Stresses that Raise Np4A Levels Induce Protective Nucleoside Tetraphosphate Capping of Bacterial RNA.

Present in all realms of life, dinucleoside tetraphosphates (Np4Ns) are generally considered signaling molecules. However, only a single pathway for Np4N signaling has been delineated in eukaryotes, and no receptor that mediates the influence of Np4Ns has ever been identified in bacteria. Here we show that, under disulfide stress conditions that elevate cellular Np4N concentrations, diverse Escherichia coli mRNAs and sRNAs acquire a cognate Np4 cap. Purified E. coli RNA polymerase and lysyl-tRNA synthetase are both capable of adding such 5' caps. Cap removal by either of two pyrophosphatases, ApaH or RppH, triggers rapid RNA degradation in E. coli. ApaH, the predominant decapping enzyme, functions as both a sensor and an effector of disulfide stress, which inactivates it. These findings suggest that the physiological changes attributed to elevated Np4N concentrations in bacteria may result from widespread Np4 capping, leading to altered RNA stability and consequent changes in gene expression.

The dinucleotide composition of the Zika virus genome is shaped by conflicting evolutionary pressures in mammalian hosts and mosquito vectors.

Most vertebrate RNA viruses show pervasive suppression of CpG and UpA dinucleotides, closely resembling the dinucleotide composition of host cell transcriptomes. In contrast, CpG suppression is absent in both invertebrate mRNA and RNA viruses that exclusively infect arthropods. Arthropod-borne (arbo) viruses are transmitted between vertebrate hosts by invertebrate vectors and thus encounter potentially conflicting evolutionary pressures in the different cytoplasmic environments. Using a newly developed Zika virus (ZIKV) model, we have investigated how demands for CpG suppression in vertebrate cells can be reconciled with potentially quite different compositional requirements in invertebrates and how this affects ZIKV replication and transmission. Mutant viruses with synonymously elevated CpG or UpA dinucleotide frequencies showed attenuated replication in vertebrate cell lines, which was rescued by knockout of the zinc-finger antiviral protein (ZAP). Conversely, in mosquito cells, ZIKV mutants with elevated CpG dinucleotide frequencies showed substantially enhanced replication compared to wild type. Host-driven effects on virus replication attenuation and enhancement were even more apparent in mouse and mosquito models. Infections with CpG- or UpA-high ZIKV mutants in mice did not cause typical ZIKV-induced tissue damage and completely protected mice during subsequent challenge with wild-type virus, which demonstrates their potential as live-attenuated vaccines. In contrast, the CpG-high mutants displayed enhanced replication in Aedes aegypti mosquitoes and a larger proportion of mosquitoes carried infectious virus in their saliva. These findings show that mosquito cells are also capable of discriminating RNA based on dinucleotide composition. However, the evolutionary pressure on the CpG dinucleotides of viral genomes in arthropod vectors directly opposes the pressure present in vertebrate host cells, which provides evidence that an adaptive compromise is required for arbovirus transmission. This suggests that the genome composition of arbo flaviviruses is crucial to maintain the balance between high-level replication in the vertebrate host and persistent replication in the mosquito vector.

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis.

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

CG dinucleotide suppression enables antiviral defence targeting non-self RNA.

Vertebrate genomes exhibit marked CG suppression-that is, lower than expected numbers of 5'-CG-3' dinucleotides. This feature is likely to be due to C-to-T mutations that have accumulated over hundreds of millions of years, driven by CG-specific DNA methyl transferases and spontaneous methyl-cytosine deamination. Many RNA viruses of vertebrates that are not substrates for DNA methyl transferases mimic the CG suppression of their hosts. This property of viral genomes is unexplained. Here we show, using synonymous mutagenesis, that CG suppression is essential for HIV-1 replication. The deleterious effect of CG dinucleotides on HIV-1 replication was cumulative, associated with cytoplasmic RNA depletion, and was exerted by CG dinucleotides in both translated and non-translated exonic RNA sequences. A focused screen using small inhibitory RNAs revealed that zinc-finger antiviral protein (ZAP) inhibited virion production by cells infected with CG-enriched HIV-1. Crucially, HIV-1 mutants containing segments whose CG content mimicked random nucleotide sequence were defective in unmanipulated cells, but replicated normally in ZAP-deficient cells. Crosslinking-immunoprecipitation-sequencing assays demonstrated that ZAP binds directly and selectively to RNA sequences containing CG dinucleotides. These findings suggest that ZAP exploits host CG suppression to identify non-self RNA. The dinucleotide composition of HIV-1, and perhaps other RNA viruses, appears to have adapted to evade this host defence.

