Polynucleobacter necessarius,a model for genome reduction in both free-living and symbiotic bacteria

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.Symbiosis, defined as a close relationship between organisms belonging to different species (1), is a ubiquitous, diverse, and important mechanism in ecology and evolution (e.g., refs. 2–4). In extreme cases, through the establishment of symbiotic relationships, quite unrelated lineages can functionally combine their genomes and generate advantageous emergent features or initiate parasite/host arms races. Ciliates, common unicellular protists of the phylum Ciliophora, are extraordinary receptacles for prokaryotic ecto- and endosymbionts (5, 6) that provide varied examples of biodiversity and ecological roles (6). Nevertheless, most of these symbionts are understudied, partially owing to the scarcity of available molecular data and the absence of sequenced genomes. Yet, thanks to their various biologies and the ease of sampling and cultivating their protist hosts, they are excellent potential models for symbioses between bacteria and heterotrophic eukaryotes. Until recently this field was dominated by studies on endosymbionts of invertebrates, especially insects (e.g., ref. 7), although unicellular systems like amoebas (e.g., refs. 8 and 9) have been shown to be suitable models.Polynucleobacter necessarius was first described as a cytoplasmic endosymbiont of the ciliate Euplotes aediculatus (10, 11). Further surveys detected its presence in a monophyletic group of fresh and brackish water Euplotes species (12, 13). All of the investigated strains of these species die soon after being cured of the endosymbiont (10, 12, 13). In the few cases in which P. necessarius is not present, a different and rarer bacterium apparently supplies the same function (12, 14). No attempt to grow symbiotic P. necessarius outside their hosts has yet been successful (15), strongly suggesting that the relationship is obligate for both partners, in contrast to most other known prokaryote/ciliate symbioses (6).Thus, the findings of many environmental 16S rRNA gene sequences similar to that of the symbiotic P. necessarius (16) but belonging to free-living freshwater bacteria came as a surprise. These free-living strains, which have been isolated and cultivated (17), are ubiquitous and abundant in the plankton of lentic environments (17, 18). They are smaller and do not show the most prominent morphological feature of the symbiotic form: the presence of multiple nucleoids, each containing one copy of the genome (10, 11). It is clear that free-living and endosymbiotic P. necessarius are not different life stages of the same organism (15). Nevertheless, these strikingly different bacteria, occupying separate ecological niches, exhibit >99% 16S rRNA gene sequence identity, and phylogenetic analyses fail to separate them into two distinct groups (15). Rather, several lines of evidence point to multiple, recent origins of symbiotic strains from the free-living bacterial pool (14, 15).Thus, the Euplotes–Polynucleobacter symbiosis provides a promising system for the study of changes promoting or caused by the shift to an intracellular lifestyle. The remarkably small (2.16 Mbp) genome of the free-living strain QLW-P1DMWA-1 has been sequenced and studied, especially for features that would explain the success of this lineage in freshwater systems worldwide (19, 20). Phylogenies based on the 16S rRNA gene (13, 14) and multiple-gene analyses (19, 21, 22) consistently cluster Polynucleobacter with bacteria of the family Burkholderiaceae (Betaproteobacteria), either in a basal position or as the sister group of Ralstonia and Cupriavidus.Here we provide the complete genomic sequence of a symbiotic P. necessarius harbored in the cytoplasm of E. aediculatus and present a comparative analysis of the two sequenced Polynucleobacter genomes, addressing the possible biological basis of the Euplotes–Polynucleobacter symbiosis. We also provide insights into the evolution of the unique two-step genome reduction in this bacterial species: the first step involving streamlining in a free-living ancestor and the second a more recent period of genome erosion confined to the symbiotic lineage.     

