The Slr0924 protein of Synechocystis sp. strain PCC 6803 resembles a subunit of the chloroplast protein import complex and is mainly localized in the thylakoid lumen

An isolated 25 kDa protein of Synechocystis sp. PCC 6803 was N-terminally sequenced and assigned to a protein encoded by the ORF slr0924. This ORF shows a certain degree of sequence similarity to a subunit from the protein Translocon at the Inner envelope of pea Chloroplasts (Tic22). The deduced amino acid sequence of Slr0924 has a N-terminal extension, that contains two possible translational start points and two possible cleavage sites for leader peptidases. Immunostaining with an antibody raised to the over-produced protein revealed two cross-reacting forms, which probably correspond to a larger intermediate and the mature protein. Immunogold labelling of thin sections showed that the protein is located mainly in the thylakoid region. This result was verified by thylakoid membrane fractionation indicating that Slr0924 is a lumenal protein. The slr0924 gene product is essential for the viability of Synechocystis sp. PCC 6803 as shown by interposon mutagenesis. The merodiploid strain showed reduced photosynthetic activity compared to the wild-type. Furthermore, growth of the merodiploid strain was found to be completely inhibited after cultivation with glucose. Accordingly, the amount of the slr0924 gene product was regulated by glucose and light intensities in wild-type cells. The potential function of the protein in Synechocystis sp. PCC 6803 will be discussed.     

Recognition of native DNA methylation by the PvuII restriction endonuclease

Recognizing the methylation status of specific DNA sequences is central to the function of many systems in eukaryotes and prokaryotes. Restriction–modification systems have to distinguish between ‘self’ and ‘non-self’ DNA and depend on the inability of restriction endonucleases to cleave their DNA substrates when the DNA is appropriately methylated. These endonucleases thus provide a model system for studying the recognition of DNA methylation by proteins. We have characterized the interaction of R·PvuII with DNA containing the physiologically relevant N4-methylcytosine modification. R·PvuII binds ^N4mC-modified DNA and cleaves it very slowly. Methylated strands in hemimethylated duplexes were cleaved at a higher rate than in fully methylated duplexes, in parallel with a higher binding affinity for hemimethylated DNA. The co-crystal structures of R·PvuII–DNA, together with a mutagenesis study, have implicated specific amino acids in recognition of the methylatable base; one of these is His84. We report that replacing His84 with Ala reduced the rate of cleavage of unmodified DNA but, in contrast, slightly increased the cleavage of ^N4mC-modified DNA.     

Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites,including expanded (CAG)n/(CTG)n repeats

Most epigenetic studies assess methylation of 5′-CpG-3′ sites but recent evidence indicates that non-CpG cytosine methylation occurs at high levels in humans and other species. This is most prevalent at 5′-CHG-3′, where H = A, C or T, and it preferentially occurs at 5′-CpA-3′ and 5′-CpT-3′ sites. With the goal of facilitating the detection of non-CpG methylation, the restriction endonucleases ApeKI, BbvI, EcoP15I, Fnu4HI, MwoI and TseI were assessed for their sensitivity to 5-methylcytosine at GpCpA, GpCpT, GpCpC or GpCpG sites, where methylation is catalyzed by the DNA 5-cytosine 5′-GpC-3′ methyltransferase M.CviPI. We tested a variety of sequences including various plasmid-based sites, a cloned disease-associated (CAG)83•(CTG)83 repeat and in vitro synthesized tracts of only (CAG)500•(CTG)500 or (CAG)800•(CTG)800. The repeat tracts are enriched for the preferred CpA and CpT motifs. We found that none of the tested enzymes can cleave their recognition sequences when they are 5′-GpC-3′ methylated. A genomic site known to convert its non-CpG methylation levels upon C2C12 differentiation was confirmed through the use of these enzymes. These enzymes can be useful in rapidly and easily determining the most common non-CpG methylation status in various sequence contexts, as well as at expansions of (CAG)n•(CTG)n repeat tracts associated with diseases like myotonic dystrophy and Huntington disease.Key words: non-CpG methylation, CpG methylation, 5-methylcytosine, trinucleotide repeats, ApeKI, BbvI, EcoP151, Fnu4HI, MwoI and TseI     

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.     

