The Uracil DNA Glycosylase UdgB of Mycobacterium smegmatis Protects the Organism from the Mutagenic Effects of Cytosine and Adenine Deamination

Spontaneous hydrolytic deamination of DNA bases represents a considerable mutagenic threat to all organisms, particularly those living in extreme habitats. Cytosine is readily deaminated to uracil, which base pairs with adenine during replication, and most organisms encode at least one uracil DNA glycosylase (UDG) that removes this aberrant base from DNA with high efficiency. Adenine deaminates to hypoxanthine approximately 10-fold less efficiently, and its removal from DNA in vivo has to date been reported to be mediated solely by alkyladenine DNA glycosylase. We previously showed that UdgB from Pyrobaculum aerophilum, a hyperthermophilic crenarchaeon, can excise hypoxanthine from oligonucleotide substrates, but as this organism is not amenable to genetic manipulation, we were unable to ascertain that the enzyme also has this role in vivo. In the present study, we show that UdgB from Mycobacterium smegmatis protects this organism against mutagenesis associated with deamination of both cytosine and adenine. Together with Ung-type uracil glycosylase, M. smegmatis UdgB also helps attenuate the cytotoxicity of the antimicrobial agent 5-fluorouracil.Spontaneous hydrolytic deamination of cytosine (C), adenine (A), and guanine (G) gives rise to uracil (U), hypoxanthine (Hx), and xanthine, respectively (3). Because the first two reactions occur at appreciable rates (31) and because their products are mutagenic, living organisms have evolved sophisticated defense mechanisms to deal with this threat to their genomic integrity. Deamination of C to U occurs with the highest frequency. Because U base pairs with A during replication, a failure to remove U from DNA prior to the next round of DNA synthesis gives rise to C→T (or, on the opposite strand, G→A) transition mutations (1). The A-to-Hx reaction is only about 10 times slower than the reaction affecting C and has biological significance due to Hx pairing with cytosine during replication.Similar to other modified DNA bases, U and Hx are removed from DNA by base excision repair. This process is initiated by one of several DNA glycosylases, which removes the aberrant base, giving rise to an abasic (apyrimidinic or apurinic) site (AP site). In the subsequent step, the AP site is cleaved by an AP endonuclease on its 5′ side, giving rise to a single-strand break. The baseless sugar phosphate is then removed by the 5′→3′ exonuclease activity of polymerase I in bacteria or by the N-terminal lyase activity of polymerase β in eukaryotes. The same polymerases then fill in the single nucleotide gap, and the remaining nick is sealed by a DNA ligase (20). Base excision repair can also proceed by mechanisms that differ somewhat from the canonical pathway described above, but these mechanisms do not alter the outcome of the studies described in this work and are not described in detail here.Uracil removal is catalyzed by uracil DNA glycosylases (UDGs). These enzymes possess two highly conserved motifs; motif A activates a catalytic water molecule for nucleophilic attack on the glycosidic bond, and motif B stabilizes the protein-DNA complex (25). To date, five UDG families with somewhat different substrate specificities have been characterized (25, 30). Enzymes belonging to family 1 are the best-characterized enzymes. They can remove uracil from U·A pairs or U·G mispairs in double-stranded DNA (dsDNA) and even more efficiently from single-stranded DNA (ssDNA) substrates (32). Family 2 enzymes excise uracil from U·G mismatches in dsDNA. Because these enzymes have most likely evolved to deal with the deamination of 5-methylcytosine to thymine, they are specific for U·G and T·G mispairs and excise neither uracil nor thymine from ssDNA (21). The active site of these enzymes is altered compared to the active site of family 1 UDGs. Family 3 consists of the single-strand-selective monofunctional UDGs, which appear to be hybrids between members of families 1 and 2 (5). As their name implies, family 3 enzymes are preferentially active on uracil-containing ssDNA substrates. Family 4 enzymes have been identified in hyperthermophiles such as Thermotoga maritima (28), Archaeoglobus fulgidus (29), and Pyrobaculum aerophilum (6). These enzymes act on U opposite A or G and exhibit maximal activity at high temperatures. Family 5 uracil glycosylases (UdgB) have been identified in a limited number of organisms, such as hyperthermophilic archaea and Mycobacterium tuberculosis (7, 30, 37). Unexpectedly, motif A of UdgB enzymes contains no polar amino acid, but motif B resembles motif B of family 1 proteins (30). In vitro assays showed that UdgB removes uracil from both ssDNA and dsDNA (30). Interestingly, this enzyme could also excise Hx from