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA–Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA–Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.All viruses must encapsidate their genomes into virions to ensure that their genetic information is transferred to the new target cells. In most, if not all, retroviruses, the virion RNA genomes are dimeric, although each RNA encodes all of the genetic information required for replication. Most HIV-1 particles contain two copies of genomes (1), indicating that RNA encapsidation is a highly regulated process. This regulation is achieved by recognizing a dimeric RNA, and not by packaging a certain mass of viral genome (2).Our previous studies showed that HIV-1 RNA dimerization is a critical step in viral RNA genome packaging and virus assembly and that the two copies of copackaged RNA genomes are dimerized before encapsidation (1, 3, 4). The dimerization initiation signal (DIS), a 6-nt palindromic sequence located at the 5′ UTR of the HIV-1 RNA genome (5), most likely initiates the interaction between two HIV-1 RNA genomes (3, 4). When two HIV-1 RNAs contain similar sequences including the same DIS, they are copackaged efficiently at a rate similar to that predicted from random distribution (1, 2). In contrast, when two HIV-1 RNAs contain discordant palindromic sequences that cannot form perfect base pairing, they are not copackaged efficiently into the same viral particle (1, 2). The ability of RNA genomes from different HIV-1 variants to dimerize has important biological consequences. For example, inefficient copackaging is known to be a major barrier for intersubtype HIV-1 recombination (4). Although DIS plays a key role in RNA dimerization, virion RNAs isolated from mutants with DIS deletions remained dimeric, suggesting that other cis-acting element(s) are also involved in the dimerization (6).Despite the importance of RNA dimerization for HIV-1 replication, many aspects of this process are unknown, including the location at which dimerization occurs. Previously, we showed that RNA dimerization leading to HIV-1 genome packaging occurs after viral RNA is exported from the nucleus (7). The viral protein Gag is known to have chaperone activity (8). Additionally, biochemical experiments showed that HIV-1 Gag can interact with viral RNAs in the cytoplasm (9, 10). Thus, it is often hypothesized that two copies of HIV-1 RNAs dimerize in the cytoplasm and that this dimeric RNA is complexed with Gag and travels to the plasma membrane (7, 11–15), the major assembly site for virus assembly. The assembly of HIV-1 RNA and Gag was demonstrated in an elegant study using total internal reflection fluorescence (TIRF) microscopy (14), which illuminates a shallow volume near the glass/cell interface and is ideal for studying events near the plasma membrane (16). However, it was difficult to address the monomeric/dimeric state of the viral RNA in this previous study because the RNA was labeled with a single type of fluorescent protein.In the present study, we sought to delineate the location at which HIV-1 RNA dimerization occurs, which leads to genome encapsidation, and whether Gag is required for RNA dimerization. We used a previously described method to label HIV-1 RNA with fluorescent proteins through interactions of sequence-specific RNA-binding proteins. We engineered HIV-1 genomes to contain RNA stem-loops that are recognized by the Escherichia coli BglG protein or the bacteriophage MS2 coat protein; because these sequences are located in the pol gene, they are present only in full-length unspliced HIV-1 RNAs. When introduced into human cells, these constructs express full-length RNAs that can serve as templates for the translation of Gag proteins and as genomes in the viral particles. Most (>90%) of the particles contain RNA genomes, indicating that the full-length viral RNAs derived from these constructs are efficiently packaged. Furthermore, RNAs derived from different constructs can dimerize and copackage at a rate close to random distribution (1), consistent with the genetic analyses from recombination studies (4, 17, 18). By using this method, we were able to detect HIV-1 RNA with single-RNA-molecule sensitivity (1) and tracked HIV-1 RNA movement in the cytoplasm by using live-cell imaging (19). In this report, we tagged two HIV-1 RNAs and Gag, each with a different fluorescent protein, and studied the RNA:RNA and RNA:Gag interactions on the plasma membrane. We found that HIV-1 RNA dimerizes on the plasma membrane and that Gag protein is required for stabilization of the dimer.     