Esp1396I restriction-modification system: structural organization and mode of regulation

Esp1396I restriction–modification (RM) system recognizes an interrupted palindromic DNA sequ ence 5′-CCA(N)₅TGG-3′. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp. strain RFL1396. The nucleotide sequence of the entire 5622 bp pEsp1396 plasmid was determined on both strands. Identified genes for DNA methyltransferase (esp1396IM) and restriction endonuclease (esp1396IR) are transcribed convergently. The restriction endonuclease gene is preceded by the small ORF (esp1396IC) that possesses a strong helix-turn-helix motif and resembles regulatory proteins found in PvuII, BamHI and few other RM systems. Gene regulation studies revealed that C.Esp1396I acts as both a repressor of methylase expression and an activator of regulatory protein and restriction endonuclease expression. Our data indicate that C protein from Esp1396I RM system activates the expression of the Enase gene, which is co-transcribed from the promoter of regulatory gene, by the mechanism of coupled translation.     

Effect of sulfhydryl group inhibitors on restriction endonuclease activities

The activity of restriction endonuclease BamHI was abolished by p-mercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid). The activity of restriction endonuclease PvuI was abolished by p-mercuribenzoate. The activity of none of the eight other restriction endonucleases tested could be abolished by the sulfhydryl group inhibitors. Despite the general practice of inclusion of sulfhydryl reducing agents in reaction mixtures containing restriction endonucleases it appears that most of these enzymes function without the active participation of a -SH moiety.     

2��-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m⁷GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.     

Isolation and characterization of restriction endonucleases from Acidiphilium sp. 16R and 22M

Restriction endonucleases (Asp16RI and Asp22MI) have been identified from the acidophilic bacteria Acidiphilium sp. 16R and 22M. The cleavage patterns with various DNAs show that both enzymes recognize the same sequence as the PvuI restriction endonuclease (5′-CGAT ↓ CG-3′), which is from Proteus vulgaris ATCC13315. Most of the catalytic properties observed for Asp16RI and Asp22MI were similar to those observed for PvuI. However, unlike PvuI both enzymes efficiently cleaved DNA in the absence of NaCl or KCl. The purification yield of Asp22MI is 60 times that of PvuI.     

Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation

In vitro compartmentalisation (IVC), a technique for selecting genes encoding enzymes based on compartmentalising gene translation and enzymatic reactions in emulsions, was used to investigate the interaction of the DNA cytosine-5 methyltransferase M.HhaI with its target DNA (5′-GCGC-3′). Crystallog raphy shows that the active site loop from the large domain of M.HhaI interacts with a flipped-out cytosine (the target for methylation) and two target recognition loops (loops I and II) from the small domain make almost all the other base-specific interactions. A library of M.HhaI genes was created by randomising all the loop II residues thought to make base-specific interactions and directly determine target specificity. The library was selected for 5′-GCGC-3′ methylation. Interestingly, in 11 selected active clones, 10 different sequences were found and none were wild-type. At two of the positions mutated (Ser252 and Tyr254) a number of different amino acids could be tolerated. At the third position, however, all active mutants had a glycine, as in wild-type M.HhaI, suggesting that Gly257 is crucial for DNA recognition and enzyme activity. Our results suggest that recognition of base pairs 3 and 4 of the target site either relies entirely on main chain interactions or that different residues from those identified in the crystal structure contribute to DNA recognition.     

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5′-CG-3′ site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 ± 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R²) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5′-CG-3′ site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.     

Characterization of a Novel Type II Restriction-Modification System, Sth368I, Encoded by the Integrative Element ICESt1 of Streptococcus thermophilus CNRZ368

A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to ST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5′-GATC-3′. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5′-GATC-3′ was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5′-GATC-3′. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5′-GATC-3′ and is related to the Sau3AI and LlaKR2I restriction systems.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

2��-O Methylation of the Viral mRNA Cap by West Nile Virus Evades Ifit1-Dependent and -Independent Mechanisms of Host Restriction In Vivo

Prior studies have shown that 2′-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2′-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2′-O methylation of the 5′ viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1 ^−/− mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD₅₀ values in Ifit1 ^−/− compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1 ^−/− brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8⁺ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2′-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.     

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N⁶-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 N⁶-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^m6AG-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-AC^m6ACC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CC^m6AGC-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^m6AG-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGC^m6AGG-3′ sites, to 100% at 5′-ACGT^m6AGG-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.     

Cloning and Characterization of the Lactococcal Plasmid-Encoded Type II Restriction/Modification System, LlaDII

The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5′-GC↓NGC-3′, where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI. Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the LlaDII and Bsp6I R/M systems are identical. Both methylases have two recognition sites (5′-GCGGC-3′ and 5′-GCCGC-3′) forming a putative stem-loop structure spanning part of the presumed −35 sequence and part of the intervening region between the −35 and −10 sequences. Alignment of the LlaDII and Bsp6I methylases with other m⁵C methylases showed that the protein primary structures possessed the same organization.     

