Similarity of genes horizontally acquired by Escherichia coli and Salmonella enterica is evidence of a supraspecies pangenome

Most bacterial and archaeal genomes contain many genes with little or no similarity to other genes, a property that impedes identification of gene origins. By comparing the codon usage of genes shared among strains (primarily vertically inherited genes) and genes unique to one strain (primarily recently horizontally acquired genes), we found that the plurality of unique genes in Escherichia coli and Salmonella enterica are much more similar to each other than are their vertically inherited genes. We conclude that E. coli and S. enterica derive these unique genes from a common source, a supraspecies phylogenetic group that includes the organisms themselves. The phylogenetic range of the sharing appears to include other (but not all) members of the Enterobacteriaceae. We found evidence of similar gene sharing in other bacterial and archaeal taxa. Thus, we conclude that frequent gene exchange, particularly that of genetic novelties, extends well beyond accepted species boundaries.     

Regulation of genetic flux between bacteria by restriction–modification systems

Restriction–modification (R-M) systems are often regarded as bacteria''s innate immune systems, protecting cells from infection by mobile genetic elements (MGEs). Their diversification has been recently associated with the emergence of particularly virulent lineages. However, we have previously found more R-M systems in genomes carrying more MGEs. Furthermore, it has been suggested that R-M systems might favor genetic transfer by producing recombinogenic double-stranded DNA ends. To test whether R-M systems favor or disfavor genetic exchanges, we analyzed their frequency with respect to the inferred events of homologous recombination and horizontal gene transfer within 79 bacterial species. Genetic exchanges were more frequent in bacteria with larger genomes and in those encoding more R-M systems. We created a recognition target motif predictor for Type II R-M systems that identifies genomes encoding systems with similar restriction sites. We found more genetic exchanges between these genomes, independently of their evolutionary distance. Our results reconcile previous studies by showing that R-M systems are more abundant in promiscuous species, wherein they establish preferential paths of genetic exchange within and between lineages with cognate R-M systems. Because the repertoire and/or specificity of R-M systems in bacterial lineages vary quickly, the preferential fluxes of genetic transfer within species are expected to constantly change, producing time-dependent networks of gene transfer.Prokaryotes evolve rapidly by acquiring genetic information from other individuals, often through the action of mobile genetic elements (MGEs) such as plasmids or phages (1). In bacterial population genetics, the events of gene transfer are usually termed horizontal gene transfer (HGT) when they result in the acquisition of new genes and homologous recombination (HR) when they result in allelic replacements. The distinction between the two evolutionary mechanisms (HGT and HR) is not always straightforward: incoming DNA may integrate the host genome by double crossovers at homologous regions, leading to allelic replacements in these regions and to the acquisition of novel genes in the intervening ones. HR takes place only between highly similar sequences, typically within species (2). As a result, it usually involves the exchange of few polymorphisms, eventually in multiple regions, between cells (3). It may also result in no change if the recombining sequences are identical, which leaves no traces and cannot be detected by sequence analysis. HGT may occur between distant species, resulting in the acquisition of many genes in a single event. The replication and maintenance of MGEs have fitness costs to the bacterial host and have led to the evolution of cellular defense systems. These systems can sometimes be counteracted by MGEs, leading to evolutionary arms races.Restriction–modification (R-M) systems are some of the best known and the most widespread bacterial defense systems (4). They encode a methyltransferase (MTase) function that modifies particular DNA sequences in function of the presence of target recognition sites and a restriction endonuclease (REase) function that cleaves them when they are unmethylated (5). R-M systems are traditionally classified into three main types. Type II systems are by far the most abundant and the best studied (6). With the exception of the subType IIC, they comprise MTase and REase functions encoded on separate genes and are able to operate independently from each other. R-M systems severely diminish the infection rate by MGEs and have been traditionally seen as bacteria''s innate immune systems (7). However, successful infection of a few cells generates methylated MGEs immune to restriction that can invade the bacterial population (8). Hence, R-M systems are effective as defense systems during short periods of time and especially when they are diverse across a population (9, 10). In particular, it has been suggested that they might facilitate colonization of new niches (11). Type II R-M systems are also addictive modules that can propagate selfishly in populations (12). Both roles of R-M systems, as defense or selfish systems, may explain why they are very diverse within species (13, 14). Accordingly, R-M systems endure selection for diversification and are rapidly replaced (15, 16).Several recent large-scale studies of population genomics have observed more frequent HR within than between lineages (17, 18). This suggests that HR might favor the generation of cohesive population structures within bacterial species (19). Specific lineages of important pathogens that have recently changed their R-M repertoires show higher sexual isolation, such as Neisseria meningitidis, Streptococcus pneumoniae, Burkholderia pseudomallei, and Staphylococcus aureus (20–22). For example, a Type I R-M system decreased transfer to and from a major methicillin-resistant S. aureus lineage (23). Diversification of R-M target recognition sites could thus reduce transfer between lineages with different systems while establishing preferential gene fluxes between those with R-M systems recognizing the same target motifs (cognate R-M). However, these results can be confounded by evolutionary distance: closely related genomes are more likely to encode similar R-M systems, inhabit the same environments (facilitating transfer between cells), and have similar sequences (that recombine at higher rates). The advantages conferred by new genes might be higher when transfer takes place between more similar genetic backgrounds.Here, we aimed at testing the effect of R-M systems on the genetic flux in bacterial populations. We concentrated on Type II R-M systems because they are the best studied, very frequent, and those for which we could predict sequence specificity. We inferred genome-wide counts of HR and HGT and tested their association with the frequency of R-M systems encoded in the genomes. We then made a more precise test of the key hypothesis that bacteria carrying similar R-M systems establish highways of gene transfer, independently of phylogenetic proximity and clade-specific traits.     

