genomic Islands.

Genomes of a given bacterial species can show great variation in gene content and thus systematic analysis of the entire gene repertoire, termed the pan-genome, is important for understanding bacterial intra-species diversity, population genetics, and evolution. Here, we analyzed the pan-genome from 30 completely sequenced strains of the human gastric pathogen Helicobacter pylori belonging to various phylogeographic groups, focusing on 991 accessory (not fully conserved) orthologous groups (OGs). We developed a method to evaluate the mobility of genes within a genome, using the gene order in the syntenically conserved regions as a reference, and classified the 991 accessory OGs into five classes: Core, Stable, Intermediate, Mobile, and Unique. Phylogenetic networks based on the gene content of Core and Stable classes are highly congruent with that created from the concatenated alignment of fully conserved core genes, in contrast to those of Intermediate and Mobile classes, which show quite different topologies. By clustering the accessory OGs on the basis of phylogenetic pattern similarity and chromosomal proximity, we identified 60 co-occurring gene clusters (CGCs). In addition to known genomic islands, including cag pathogenicity island, bacteriophages, and integrating conjugative elements, we identified some novel ones. One island encodes TerY-phosphorylation triad, which includes the eukaryote-type protein kinase/phosphatase gene pair, and components of type VII secretion system. Another one contains a reverse-transcriptase homolog, which may be involved in the defense against phage infection through altruistic suicide. Many of the CGCs contained restriction-modification (RM) genes. Different RM systems sometimes occupied the same (orthologous) locus in the strains. We anticipate that our method will facilitate pan-genome studies in general and help identify novel genomic islands in various bacterial species.

Microevolution of Virulence-Related Genes in Helicobacter pylori Familial Infection.

Helicobacter pylori, a bacterial pathogen that can infect human stomach causing gastritis, ulcers and cancer, is known to have a high degree of genome/epigenome diversity as the result of mutation and recombination. The bacteria often infect in childhood and persist for the life of the host. One of the reasons of the rapid evolution of H. pylori is that it changes its genome drastically for adaptation to a new host. To investigate microevolution and adaptation of the H. pylori genome, we undertook whole genome sequencing of the same or very similar sequence type in multi-locus sequence typing (MLST) with seven genes in members of the same family consisting of parents and children in Japan. Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous (amino acid changing) mutations were found in virulence-related genes (cag genes, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes. We reconstructed various pathways by which H. pylori can adapt to a new human host, and our results raised the possibility that the mutational changes in virulence-related genes have a role in adaptation to a child host. Changes in restriction-modification genes might remodel the methylome and transcriptome to help adaptation. This study has provided insights into H. pylori transmission and virulence and has implications for basic research as well as clinical practice.

Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006.

Restriction-modification (RM) systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT) sequencing. This analysis was also performed on a strain of H. volcanii in which an annotated DNA methyltransferase gene HVO_A0006 was deleted from the genome. Sequence analysis of H26 revealed two motifs which are modified in the genome: Cm4TAG and GCAm6BN6VTGC. Analysis of the ΔHVO_A0006 strain indicated that it exhibited reduced adenine methylation compared to the parental strain and altered the detected adenine motif. However, protein domain architecture analysis and amino acid alignments revealed that HVO_A0006 is homologous only to the N-terminal endonuclease region of Type IIG RM proteins and contains a PD-(D/E)XK nuclease motif, suggesting that HVO_A0006 is a PD-(D/E)XK nuclease family protein. Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria. It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s). Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

A bistable hysteretic switch in an activator–repressor regulated restriction–modification system

Restriction–modification (RM) systems are extremely widespread among bacteria and archaea, and are often specified by mobile genetic elements. In type II RM systems, where the restriction endonuclease (REase) and protective DNA methyltransferase (MTase) are separate proteins, a major regulatory challenge is delaying expression of the REase relative to the MTase after RM genes enter a new host cell. Basic understanding of this regulation is available for few RM systems, and detailed understanding for none. The PvuII RM system is one of a large subset in which the central regulatory role is played by an activator–repressor protein (called C, for controller). REase expression depends upon activation by C, whereas expression of the MTase does not. Thus delay of REase expression depends on the rate of C-protein accumulation. This is a nonlinear process, as C also activates transcription of its own gene. Mathematical modeling of the PvuII system led to the unexpected predictions of responsiveness to a factor not previously studied in RM system control—gene copy number—and of a hysteretic response. In this study, those predictions have been confirmed experimentally. The results may apply to many other C-regulated RM systems, and help explain their ability to spread so widely.