Functional Characterization of the slr1944 Gene of Cyanobacterium Synechocystis sp. PCC 6803

The properties of Slr1944 protein encoded by the slr1944 gene and participating in the metabolism of lipophilic compounds in a cyanobacterium Synechocystis were under study. Located in the periplasm, this protein comprises a conserved pentapeptide G-X-S-X-G characteristic of lipases, acetylcholinesterases, and thioesterases. An attempt to delete the gene from the cyanobacterial genome failed; this fact presumes an essential function of Slr1944 protein under the optimum growth conditions. Expression of the slr1944 gene in Escherichia coli cells demonstrated a high affinity of the product for lipophilic compounds. An enhanced slr1944 expression deprived Synechocystis cells of the ability to restore the activity of the photosynthetic electron-transport chain following photoinactivation. The authors believe that Slr1944 participates in the biogenesis of the lipophilic components of photosynthetic complexes.     

Molecular Cloning of the Fujinami Sarcoma Virus Genome and Its Comparison with Sequences of Other Related Transforming Viruses

Full-length proviral DNA of Fujinami sarcoma virus (FSV) of chickens was molecularly cloned and characterized. An analysis of FSV DNA integrated in mammalian cells showed that restriction endonuclease SacI has a single cleavage site on FSV DNA. Unintegrated closed circular FSV DNA obtained from newly infected cells was linearized by digestion with SacI and cloned into λgtWES·λB. The following three different molecules were isolated: FSV-1 (4.4 kilobases [kb]) and FSV-2 (4.7 kb), which appeared to be full-length FSV DNA molecules containing either one or two copies of the long terminal repeat structure, and FSV-3 (6 kb), which consisted of part FSV DNA and part DNA of unknown origin. An analysis of the structure of cloned FSV-1 and FSV-2 DNA molecules by restriction endonuclease mapping and hybridization with appropriate probes showed that about 2.6 kb of the FSV-unique sequence called FSV-fps is located in the middle of the FSV genome and is flanked by helper virus-derived sequences of about 1.3 kb at the 5′ end and 0.5 kb at the 3′ end. The long terminal repeats of FSV were found to have no cleavage site for either EcoRI or PvuI. Upon transfection, both FSV-1 DNA and FSV-2 DNA were able to transform mammalian fibroblasts. Four ³²P-labeled DNA fragments derived from different portions of the FSV-fps sequence were used for hybridization to viral RNAs. We found that sequences within the 3′ half of the FSV-fps gene are homologous to RNAs of PRCII avian sarcoma virus and the Snyder-Theilen strain of feline sarcoma virus, both of which were previously shown to contain transforming genes related to FSV-fps. These results suggest that the 3′ portion of the FSV-fps sequence may be crucial for the transforming activity of fps-related oncogenic sequences.     

Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family

The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5′-CCGC/GG-3′ (‘/’ indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies provided here demonstrate that the Cfr42I tetramer is catalytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the homotetrameric Type IIF restriction enzymes that belong to the distinct PD-(E/D)XK nuclease superfamily. Unlike the PD-(E/D)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal-ion cofactors, including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺, Cu²⁺ and Ca²⁺. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar structural arrangement and phenotypes displayed by restriction enzymes of the PD-(E/D)XK and GIY-YIG nuclease families point to the functional significance of tetramerization.     

Structure–function of cyanobacterial outer-membrane protein,Slr1270: Homolog of Escherichia coli drug export/colicin import protein,TolC

Compared to thylakoid and inner membrane proteins in cyanobacteria, no structure–function information is available presently for integral outer-membrane proteins (OMPs). The Slr1270 protein from the cyanobacterium Synechocystis 6803, over-expressed in Escherichia coli, was refolded, and characterized for molecular size, secondary structure, and ion-channel function. Refolded Slr1270 displays a single band in native-electrophoresis, has an α-helical content of 50–60%, as in E. coli TolC with which it has significant secondary-structure similarity, and an ion-channel function with a single-channel conductance of 80–200 pS, and a monovalent ion (K⁺:Cl⁻) selectivity of 4.7:1. The pH-dependence of channel conductance implies a role for carboxylate residues in channel gating, analogous to that in TolC.