oligonucleotide substrates (30, 36) and is thus the only glycosylase that removes both aberrant pyrimidines and purines from DNA in vitro. One of the goals of the present study was to learn whether this enzyme also processes U and Hx in vivo.As discussed above, uracil is generated in DNA by spontaneous hydrolytic deamination of cytosine. However, it can also be directly incorporated into DNA during replication in the form of dUMP. This process is not mutagenic, because dUMP is incorporated opposite adenine. However, it can be cytotoxic, particularly in cells with an imbalanced dUTP-dTTP pool, such as mutants lacking dUTPase, or in cells in which thymidylate synthase is inhibited by, e.g., 5-fluorodeoxyuridine (5FdUrd) treatment. Deoxyuridine incorporated during DNA synthesis is processed primarily by family 1 UDGs, which have been shown to associate with replicating polymerase complexes. Under normal circumstances, this repair process affects only the newly synthesized strand. However, at high dUTP/dTTP ratios, uracil repair might become saturated, so that some uracil remains in the DNA until the following round of DNA replication. In this scenario, processing of uracil in both template and nascent strands would give rise to double-strand breaks and thus to genomic instability (14, 15). This hypothesis is supported by the finding that organisms lacking an efficient repair system can tolerate considerable replacement of thymine with uracil in their DNA (40). The second goal of the present study was to test whether UdgB, like family 1 UDGs, contributed to the cytotoxicity associated with a dUTP-dTTP pool imbalance.We wanted to study whether the substrates processed by UdgB in vitro are also addressed in vivo by this enzyme. As we were unable to do this with P. aerophilum, we set out to find an organism encoding UdgB that would be more amenable to genetic manipulation. M. tuberculosis encodes two potential UDGs, one belonging to family 1 (ung, M. tuberculosis Rv2976c) and one belonging to family 5 (udgB, M. tuberculosis Rv1259) (30). However, to avoid complications linked to the pathogenicity of this organism, we decided to investigate the role of Ung and UdgB in the nonpathogenic organism Mycobacterium smegmatis, which is, unlike P. aerophilum, amenable to genetic manipulation. Our results provide novel insights into the coordinated action of family 1 and family 5 glycosylases.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).     

Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject''s stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.Helicobacter pylori, a gram-negative bacterium, is remarkable for its ability to persist in the human stomach for decades. Colonization with H. pylori increases risk for peptic ulcer disease and gastric adenocarcinoma (53, 70) and elicits a vigorous immune response (15). The persistence of H. pylori occurs in a niche in the human body previously considered inhospitable to microbial colonization: the acidic stomach replete with proteolytic enzymes.H. pylori strains exhibit substantial genetic diversity, including extensive variation in the presence, arrangement, order, and identity of genes (2, 4-7, 25, 51, 74). Furthermore, analyses of multiple single-colony H. pylori isolates from separate stomach biopsy specimens of individual patients have demonstrated diversity, both within hosts (27, 65), and over time (36). The mechanisms that generate H. pylori genetic diversity may be among the factors that enable persistence in this environment (3, 28).While the natural ability of H. pylori for transformation and recombination may explain some of the intra- and interhost genetic variation observed in this bacterium (43), point mutations and interspecies recombination alone are not sufficient for explaining the extent of the variation in H. pylori (14, 32). The initial genomic sequencing of H. pylori strains 26695 and J99 (6, 72) revealed large amounts of repetitive DNA (1, 59). DNA repeats in bacteria are associated with mechanisms of plasticity, such as phase variation (49, 67); slipped-strand mispairing (41, 46); and increased rates of recombination, deletion, and insertion (17, 60, 62). Because many of the recombination repair and mismatch repair mechanisms common in bacteria are absent or modified in H. pylori (28-30, 56, 76), this organism may be particularly susceptible to the diversifying effects of repetitive DNA. In fact, loci in the H. pylori genome containing repetitive DNA have been shown to exhibit extensive inter- and intrahost variation (9, 10, 28, 37).We hypothesized that identification of repetitive DNA hotspots in H. pylori would allow the recognition of genes whose variation could aid in persistence. To examine this hypothesis, we conducted in silico analyses to identify open reading frames (ORFs) enriched for DNA repeats and then used a combination of sequence analyses and immunoassays to examine the patterns associated with the specific repetitive DNA observed. Our approach led to the realization that a previously identified H. pylori-specific gene family (19, 52) exhibits extensive genetic variation at multiple levels.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