Molecular identification and characterization of type III restriction-modification (R-M) gene cluster in Campylobacter lari

Although the human clinical strain of Campylobacter lari (RM2100) has been shown not to carry any type III restriction-modification (R-M) systems, an R-M genes cluster was identified downstream of the full-length cytolethal distending toxin gene operon in the urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain. Two possible open reading frames (ORFs) for restriction endonuclease and methyltransferase were predicted to encode peptides of 947 and 613 amino acid residues with calculated molecular weights of 111 and 70.8 kDa, respectively. Two putative promoters consisting of the consensus sequences and two probable ribosome binding sites for the two ORFs were also identified. Reverse transcription PCR identified co-transcription of the R-M genes in the cells. The existence of an S-adenosyl methionine-binding motif in the N-terminal conserved region of the possible ORF for the M gene, and seven conserved helicase motifs in the R gene were also identified. PCR and Southern blot hybridization analyses for type III R-M enzyme genes with some of the C. lari isolates including UPTC gave positive signals. UPTC isolates were shown to carry type III R-M enzyme genes, with a relatively high frequency.

Epidemiology of methicillin resistant Staphylococcus aureus (MRSA) isolates from Pakistan

In order to have an understanding of the distribution of MRSA clones in Pakistan, where unregulated antibiotic use is widespread and the distribution of MRSA is thought to be high, an epidemiological study was designed. In this study, epidemiological relationships between 123 methicillin resistant Staphylococcus aureus strains, isolated between 2006 and 2008 from three tertiary care hospitals of Rawalpindi and Islamabad, were examined using six loci in a multilocus variable number tandem repeat analysis (MLVA). A total of 63 haplotypes were obtained by MLVA. Analysis of restriction modification (RM) genes detected an RM3 type, associated with CC8 in 98% of strains and an RM1 type, associated with CC30, in only two strains. On further typing of selected strains by Spa typing and MLST, it was found that the RM3/CC8 isolates were ST113-t064, ST113-t451 or ST239, with one of four spa types, while the RM1/CC30 isolates were ST30-t021. We concluded that there is a high prevalence of CC8 MRSA strains in Rawalpindi/Islamabad, Pakistan and frequent exchange of strains between closely-linked hospitals. Key words: Epidemiology, MRSA, MLVA, MLST, Pakistan, spa

Naturally-occurring, dually-functional fusions between restriction endonucleases and regulatory proteins

BackgroundRestriction-modification (RM) systems appear to play key roles in modulating gene flow among bacteria and archaea. Because the restriction endonuclease (REase) is potentially lethal to unmethylated new host cells, regulation to ensure pre-expression of the protective DNA methyltransferase (MTase) is essential to the spread of RM genes. This is particularly true for Type IIP RM systems, in which the REase and MTase are separate, independently-active proteins. A substantial subset of Type IIP RM systems are controlled by an activator-repressor called C protein. In these systems, C controls the promoter for its own gene, and for the downstream REase gene that lacks its own promoter. Thus MTase is expressed immediately after the RM genes enter a new cell, while expression of REase is delayed until sufficient C protein accumulates. To study the variation in and evolution of this regulatory mechanism, we searched for RM systems closely related to the well-studied C protein-dependent PvuII RM system. Unexpectedly, among those found were several in which the C protein and REase genes were fused.ResultsThe gene for CR.NsoJS138I fusion protein (nsoJS138ICR, from the bacterium Niabella soli) was cloned, and the fusion protein produced and partially purified. Western blots provided no evidence that, under the conditions tested, anything other than full-length fusion protein is produced. This protein had REase activity in vitro and, as expected from the sequence similarity, its specificity was indistinguishable from that for PvuII REase, though the optimal reaction conditions were different. Furthermore, the fusion was active as a C protein, as revealed by in vivo activation of a lacZ reporter fusion to the promoter region for the nsoJS138ICR gene.ConclusionsFusions between C proteins and REases have not previously been characterized, though other fusions have (such as between REases and MTases). These results reinforce the evidence for impressive modularity among RM system proteins, and raise important questions about the implications of the C-REase fusions on expression kinetics of these RM systems.