Giant virus with a remarkable complement of genes infects marine zooplankton

As major consumers of heterotrophic bacteria and phytoplankton, microzooplankton are a critical link in aquatic foodwebs. Here, we show that a major marine microflagellate grazer is infected by a giant virus, Cafeteria roenbergensis virus (CroV), which has the largest genome of any described marine virus (≈730 kb of double-stranded DNA). The central 618-kb coding part of this AT-rich genome contains 544 predicted protein-coding genes; putative early and late promoter motifs have been detected and assigned to 191 and 72 of them, respectively, and at least 274 genes were expressed during infection. The diverse coding potential of CroV includes predicted translation factors, DNA repair enzymes such as DNA mismatch repair protein MutS and two photolyases, multiple ubiquitin pathway components, four intein elements, and 22 tRNAs. Many genes including isoleucyl-tRNA synthetase, eIF-2γ, and an Elp3-like histone acetyltransferase are usually not found in viruses. We also discovered a 38-kb genomic region of putative bacterial origin, which encodes several predicted carbohydrate metabolizing enzymes, including an entire pathway for the biosynthesis of 3-deoxy-d-manno-octulosonate, a key component of the outer membrane in Gram-negative bacteria. Phylogenetic analysis indicates that CroV is a nucleocytoplasmic large DNA virus, with Acanthamoeba polyphaga mimivirus as its closest relative, although less than one-third of the genes of CroV have homologs in Mimivirus. CroV is a highly complex marine virus and the only virus studied in genetic detail that infects one of the major groups of predators in the oceans.     

Impaired induction of DNA lesions during immunoglobulin class-switch recombination in humans influences end-joining repair

Ig class-switch recombination (CSR) is a region-specific process that exchanges the constant Ig heavy-chain region and thus modifies an antibody's effector function. DNA lesions in switch (S) regions are induced by activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase 2 (UNG2), subsequently processed to DNA breaks, and resolved by either the classical nonhomologous end-joining pathway or the alternative end-joining pathway (XRCC4/DNA ligase 4- and/or Ku70/Ku80-independent and prone to increased microhomology usage). We examined whether the induction of DNA lesions influences DNA end-joining during CSR by analyzing Sμ-Sα recombination junctions in various human Ig CSR defects of DNA lesion induction. We observed a progressive trend toward the usage of microhomology in Sμ-Sα recombination junctions from AID-heterozygous to AID-autosomal dominant to UNG2-deficient B lymphocytes. We thus hypothesize that impaired induction of DNA lesions in S regions during CSR leads to unusual end-processing of the DNA breaks, resulting in microhomology-mediated end-joining, which could be an indication for preferential processing by alternative end-joining rather than by classical nonhomologous end-joining.     