High Abundance of Ammonia-Oxidizing Archaea in Coastal Waters,Determined Using a Modified DNA Extraction Method

Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.Efficient DNA extraction from environmental samples is fundamental to many culture-independent characterizations (10). Thus, there was an early and concerted effort to establish appropriate methods of DNA extraction from different types of environmental samples (14, 19, 25, 30, 34, 43, 47). DNA extraction efficiency is particularly important for quantitative PCR (qPCR), because poor DNA extraction efficiency results in the underestimation of gene copy numbers in the samples examined (6, 42).Most methodological developments addressed DNA extraction from soil and sediment samples, with fewer comparative studies of the efficiency of collection and extraction from water samples (4, 13, 40). In part, a methodological focus on soils reflected the simplicity of filtration to collect aquatic populations and the generally good recovery of DNA from the Gram-negative bacteria making up a significant fraction of aquatic communities. However, small Archaea are now known to constitute a substantial fraction of the prokaryotic populations in marine and terrestrial systems (2, 7, 9, 20, 26, 31, 33, 45). Since the archaeal cell wall and membrane structures are distinct from those of bacteria, there is no assurance that commonly used extraction methods are adequate. With increasing reliance on commercially available bead-beating-type DNA extraction kits, these methods are now often used for different water samples (1, 5-7, 14, 19, 36). Although most protocols incorporate mechanical disruption to ensure more-uniform extraction than is possible by using methods that rely entirely on enzymatic digestion and/or chemical disruption (4, 13, 40), the suitability of these protocols for the concerted analysis of archaeal and bacterial populations has not been fully evaluated.In the studies reported here, the recently isolated marine archaeon Nitrosopumilus maritimus strain SCM1 (22) was therefore used as a reference standard for evaluation of the commonly employed DNA extraction methods by using qPCR. This archaeon was then used as a reference for the development of a simple, rapid, and efficient method of extracting DNA from both archaeal and bacterial cells. The modified protocol was subsequently employed to characterize the vertical distribution of ammonia-oxidizing Archaea in a fjord (Hood Canal) in Puget Sound (Washington State), revealing a high fractional representation of Archaea relative to Bacteria not observed previously in coastal waters.     

Mitochondrial DNA Toxicity in Forebrain Neurons Causes Apoptosis,Neurodegeneration, and Impaired Behavior

Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIα-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration.A variety of both exogenous and endogenous reactive compounds present a constant threat to the integrity of DNA in living cells. DNA damage introduced by such compounds can lead to high and deleterious mutation rates as well as DNA cytotoxicity, both to the nuclear and the mitochondrial genome. This has triggered the evolution of several different DNA repair pathways (28). One is the base excision repair (BER) pathway, which repairs small base alterations that do not distort the DNA helix. Repair of such highly abundant lesions by BER is performed by a multistep process that is initiated by a damage-specific DNA glycosylase, which removes the damaged base. One of these glycosylases is uracil-DNA glycosylase (UDG), which acts to preserve the genome by removing mutagenic uracil residues from the DNA. This glycosylase, as well as the OGG1 glycosylase that is specialized for the removal of oxidized bases, exists in a nuclear and mitochondrial splice form (1, 11, 37, 45). Accordingly, BER of a variety of lesions has been observed in mitochondria (26, 31).Damage to the mitochondrial DNA (mtDNA) can cause respiratory chain deficiency and lead to disorders that have varied phenotypes (35, 41). Many involve neurological features that are often associated with cell loss within specific brain regions. These pathologies, along with the increasing evidence of a decline in mitochondrial function with aging, have raised speculation that key changes in mitochondrial DNA sequences and functions could have a vital role in age-related neurodegenerative diseases (41). This has also been studied in several model organisms. Mouse models with respiratory chain deficient