Structural basis for glucocorticoid receptor recognition of both unmodified and methylated binding sites, precursors of a modern recognition element.

The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS.

Np4A alarmones function in bacteria as precursors to RNA caps.

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5' end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Alarmone Ap4A is elevated by aminoglycoside antibiotics and enhances their bactericidal activity.

Second messenger molecules play important roles in the responses to various stimuli that can determine a cell's fate under stress conditions. Here, we report that lethal concentrations of aminoglycoside antibiotics result in the production of a dinucleotide alarmone metabolite-diadenosine tetraphosphate (Ap4A), which promotes bacterial cell killing by this class of antibiotics. We show that the treatment of Escherichia coli with lethal concentrations of kanamycin (Kan) dramatically increases the production of Ap4A. This elevation of Ap4A is dependent on the production of a hydroxyl radical and involves the induction of the Ap4A synthetase lysyl-tRNA synthetase (LysU). Ectopic alteration of intracellular Ap4A concentration via the elimination of the Ap4A phosphatase diadenosine tetraphosphatase (ApaH) and the overexpression of LysU causes over a 5,000-fold increase in bacterial killing by aminoglycosides. This increased susceptibility to aminoglycosides correlates with bacterial membrane disruption. Our findings provide a role for the alarmone Ap4A and suggest that blocking Ap4A degradation or increasing its synthesis might constitute an approach to enhance aminoglycoside killing potency by broadening their therapeutic index and thereby allowing lower nontoxic dosages of these antibiotics to be used in the treatment of multidrug-resistant infections.

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms.

CpG dinucleotides are suppressed in the genomes of many vertebrate RNA viruses, including HIV-1. The cellular antiviral protein ZAP (zinc finger antiviral protein) binds CpGs and inhibits HIV-1 replication when CpGs are introduced into the viral genome. However, it is not known if ZAP-mediated restriction is the only mechanism driving CpG suppression. To determine how CpG dinucleotides affect HIV-1 replication, we increased their abundance in multiple regions of the viral genome and analyzed the effect on RNA expression, protein abundance, and infectious-virus production. We found that the antiviral effect of CpGs was not correlated with their abundance. Interestingly, CpGs inserted into some regions of the genome sensitize the virus to ZAP antiviral activity more efficiently than insertions into other regions, and this sensitivity can be modulated by interferon treatment or ZAP overexpression. Furthermore, the sensitivity of the virus to endogenous ZAP was correlated with its sensitivity to the ZAP cofactor KHNYN. Finally, we show that CpGs in some contexts can also inhibit HIV-1 replication by ZAP-independent mechanisms, and one of these is the activation of a cryptic splice site at the expense of a canonical splice site. Overall, we show that the location and sequence context of the CpG in the viral genome determines its antiviral activity.IMPORTANCE Some RNA virus genomes are suppressed in the nucleotide combination of a cytosine followed by a guanosine (CpG), indicating that they are detrimental to the virus. The antiviral protein ZAP binds viral RNA containing CpGs and prevents the virus from multiplying. However, it remains unknown how the number and position of CpGs in viral genomes affect restriction by ZAP and whether CpGs have other antiviral mechanisms. Importantly, manipulating the CpG content in viral genomes could help create new vaccines. HIV-1 shows marked CpG suppression, and by introducing CpGs into its genome, we show that ZAP efficiently targets a specific region of the viral genome, that the number of CpGs does not predict the magnitude of antiviral activity, and that CpGs can inhibit HIV-1 gene expression through a ZAP-independent mechanism. Overall, the position of CpGs in the HIV-1 genome determines the magnitude and mechanism through which they inhibit the virus.

The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides.

Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses.

The Bateman domain of IMP dehydrogenase is a binding target for dinucleoside polyphosphates.