The evolution of modularity in bacterial metabolic networks

Deciphering the modular organization of metabolic networks and understanding how modularity evolves have attracted tremendous interest in recent years. Here, we present a comprehensive large scale characterization of modularity across the bacterial tree of life, systematically quantifying the modularity of the metabolic networks of >300 bacterial species. Three main determinants of metabolic network modularity are identified. First, network size is an important topological determinant of network modularity. Second, several environmental factors influence network modularity, with endosymbionts and mammal-specific pathogens having lower modularity scores than bacterial species that occupy a wider range of niches. Moreover, even among the pathogens, those that alternate between two distinct niches, such as insect and mammal, tend to have relatively high metabolic network modularity. Third, horizontal gene transfer is an important force that contributes significantly to metabolic modularity. We additionally reconstruct the metabolic network of ancestral bacterial species and examine the evolution of modularity across the tree of life. This reveals a trend of modularity decrease from ancestors to descendants that is likely the outcome of niche specialization and the incorporation of peripheral metabolic reactions.     

Integrons in Xanthomonas: a source of species genome diversity

Integrons are best known for assembling antibiotic resistance genes in clinical bacteria. They capture genes by using integrase-mediated site-specific recombination of mobile gene cassettes. Integrons also occur in the chromosomes of many bacteria, notably beta- and gamma-Proteobacteria. In a survey of Xanthomonas, integrons were found in all 32 strains representing 12 pathovars of two species. Their chromosomal location was downstream from the acid dehydratase gene, ilvD, suggesting that an integron was present at this site in the ancestral xanthomonad. There was considerable sequence and structural diversity among the extant integrons. The majority of integrase genes were predicted to be inactivated by frameshifts, stop codons, or large deletions, suggesting that the associated gene cassettes can no longer be mobilized. In support, groups of strains with the same deletions or stop codons/frameshifts in their integrase gene usually contained identical arrays of gene cassettes. In general, strains within individual pathovars had identical cassettes, and these exhibited no similarity to cassettes detected in other pathovars. The variety and characteristics of contemporary gene cassettes suggests that the ancestral integron had access to a diverse pool of these mobile elements, and that their genes originated outside the Xanthomonas genome. Subsequent inactivation of the integrase gene in particular lineages has largely fixed the gene cassette arrays in particular pathovars during their differentiation and specialization into ecological niches. The acquisition of diverse gene cassettes by different lineages within Xanthomonas has contributed to the species-genome diversity of the genus. The role of gene cassettes in survival on plant surfaces is currently unknown.     

Biogeography of the Sulfolobus islandicus pan-genome

Variation in gene content has been hypothesized to be the primary mode of adaptive evolution in microorganisms; however, very little is known about the spatial and temporal distribution of variable genes. Through population-scale comparative genomics of 7 Sulfolobus islandicus genomes from 3 locations, we demonstrate the biogeographical structure of the pan-genome of this species, with no evidence of gene flow between geographically isolated populations. The evolutionary independence of each population allowed us to assess genome dynamics over very recent evolutionary time, beginning ≈910,000 years ago. On this time scale, genome variation largely consists of recent strain-specific integration of mobile elements. Localized sectors of parallel gene loss are identified; however, the balance between the gain and loss of genetic material suggests that S. islandicus genomes acquire material slowly over time, primarily from closely related Sulfolobus species. Examination of the genome dynamics through population genomics in S. islandicus exposes the process of allopatric speciation in thermophilic Archaea and brings us closer to a generalized framework for understanding microbial genome evolution in a spatial context.     

Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation

Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3′ end of the intron RNA to the 5′ end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5′ junctions with 5′ exon resections, 5′ intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting.     