Investigation of terpene diversification across multiple sequenced plant genomes

Plants produce an array of specialized metabolites, including chemicals that are important as medicines, flavors, fragrances, pigments and insecticides. The vast majority of this metabolic diversity is untapped. Here we take a systematic approach toward dissecting genetic components of plant specialized metabolism. Focusing on the terpenes, the largest class of plant natural products, we investigate the basis of terpene diversity through analysis of multiple sequenced plant genomes. The primary drivers of terpene diversification are terpenoid synthase (TS) “signature” enzymes (which generate scaffold diversity), and cytochromes P450 (CYPs), which modify and further diversify these scaffolds, so paving the way for further downstream modifications. Our systematic search of sequenced plant genomes for all TS and CYP genes reveals that distinct TS/CYP gene pairs are found together far more commonly than would be expected by chance, and that certain TS/CYP pairings predominate, providing signals for key events that are likely to have shaped terpene diversity. We recover TS/CYP gene pairs for previously characterized terpene metabolic gene clusters and demonstrate new functional pairing of TSs and CYPs within previously uncharacterized clusters. Unexpectedly, we find evidence for different mechanisms of pathway assembly in eudicots and monocots; in the former, microsyntenic blocks of TS/CYP gene pairs duplicate and provide templates for the evolution of new pathways, whereas in the latter, new pathways arise by mixing and matching of individual TS and CYP genes through dynamic genome rearrangements. This is, to our knowledge, the first documented observation of the unique pattern of TS and CYP assembly in eudicots and monocots.Plants produce a rich and diverse array of specialized metabolites (1, 2). These compounds have important ecological functions, providing protection against pests, diseases, UV-B damage and other environmental stresses, and serve as attractants for pollinators and seed dispersal agents. They are exploited by humans as pharmaceutics, agrochemicals, and in a wide variety of other industrial applications. Metabolic diversification in higher plants is likely to have been driven by the need to adapt and survive in different ecological niches (3, 4). Although a considerable proportion of the genes in higher plant genomes are predicted to encode enzymes with roles in metabolism (∼20% in Arabidopsis thaliana; ref. 5), most of these are as yet uncharacterized. The availability of a growing number of sequenced plant genomes now makes it possible to exploit knowledge extracted from multiple diverse species to take a more holistic approach toward understanding mechanisms of metabolic diversification in plants (1, 2).The terpenes are the largest class of plant-derived natural products, with over 40,000 structures reported to date (6–8). As such they provide an excellent entrée for investigation of mechanisms of metabolic diversification. Terpenes range from simple flavor and fragrance compounds such as limonene and cymene to complex triterpenes, and have numerous potential applications across the food and beverage, pharmaceutical, cosmetic and agriculture industries. They include taxol (one of the most widely prescribed anticancer drugs) and artemisinin (the most potent antimalarial compound). This major class of compounds represents tremendous chemical diversity of which only a relatively small fraction has so far been accessed and used by industry (9). This is because the biosynthetic pathways for the vast majority of these compounds are unknown due to the challenges associated with mining large and complex genomes and establishing the function of genes implicated in specialized metabolism. Many of these genes are divergent members of multigene families, making the delineation of new metabolic pathways extremely difficult (10–13).The primary drivers of terpene diversification are the terpenoid synthase (TS) “signature” enzymes (which generate scaffold diversity), and the cytochrome P450-dependent monooxygenases (CYPs), which modify and further diversify these scaffolds, also paving the way for subsequent downstream modifications (10–15). TSs are defined as the related superfamily of biosynthetic enzymes involved in construction of the basic backbone structure of terpene natural products (16). As such, this includes the trans-isoprenyl diphosphate synthases and squalene synthases (SSs) that form the basic linear chains, as well as terpene synthases (TPSs) and triterpene cyclases (TTCs) that cyclize and rearrange these (16). Our knowledge of how the genes for terpene biosynthetic pathways are organized in plant genomes is limited, because the genomes of plants that produce some of the best characterized terpenoids (e.g., artemisinin and taxol) have not yet been sequenced. However, in a number of cases the genes for terpene biosynthetic pathways have been shown to be organized as metabolic gene clusters (14, 17). These include two diterpene clusters from Oryza sativa (rice) [the momilactone and phytocassane clusters (18, 19)], three triterpene clusters [the thalianol and marneral clusters from A. thaliana (20, 21), the avenacin cluster from Avena strigosa (oat) (22, 23)], and clusters for steroidal glycoalkaloids and other terpenes in the Solanaceae (24, 25). Potential new clusters implicated in terpene synthesis have also been reported in A. thaliana (20, 26–28) and cucumber (29). The available evidence indicates that the characterized clusters have arisen within recent evolutionary history by gene duplication, acquisition of new function and genome reorganization, and that they are not products of horizontal gene transfer from microbes (reviewed in refs. 14, 17, and 30). Clustering has also been shown for other classes of plant natural products and is likely to facilitate coinheritance of beneficial gene combinations and also regulation at the level of chromatin (14, 17, 30–32).TSs and CYPs are the core components of terpene biosynthetic pathways and together are responsible for the generation of a vast array of diverse terpene structures (10–13, 15, 33). Here we have selected these two enzyme superfamilies as markers to investigate the foundations of terpene synthesis and evolution across 17 sequenced plant genomes. Our analyses shed light on the roots of terpene biosynthesis and diversification in plants. They also reveal that different genomic mechanisms of pathway assembly predominate in eudicots and monocots.     