dopamine neurons have demonstrated adult onset Parkinsonism phenotype (16), and cell death induced by mitochondrial toxicity is likely to underlie Alzheimer disease (32). Mitochondrial oxidative stress and accumulation of mtDNA damage are believed to be particularly devastating to postmitotic differentiated tissue, including neurons (30). The mtDNA contains genetic information for 13 polypeptides that are a part of the electron transport chain and for rRNAs and tRNAs that are necessary for mitochondrial protein synthesis. Thus, damage to the mtDNA genome will affect the energetic capacities of the mitochondria and also influence the level of reactive oxygen species (ROS) and ultimately the susceptibility to apoptosis (30, 35).Some recent influential studies have assessed the effect of mtDNA mutagenesis, including small base-pair substitutions and larger mtDNA deletions, on the life span of mice. It was concluded that a massive increase in the frequency of mtDNA base-pair substitutions are required for inducing premature aging, whereas the number of mtDNA deletions coincides better with natural aging (25, 47-49).In the present study, we have combined two novel transgenic mouse models, which allow the induction of a high number of apyrimidinic (AP) sites specifically to the mitochondrial genome in adults simply by the addition of doxycycline to the diet. Such AP sites are created by the expression of a mutated version of mitochondrion-targeted human UDG (abbreviated here as mutUNG1), whereby an amino acid substitution results in an enzyme that removes thymine, in addition to uracil, from DNA (23). The CaMKIIα promoter restricts expression of the mutUNG1 to forebrain neurons (34). We demonstrate that a continuous generation of AP sites leads to apoptosis, accelerated neurodegeneration, and impaired behavior.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant. In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence-mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to comEA, comEC, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms.The genera Thermoanaerobacter and Thermoanaerobacterium contain bacteria which are thermophilic, obligate anaerobes that specialize in polysaccharide and carbohydrate fermentation, producing primarily l-lactic acid, acetic acid, ethanol, CO₂, and H₂ (24, 27, 49). Taxonomically, they are distinguished from other anaerobic thermophilic clostridia by the ability to reduce thiosulfate to hydrogen sulfide or elemental sulfur (21). The majority of characterized Thermoanaerobacter and Thermoanaerobacterium strains have been isolated from hot springs and other thermal environments (20-22, 38, 47); however, they have also been isolated from canned foods (4, 10), soil (48), paper mills and breweries (41, 43), and deep subsurface environments (5, 13, 35), suggesting a somewhat ubiquitous environmental presence.Representatives of the Thermoanaerobacter and Thermoanaerobacterium genera have been considered for biotechnological applications, such as conversion of lignocellulosic biomass to ethanol (8, 27) or other fuels and chemicals (3, 24). However, the branched fermentation pathways of these organisms generally require modification for industrial application. Several studies have investigated manipulating bioprocess and growth conditions to alter end product ratios and yields, but this has not resulted in reliable conditions to maximize the yield of a single end product (18, 25). Genetic engineering is likely necessary for commercial application of Thermanaerobacter or Thermoanaerobacterium species (26, 27, 44). As genetic systems for these bacteria have emerged (28, 45), increased product yields have been demonstrated by gene knockout of l-lactate dehydrogenase (9, 14), phosphotransacetylase and acetate kinase (40), and hydrogenase (39). Despite this recent progress, genetic transformation is still considered the greatest barrier for engineering these organisms (44).In contrast, some of the bacteria most amenable to genetic manipulation are those exhibiting natural competence; for example, work with the naturally competent Streptococcus pneumoniae first established DNA as the molecule containing inheritable information (42). Naturally competent organisms are found in many bacterial phyla, although the overall number of bacteria known to be naturally competent is relatively small (16).The molecular mechanisms of natural competence are often divided into two stages: early-stage genes that encode regulatory and signal cascades to control competence induction, and late-stage genes that encode the machinery of DNA uptake and integration (16). The Gram-positive late-stage consensus mechanism for DNA uptake and assimilation, elucidated primarily through work with Bacillus subtilis, occurs through several molecular machinery steps. First, DNA is believed to interact