A collective mechanism for phase variation in biofilms

Understanding how microbes gather into biofilm communities and maintain diversity remains one of the central questions of microbiology, requiring an understanding of microbes as communal rather then individual organisms. Phase variation plays an integral role in the formation of diverse phenotypes within biofilms. We propose a collective mechanism for phase variation based on gene transfer agents, and apply the theory to predict the population structure and growth dynamics of a biofilm. Our results describe quantitatively recent experiments, with the only adjustable parameter being the rate of intercellular horizontal gene transfer. Our approach derives from a more general picture for the emergence of cooperation between microbes.     

Bacterial natural transformation by highly fragmented and damaged DNA

DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old.DNA molecules are continuously released into the surroundings through decomposition of organic matter and are ubiquitous in most environments (1). DNA degradation is initiated at cell death by coreleased cellular nucleases and continued by microbes feeding on organic matter (1). Because fragmentation proceeds quickly, larger (gene-length) fragments are not expected to persist in the environment (1). Extracellular DNA is further modified by spontaneous biochemical, chemical, or physical processes, of which the most important are hydrolysis and oxidation (2). These result in apurinic sites (depurination) and in loss of amino groups at the base-residues (deamination). Depurination affects the stability of the DNA backbone and leads to nicks and single-strand overhangs of the DNA fragments (2). Consequently, most free DNA fragments in the environment are <100 bp in size (3–8). Deamination particularly affects cytosine, creating uracil residues that can lead to cytosine-to-thymine exchanges, which result in DNA sequencing errors (9). Other chemical modifications of the DNA backbone and base residues also occur, but normally at lower rates than those of depurination and cytosine deamination (10–13). Despite continuous degradation of free DNA, short fragments often persist for thousands of years and may survive for more than a million years in the environment given optimal preservation conditions (4, 14–17). However, physical disturbance of preservation conditions may lead to release of such environmental ancient DNA. For instance, estimates suggest that 859–14,500 tons of sedimentary DNA are released per year from rivers alone (SI Text).Fragmented and chemically damaged DNA is recognized as an important microbial nutrient source but has so far not been considered to contribute to bacterial genome evolution (1, 18–20). We decided to assess whether bacterial cells can acquire such degraded DNA, including genetic signatures of the deep past, by horizontal gene transfer—a driving force in prokaryotic evolution (21–24). Horizontal gene transfer by natural transformation is a process by which cells take up free (donor) DNA from the environment and integrate it into their own genomes (25, 26). The process likely occurs in most environments and is well described for high–molecular-weight DNA (chromosomal fragments typically ≥10,000 bp). Taken-up homologous DNA is efficiently incorporated into the genome by RecA-mediated homologous recombination. Successful natural transformation has not been described for DNA shorter than 250 bp (19, 20, 23). In this study we investigate (i) experimentally, to what extent is short (down to 20 bp) and damaged DNA taken up by natural transformation and integrated into the genome of the model organism Acinetobacter baylyi, a Gram-negative soil bacterium (27, 28); and (ii) in silico, the effect such recombination events have in genome evolution of transformable species.     

Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms

Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of “Marseille” virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are “melting pots” of microbial evolution where diverse forms emerge, including giant viruses with complex gene repertoires of various origins.     

Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli

Basic ideas about the constancy and randomness of mutagenesis that drives evolution were challenged by the discovery of mutation pathways activated by stress responses. These pathways could promote evolution specifically when cells are maladapted to their environment (i.e., are stressed). However, the clearest example--a general stress-response-controlled switch to error-prone DNA break (double-strand break, DSB) repair--was suggested to be peculiar to an Escherichia coli F' conjugative plasmid, not generally significant, and to occur by an alternative stress-independent mechanism. Moreover, mechanisms of spontaneous mutation in E. coli remain obscure. First, we demonstrate that this same mechanism occurs in chromosomes of starving F(-) E. coli. I-SceI endonuclease-induced chromosomal DSBs increase mutation 50-fold, dependent upon general/starvation- and DNA-damage-stress responses, DinB error-prone DNA polymerase, and DSB-repair proteins. Second, DSB repair is also mutagenic if the RpoS general-stress-response activator is expressed in unstressed cells, illustrating a stress-response-controlled switch to mutagenic repair. Third, DSB survival is not improved by RpoS or DinB, indicating that mutagenesis is not an inescapable byproduct of repair. Importantly, fourth, fully half of spontaneous frame-shift and base-substitution mutation during starvation also requires the same stress-response, DSB-repair, and DinB proteins. These data indicate that DSB-repair-dependent stress-induced mutation, driven by spontaneous DNA breaks, is a pathway that cells usually use and a major source of spontaneous mutation. These data also rule out major alternative models for the mechanism. Mechanisms that couple mutagenesis to stress responses can allow cells to evolve rapidly and responsively to their environment.     