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the de novo guanine nucleotide biosynthetic pathway. Because of its involvement in the control of cell division and proliferation, IMPDH represents a therapeutic for managing several diseases, including microbial infections and cancer. IMPDH must be tightly regulated, but the molecular mechanisms responsible for its physiological regulation remain unknown. To this end, we recently reported an important role of adenine and guanine mononucleotides that bind to the regulatory Bateman domain to allosterically modulate the catalytic activity of eukaryotic IMPDHs. Here, we have used enzyme kinetics, X-ray crystallography, and small-angle X-ray scattering (SAXS) methodologies to demonstrate that adenine/guanine dinucleoside polyphosphates bind to the Bateman domain of IMPDH from the fungus Ashbya gossypii with submicromolar affinities. We found that these dinucleoside polyphosphates modulate the catalytic activity of IMPDHs in vitro by efficiently competing with the adenine/guanine mononucleotides for the allosteric sites. These results suggest that dinucleoside polyphosphates play important physiological roles in the allosteric regulation of IMPDHs by adding an additional mechanism for fine-tuning the activities of these enzymes. We propose that these findings may have important implications for the design of therapeutic strategies to inhibit IMPDHs.

Unusual sequence characteristics of human chromosome 19 are conserved across 11 nonhuman primates.

BACKGROUND: Human chromosome 19 has many unique characteristics including gene density more than double the genome-wide average and 20 large tandemly clustered gene families. It also has the highest GC content of any chromosome, especially outside gene clusters. The high GC content and concomitant high content of hypermutable CpG sites raises the possibility chromosome 19 exhibits higher levels of nucleotide diversity both within and between species, and may possess greater variation in DNA methylation that regulates gene expression. RESULTS: We examined GC and CpG content of chromosome 19 orthologs across representatives of the primate order. In all 12 primate species with suitable genome assemblies, chromosome 19 orthologs have the highest GC content of any chromosome. CpG dinucleotides and CpG islands are also more prevalent in chromosome 19 orthologs than other chromosomes. GC and CpG content are generally higher outside the gene clusters. Intra-species variation based on SNPs in human common dbSNP, rhesus, crab eating macaque, baboon and marmoset datasets is most prevalent on chromosome 19 and its orthologs. Inter-species comparisons based on phyloP conservation show accelerated nucleotide evolution for chromosome 19 promoter flanking and enhancer regions. These same regulatory regions show the highest CpG density of any chromosome suggesting they possess considerable methylome regulatory potential. CONCLUSIONS: The pattern of high GC and CpG content in chromosome 19 orthologs, particularly outside gene clusters, is present from human to mouse lemur representing 74 million years of primate evolution. Much CpG variation exists both within and between primate species with a portion of this variation occurring in regulatory regions.

Use of a small DNA virus model to investigate mechanisms of CpG dinucleotide-induced attenuation of virus replication.

Suppression of the CpG dinucleotide is widespread in RNA viruses infecting vertebrates and plants, and in the genomes of retroviruses and small mammalian DNA viruses. The functional basis for CpG suppression in the latter was investigated through the construction of mutants of the parvovirus, minute virus of mice (MVM) with increased CpG or TpA dinucleotides in the VP gene. CpG-high mutants displayed extraordinary attenuation in A9 cells compared to wild-type MVM (>six logs), while TpA elevation showed no replication effect. Attenuation was independent of Toll-like receptor 9 and STING-mediated DNA recognition pathways and unrelated to effects on translation efficiency. While translation from codon-optimized VP RNA was enhanced in a cell-free assay, MVM containing this sequence was highly attenuated. Further mutational analysis indicated that this arose through its increased numbers of CpG dinucleotides (7→70) and separately from its increased G+C content (42.3→57.4 %), which independently attenuated replication. CpG-high viruses showed impaired NS mRNA expression by qPCR and reduced NS and particularly VP protein expression detected by immunofluorescence and replication in A549 cells, effects reversed in zinc antiviral protein (ZAP) knockout cells, even though nuclear relocalization of VP remained defective. The demonstrated functional basis for CpG suppression in MVM and potentially other small DNA viruses and the observed intolerance of CpGs in coding sequences, even after codon optimization, has implications for the use of small DNA virus vectors in gene therapy and immunization.

Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis.

Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in Saccharomyces cerevisiae using DNA polymerase active-site mutants as a "sensitized mutator background." Among the genes identified in our screen, three metabolism-related genes (GLN3, URA7, and SHM2) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.

Solution Structures of a G-Quadruplex Bound to Linear- and Cyclic-Dinucleotides.

Cyclic dinucleotides have emerged as important secondary messengers and cell signaling molecules that regulate several cell responses. A guanine-deficit G-quadruplex structure formation by a sequence containing (4n - 1) guanines, n denoting the number of G-tetrad layers, was previously reported. Here, a (4n - 1) G-quadruplex structure is shown to be capable of binding guanine-containing dinucleotides in micromolar affinity. The guanine base of the dinucleotides interacts with a vacant G-triad, forming four additional Hoogsteen hydrogen bonds to complete a G-tetrad. Solution structures of two complexes, both comprised of a (4n - 1) G-quadruplex structure, one bound to a linear dinucleotide (d(AG)) and the other to a cyclic dinucleotide (cGAMP), are solved using NMR spectroscopy. The latter suggests sufficiently strong interaction between the guanine base of the dinucleotide and the vacant G-triad, which acts as an anchor point of binding. The binding interfaces from the two solution structures provide useful information for specific ligand design. The results also infer that other guanine-containing metabolites of a similar size have the capability of binding G-quadruplexes, potentially affecting the expression of the metabolites and functionality of the bound G-quadruplexes.

HTLV-1 contains a high CG dinucleotide content and is susceptible to the host antiviral protein ZAP.

BACKGROUND: Human T cell leukaemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases such as adult T-cell leukaemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. In contrast to another human retrovirus, human immunodeficiency virus type 1 (HIV-1), HTLV-1 persists in the host not via vigorous virus production but mainly via proliferation and/or long-term survival in the form of silent proviruses in infected host cells. As a result, HTLV-1-infected cells rarely produce virus particles in vivo even without anti-retroviral treatment. That should be an advantage for the virus to escape from the host immune surveillance by minimizing the expression of viral antigens in host cells. However, why HIV-1 and HTLV-1 behave so differently during natural infection is not fully understood. RESULTS: We performed cap analysis of gene expression (CAGE) using total RNAs and nascent, chromatin-associated, RNAs in the nucleus and found that HTLV-1 RNAs were processed post-transcriptionally in infected cells. RNA processing was evident for the sense viral transcripts but not the anti-sense ones. We also found a higher proportion of CG di-nucleotides in proviral sequences of HTLV-1-infected cells, when compared to the HIV-1 genomic sequence. It has been reported recently that CG dinucleotide content of viral sequence is associated with susceptibility to the antiviral ZC3HAV1 (ZAP), suggesting the involvement of this protein in the regulation of HTLV-1 transcripts. To analyse the effect of ZAP on HTLV-1 transcripts, we over-expressed it in HTLV-1-infected cells. We found there was a dose-dependent reduction in virus production with ZAP expression. We further knocked down endogenous ZAP with two independent targeting siRNAs and observed a significant increase in virus production in the culture supernatant. Other delta-type retroviruses such as simian T-cell leukaemia virus and bovine leukaemia virus, also contain high CG-dinucleotide contents in their viral genomes, suggesting that ZAP-mediated suppression of viral transcripts might be a common feature of delta-type retroviruses, which cause minimal viremia in their natural hosts. CONCLUSIONS: The post-transcriptional regulatory mechanism involving ZAP might allow HTLV-1 to maintain a delicate balance required for prolonged survival in infected individuals.

Differential Methylation Levels in CpGs of the BIN1 Gene in Individuals With Alzheimer Disease.