The structural basis of differential DNA sequence recognition by restriction–modification controller proteins

Controller (C) proteins regulate the expression of restriction–modification (RM) genes in a wide variety of RM systems. However, the RM system Esp1396I is of particular interest as the C protein regulates both the restriction endonuclease (R) gene and the methyltransferase (M) gene. The mechanism of this finely tuned genetic switch depends on differential binding affinities for the promoters controlling the R and M genes, which in turn depends on differential DNA sequence recognition and the ability to recognize dual symmetries. We report here the crystal structure of the C protein bound to the M promoter, and compare the binding affinities for each operator sequence by surface plasmon resonance. Comparison of the structure of the transcriptional repression complex at the M promoter with that of the transcriptional activation complex at the R promoter shows how subtle changes in protein–DNA interactions, underpinned by small conformational changes in the protein, can explain the molecular basis of differential regulation of gene expression.

Comparative Transcriptomics of H. pylori Strains AM5, SS1 and Their hpyAVIBM Deletion Mutants: Possible Roles of Cytosine Methylation

Helicobacter pylori is an important human pathogen and one of the most successful chronic colonizers of the human body. H. pylori uses diverse mechanisms to modulate its interaction with the host in order to promote chronic infection and overcome host immune response. Restriction-modification genes are a major part of strain-specific genes present in H. pylori. The role of N6 - adenine methylation in bacterial gene regulation and virulence is well established but not much is known about the effect of C5 -cytosine methylation on gene expression in prokaryotes. In this study, it was observed by microarray analysis and RT-PCR, that deletion of an orphan C5 -cytosine methyltransferase, hpyAVIBM in H. pylori strains AM5and SS1 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. AM5ΔhpyAVIBM mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. hpyAVIBM from strain 26695 is able to complement mutant SS1 and AM5 strains. This study highlights a possible significance of cytosine methylation in the physiology of H. pylori.

Conservation of Genomic Localization and Sequence Content of Sau3AI-Like Restriction-Modification Gene Cassettes amongListeria monocytogenesEpidemic Clone I and Selected Strains of Serotype 1/2a

Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.

Genome comparison and context analysis reveals putative mobile forms of restriction–modification systems and related rearrangements

The mobility of restriction–modification (RM) gene complexes and their association with genome rearrangements is a subject of active investigation. Here we conducted systematic genome comparisons and genome context analysis on fully sequenced prokaryotic genomes to detect RM-linked genome rearrangements. RM genes were frequently found to be linked to mobility-related genes such as integrase and transposase homologs. They were flanked by direct and inverted repeats at a significantly high frequency. Insertion by long target duplication was observed for I, II, III and IV restriction types. We found several RM genes flanked by long inverted repeats, some of which had apparently inserted into a genome with a short target duplication. In some cases, only a portion of an apparently complete RM system was flanked by inverted repeats. We also found a unit composed of RM genes and an integrase homolog that integrated into a tRNA gene. An allelic substitution of a Type III system with a linked Type I and IV system pair, and allelic diversity in the putative target recognition domain of Type IIG systems were observed. This study revealed the possible mobility of all types of RM systems, and the diversity in their mobility-related organization.

Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator

BackgroundMost type II restriction-modification (RM) systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease) initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene.ResultsExpression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans). In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting the putative hairpins had no significant effects.ConclusionsThe initiation of translation of pvuIIR appears to be independent of that for pvuIIC. Direct tests failed to detect regulatory rules for either gene overlap or the putative hairpins. Thus, at least during balanced growth, transcriptional control appears to be sufficiently robust for proper regulation of this RM system.