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20–60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6–12 bp perfect repeats, or 10–16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomology-mediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle''s ability to repair broken chromosomes.     

Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli

AlkB repairs 1-alkyladenine and 3-methylcytosine lesions in DNA by directly reversing the base damage. Although repair studies with randomly alkylated substrates have been performed, the miscoding nature of these and related individually alkylated bases and the suppression of mutagenesis by AlkB within cells have not yet been explored. Here, we address the miscoding potential of 1-methyldeoxyadenosine (m1A), 3-methyldeoxycytidine (m3C), 3-ethyldeoxycytidine (e3C), 1-methyldeoxyguanosine (m1G), and 3-methyldeoxythymidine (m3T) by synthesizing single-stranded vectors containing each alkylated base, followed by vector passage through Escherichia coli. In SOS(-), AlkB-deficient cells, m1A was only 1% mutagenic; however, m3C and e3C were 30% mutagenic, rising to 70% in SOS(+) cells. In contrast, the mutagenicity of m1G and m3T in AlkB(-) cells dropped slightly when SOS polymerases were expressed (m1G from 80% to 66% and m3T from 60% to 53%). Mutagenicity was abrogated for m1A, m3C, and e3C in wild-type (AlkB(+)) cells, whereas m3T mutagenicity was only partially reduced. Remarkably, m1G mutagenicity was also eliminated in AlkB(+) cells, establishing it as a natural AlkB substrate. All lesions were blocks to replication in AlkB-deficient cells. The m1A, m3C, and e3C blockades were completely removed in wild-type cells; the m1G blockade was partially removed and that for m3T was unaffected by the presence of AlkB. All lesions demonstrated enhanced bypass when SOS polymerases were induced. This work provides direct evidence that AlkB suppresses both genotoxicity and mutagenesis by physiologically realistic low doses of 1-alkylpurine and 3-alkylpyrimidine DNA damage in vivo.     

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.Several dozen oxidatively modified, and mostly mutagenic, bases are induced in the genomes of aerobic organisms by endogenous and environmentally induced reactive oxygen species (ROS) (1, 2). For example, 5-hydroxyuracil (5-OHU), a predominant lesion generated by oxidative deamination of C, is mutagenic because of its mispairing with A (3). The bases in the single-stranded (ss) replicating DNA template are particularly prone to oxidation (4); the lack of their repair before replication could fix the mutations. The bulky base adducts if formed in the template strand would block replication and trigger DNA damage-response signaling. In contrast, oxidized bases with minor modifications, which are continuously formed in much higher abundance than the bulky adducts, would mostly allow replication. This raises the question of how these bases are marked for repair before replication to avoid mutagenic consequences. Oxidized base repair in mammalian genomes occurs primarily via the base excision repair (BER) pathway which is initiated with lesion base excision mediated by one of five major DNA glycosylases belonging to the Nth or Nei families, with distinct structural features and reaction mechanisms (1). Nei-like (NEIL) 1 and NEIL2 DNA glycosylases (5, 6) of the Nei family (which also contains the less characterized NEIL3; ref. 7) are distinct from NTH1 and OGG1 of the Nth family because the NEILs can excise damaged bases from ssDNA substrates (8). Furthermore, NEIL1 is activated during the S phase (5). Our earlier studies also showed that NEIL1 functionally interacts with many DNA replication proteins including sliding clamp proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN-1), and Werner RecQ helicase (WRN) via its disordered C-terminal segment (9–12). Importantly, mammalian ssDNA-binding replication protein A (RPA), essential for DNA replication and most other DNA transactions, inhibits NEIL1 or NEIL2 activity with primer-template DNA substrates mimicking the replication fork, presumably to prevent double-strand break formation (13). Although they collectively implicate NEIL1 in the repair of replicating DNA, those observations did not provide direct evidence for NEIL1’s role in prereplicative repair, nor did they address whether NEIL1 is unique for this function. In this report, we document that NEIL1 binds to the lesion base in an RPA-coated ssDNA template in vitro, without excising the lesion and cleaving the DNA strand, and blocks primer elongation by the replicative DNA polymerase δ (Polδ). This strongly suggests that the replication complex at the lesion site is stalled in vivo in the presence of NEIL1, which provides the signal for repair of lesions in the template strand before replication.     

Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi

A notable characteristic of fungal genomes is that genes involved in successive steps of a metabolic pathway are often physically linked or clustered. To investigate how such clusters of functionally related genes are assembled and maintained, we examined the evolution of gene sequences and order in the galactose utilization (GAL) pathway in whole-genome data from 80 diverse fungi. We found that GAL gene clusters originated independently and by different mechanisms in three unrelated yeast lineages. Specifically, the GAL cluster found in Saccharomyces and Candida yeasts originated through the relocation of native unclustered genes, whereas the GAL cluster of Schizosaccharomyces yeasts was acquired through horizontal gene transfer from a Candida yeast. In contrast, the GAL cluster of Cryptococcus yeasts was assembled independently from the Saccharomyces/Candida and Schizosaccharomyces GAL clusters and coexists in the Cryptococcus genome with unclustered GAL paralogs. These independently evolved GAL clusters represent a striking example of analogy at the genomic level. We also found that species with GAL clusters exhibited significantly higher rates of GAL pathway loss than species with unclustered GAL genes. These results suggest that clustering of metabolic genes might facilitate fungal adaptation to changing environments both through the acquisition and loss of metabolic capacities.     

Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10⁻³ mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold.A complete understanding of the evolution and stability of the genome requires that the determinants of spontaneous mutation be identified and characterized. Among the variety of mistakes that can occur during DNA transactions, four sources of sequence variation appear to dominate in prokaryotes: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage induced by exogenous agents, and the activities of error-prone polymerases. This conclusion is based on changes in the rates and spectra of mutations that occur when genes affecting these processes are deleted or amplified. In particular, loss of a DNA repair pathway often gives a mutator phenotype, indicating that the pathway of interest exerts an important limitation on spontaneous mutation (1). However, investigations of the mutagenic impact of various DNA repair pathways have relied almost exclusively on reporter genes, leaving open the possibility that the results are biased by the particular features of the selected loci. This concern can be avoided by allowing mutations to accumulate nonselectively in DNA repair-defective strains and identifying the resulting sequence changes by whole-genome sequencing (WGS). Although this approach may miss rare but interesting mutational processes, it can reveal the overall threats to genomic stability and identify features, such as local sequence context, that influence mutational frequencies. Surprisingly, this technique has been used with the eukaryote Caenorhabditis elegans (2) but has not been extensively applied to prokaryotes.The mutation accumulation (MA) protocol involves establishing multiple clonal populations from a single founder and then repeatedly passing the lines through single-individual bottlenecks (3, 4), which in bacteria is achieved easily by streaking for single colonies on agar medium. This procedure allows mutations to accumulate in an unbiased manner with a minimum of selective pressure. After a sufficient number of generations have occurred, the genomes are sequenced, and mutations are identified. Using this technique, we recently determined the intrinsic mutation rates and mutational spectra of repair-proficient strains of Escherichia coli and Bacillus subtilis and documented the mutational impact of the loss of the major error-correcting system, mismatch repair (MMR) (5–7). In the studies reported here we concentrate on E. coli, first asking if other commensal strains of E. coli have the same mutation rate and spectrum as our K12 strain and whether changing the growth medium influences mutation. Then we determined the mutational effects of the loss of several important DNA repair pathways. Our major conclusion is that, under the conditions of our experiments, mutation rates and spectra are nearly impervious to the loss of DNA repair functions except for those that deal with oxidative DNA damage. We also show that the mutagenicity of a major oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), is highly dependent on the local sequence context.