with a type IV pilus (T4P) or pseudopilus that brings it into close proximity of the cell membrane. The precise mechanism of this phenomenon is unclear; although components of the T4P in both Gram-positive and Gram-negative bacteria have been shown to bind DNA (7, 19), in specific studies, a full pilus structure has been either not observed or shown not to be essential during natural competence (6, 36). Two proteins, ComEA and ComEC, are then involved in creation and transport of single-stranded DNA across the membrane, where it is subsequently bound by CinA-localized RecA and either integrated into the genome or replicated at an independent origin, as for plasmid DNA (6).Here, we report that several Thermoanaerobacter and Thermoanaerobacterium strains are naturally competent, characterize growth conditions conducive to natural competence, and identify genes in Thermoanaerobacterium saccharolyticum JW/SL-YS485 required for competence exhibition.     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

Vaccinia Virus D4 Mutants Defective in Processive DNA Synthesis Retain Binding to A20 and DNA

Genome replication is inefficient without processivity factors, which tether DNA polymerases to their templates. The vaccinia virus DNA polymerase E9 requires two viral proteins, A20 and D4, for processive DNA synthesis, yet the mechanism of how this tricomplex functions is unknown. This study confirms that these three proteins are necessary and sufficient for processivity, and it focuses on the role of D4, which also functions as a uracil DNA glycosylase (UDG) repair enzyme. A series of D4 mutants was generated to discover which sites are important for processivity. Three point mutants (K126V, K160V, and R187V) which did not function in processive DNA synthesis, though they retained UDG catalytic activity, were identified. The mutants were able to compete with wild-type D4 in processivity assays and retained binding to both A20 and DNA. The crystal structure of R187V was resolved and revealed that the local charge distribution around the substituted residue is altered. However, the mutant protein was shown to have no major structural distortions. This suggests that the positive charges of residues 126, 160, and 187 are required for D4 to function in processive DNA synthesis. Consistent with this is the ability of the conserved mutant K126R to function in processivity. These mutants may help unlock the mechanism by which D4 contributes to processive DNA synthesis.Poxviruses are large, double-stranded DNA viruses that replicate exclusively in the cell cytoplasm in granular structures known as virosomes (31). Separated from the host nucleus, they rely on their own encoded gene products for DNA synthesis and replication (43). To efficiently synthesize its ∼200,000-base genome, the poxvirus DNA polymerase must be tethered to the DNA template by its processivity factor. DNA processivity factors are proteins that stabilize polymerases onto their templates for effective genome replication (1, 22). Processivity factors are synthesized by nearly all replicating systems, ranging from bacteriophages to eukaryotes, yet each one is specific to its cognate polymerase. In the presence of these factors, polymerases are able to incorporate a great number of nucleotides per template binding event; in their absence, polymerases detach from their templates too frequently to successfully replicate the genome (14, 20). E9, the DNA polymerase of the prototypical poxvirus, vaccinia virus, synthesizes approximately 10 nucleotides before dissociating from the viral DNA template (28). However, it can incorporate thousands of nucleotides when it is associated with its processivity factor (29). This extended strand synthesis, known as processivity, is necessary for vaccinia virus to effectively replicate its 192-kb genome.The protein A20 was first reported to be a component of the vaccinia virus processive DNA polymerase (19, 37), yet we were unable to establish processivity in vitro using only A20 and E9. To identify which other proteins were required for processivity, we assessed six in vitro-synthesized proteins known to be involved in vaccinia virus replication (E9, A20, B1, D4, D5, and H5). We found that the protein D4, a uracil DNA glycosylase (UDG), was required in addition to A20 and E9 and that these three proteins are both necessary and sufficient for vaccinia virus processivity. Indeed, A20 and D4 have been shown to interact with each other (15, 26), and our finding supports a report identifying A20 and D4 as forming a heterodimeric processivity factor for E9 (41). Here, we use mutational analysis to examine the role of D4 in processive DNA synthesis. We report the finding of three D4 mutants which are unable to function in processivity yet retain their UDG enzymatic activity and their ability to bind both A20 and DNA.