INTRODUCTION: Late-onset Alzheimer disease (LOAD) is the most common dementia worldwide. APOE-[Latin Small Letter Open E]4 and BIN1 (Bridging Integrator 1) have been implicated in the pathogenesis of this disease, but, although DNA methylation of dinucleotide CpGs in the BIN1 gene influences alterations, it has not been studied in Hispanics. OBJECTIVE: The objective of this study was to evaluate the BIN1 3' intergenic region DNA methylation patterns in a Colombian sample of LOAD patients. METHODS: A case-control study was conducted in 50 individuals with LOAD and 50 age-sex matched controls to determine associations of LOAD with DNA methylation. DNA was isolated from peripheral blood, and methylation levels of 8 CpGs were estimated by bisulfite conversion followed by Sanger sequencing with direct PCR analysis. Logistic regression models adjusted by age, sex, and APOE were used to calculate risk associations between methylation levels and LOAD. RESULTS: Overall, participants with LOAD had significantly lower methylation levels on CpG26 (0.86±0.11 vs. 0.95±0.05; P>0.001), CpG44 (0.84±0.09 vs. 0.94±0.06; P=0.001), and CpG87 (0.64±0.12 vs. 0.82±0.10; P>0.001). Adjusted regression models showed that decreased methylation levels of these CpGs remained as risk factors for LOAD (P<0.05). CONCLUSIONS: Hypomethylation of CpGs in BIN1 might play an important role in the expression of BIN1 and may be a biomarker for identifying individuals at high risk of developing LOAD.

Statistical modelling of CG interdistance across multiple organisms.

BACKGROUND: Statistical approaches to genetic sequences have revealed helpful to gain deeper insight into biological and structural functionalities, using ideas coming from information theory and stochastic modelling of symbolic sequences. In particular, previous analyses on CG dinucleotide position along the genome allowed to highlight its epigenetic role in DNA methylation, showing a different distribution tail as compared to other dinucleotides. In this paper we extend the analysis to the whole CG distance distribution over a selected set of higher-order organisms. Then we apply the best fitting probability density function to a large range of organisms (>4400) of different complexity (from bacteria to mammals) and we characterize some emerging global features. RESULTS: We find that the Gamma distribution is optimal for the selected subset as compared to a group of several distributions, chosen for their physical meaning or because recently used in literature for similar studies. The parameters of this distribution, when applied to our larger set of organisms, allows to highlight some biologically relavant features for the considered organism classes, that can be useful also for classification purposes. CONCLUSIONS: The quantification of statistical properties of CG dinucleotide positioning along the genome is confirmed as a useful tool to characterize broad classes of organisms, spanning the whole range of biological complexity.

c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation.

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the ß-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.

Identification of Major Enzymes Involved in the Synthesis of Diadenosine Tetraphosphate and/or Adenosine Tetraphosphate in Myxococcus xanthus.

Myxococcus xanthus generates diadenosine tetraphosphates (Ap4A) and diadenosine pentaphosphates (Ap5A) under various stress conditions. M. xanthus lysyl-tRNA synthetase (LysS) efficiently synthesizes Ap4A from ATP, Ap5A from ATP and adenosine tetraphosphate (Ap4), and Ap4 from ATP and triphosphate. To identify other M. xanthus enzymes that can catalyze Ap4A and Ap4 synthesis, 15 M. xanthus aminoacyl-tRNA synthetases (aaRSs), four acyl-CoA synthetases (Acys), three acetyl-CoA synthetases (Aces), phosphoglycerate kinase (Pgk), and adenylate kinase (Adk) were expressed in Escherichia coli and examined for Ap4A or Ap4 synthetase activity using ATP or ATP and triphosphate as substrates. Among the tested enzymes, LysS had the highest Ap4A synthetase activity. AlaRS, SerRS, and LeuRS1 showed high ADP synthetase activity with ATP as a substrate in the presence of pyrophosphatase, and also demonstrated the ability to produce Ap4 from ATP and triphosphate in the absence of pyrophosphatase. Ap4 formation by AlaRS, SerRS, and LeuRS1 was approximately 4- to 13-fold higher compared with that of Ap4A, suggesting that these enzymes prefer triphosphate over ATP as a substrate in the second reaction. Some of the recombinant M. xanthus Acys and Aces also synthesized Ap4 from ATP and triphosphate. However, Pgk was capable of catalyzing the production of Ap4 from ATP and 3-phosphoglycerate in the presence of Mg2+ and did not require triphosphate, suggesting that this enzyme is mainly responsible for Ap4 synthesis in M. xanthus.