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

Rapid diversification of five Oryza AA genomes associated with rice adaptation

Comparative genomic analyses among closely related species can greatly enhance our understanding of plant gene and genome evolution. We report de novo-assembled AA-genome sequences for Oryza nivara, Oryza glaberrima, Oryza barthii, Oryza glumaepatula, and Oryza meridionalis. Our analyses reveal massive levels of genomic structural variation, including segmental duplication and rapid gene family turnover, with particularly high instability in defense-related genes. We show, on a genomic scale, how lineage-specific expansion or contraction of gene families has led to their morphological and reproductive diversification, thus enlightening the evolutionary process of speciation and adaptation. Despite strong purifying selective pressures on most Oryza genes, we documented a large number of positively selected genes, especially those genes involved in flower development, reproduction, and resistance-related processes. These diversifying genes are expected to have played key roles in adaptations to their ecological niches in Asia, South America, Africa and Australia. Extensive variation in noncoding RNA gene numbers, function enrichment, and rates of sequence divergence might also help account for the different genetic adaptations of these rice species. Collectively, these resources provide new opportunities for evolutionary genomics, numerous insights into recent speciation, a valuable database of functional variation for crop improvement, and tools for efficient conservation of wild rice germplasm.Drawing the landscape of genomic divergence among multiple lineages is fundamental to understanding plant gene and genome evolution (1, 2). The comprehensive comparison of closely related genomes in different chronologically ordered stages under a well-resolved phylogenetic framework could dramatically improve the inference precision and sensitivity of gene evolution studies and should allow more robust results for investigating broad-scale patterns of genomic architecture in the course of the speciation process compared with analyses of single genomes (3, 4). For instance, studies of yeast, Drosophila, and human genomes have demonstrated how comparisons of closely related genome sequences can reveal mechanisms of gene and genome evolution in fungi and animals (5–7). In plants, however, we know little about broad-scale patterns of evolutionary dynamics, differentiation, and consequences. Studies are needed of very closely related plant species that span the speciation continuum and have well-characterized biogeographic histories.The genus Oryza, consisting of 24 species, provides a uniquely powerful system for studying comparative genomics and evolutionary biology, and can contribute to the improvement of rice, which is of pivotal significance in worldwide food production and security (8–10). Many genes involved in rice improvement are derived from wild AA-genome species, and broadening the gene pool of cultivated rice through introgression from other wild relatives of Oryza has attracted increasing attention (11). Phylogenetic analysis of the diploid AA-genome species indicated a closely spaced series of recent speciation events in this genus (12). These species span a wide range of global pantropical regions and are disjunctively distributed in Asia, Africa, Australia, and South America (13). Having diverged less than 3 Mya from a common AA-genome ancestor (12), these eight species have generated extensive adaptive and breeding traits (14, 15).By placing multiple Oryza genome comparisons in a phylogenetic context, previous studies have recorded some of the genomic changes associated with the diversification of the rice genus (16–20). Several studies have compared orthologous genomic segments of Oryza species that represent the different genome types that have diverged over long time scales (18–20). However, none of these analyses provide the full-genome, multispecies perspective that allows comprehensive analysis of gene, genome, or trait evolution.Here, we fully sequenced and assembled de novo the five biogeographically representative AA genomes. In comparison to the high-quality assembly of the O. sativa ssp. japonica cv. Nipponbare (SAT) genome (21), we provide a starting point for studies in the emerging field of comparative and evolutionary genomics. Despite the wealth of phenotypic diversity and adaptive differences, it seems that many attributes of these rice genomes are remarkably conserved across species. Thus, in addition to examining the relationship between sequence and phenotypic diversity, the genomes of these species provide an excellent model for studying, from specific loci to divergent genomic regions, how the evolutionary dynamics of genes and genomes can facilitate speciation processes on a genomic scale. Access to the unprecedented dataset of these Oryza genome sequences will accelerate the pace at which the untapped reservoir of agronomically important genes can be exploited for rice improvement.     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira

Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO₂. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.Nitrification, a key aerobic process of the biogeochemical nitrogen (N) cycle, is catalyzed by two guilds of chemolithoautotrophic microorganisms. The ammonia-oxidizing microorganisms (bacteria and archaea; AOM) oxidize ammonia to nitrite, which is subsequently oxidized to nitrate by nitrite-oxidizing bacteria (NOB). Nitrification links aerobic and anaerobic pathways of the N cycle by providing nitrate or nitrite as electron acceptors for dissimilatory nitrate reduction, denitrification, respiratory ammonification, and anaerobic ammonium oxidation (1, 2). The end product of nitrification, nitrate, is an important source of nitrogen for assimilation by many microorganisms and plants. Moreover, nitrification is a key step of biological wastewater treatment but also contributes to N losses from fertilized agricultural soils (3). Being a two-step process that involves two functional groups, nitrification is a prime example of a tight metabolic interaction between free-living microorganisms.Current insights into the ecology of chemolithoautotrophic NOB suggest that two of the six known NOB genera are restricted to marine ecosystems (Nitrospina and Nitrococcus) (4). The recently identified Nitrolancea was enriched from activated sludge from a wastewater treatment plant (WWTP) (5), whereas Nitrotoga occur in soils and WWTPs (6, 7). Nitrobacter are generally common in terrestrial and limnic habitats. Analyses of Nitrobacter genome sequences provided first insights into the genomic makeup of NOB and revealed a greater metabolic flexibility than anticipated earlier, which included the mixotrophic utilization of various organic substrates (8, 9). However, Nitrobacter require high nitrite concentrations (10, 11), and molecular surveys indicated that Nitrobacter are not the primary NOB in ecosystems with low ambient nitrite levels such as unfertilized soils (12), freshwater habitats (13), and most WWTPs (14).Among all known NOB, the genus Nitrospira appears to be most widespread in different habitat types and is phylogenetically most diverse. Nitrospira are well adapted to low nitrite concentrations (10, 11) and form at least six phylogenetic lineages (15, 16) that are globally distributed in soils (17, 18), the oceans (19), freshwater habitats (20), hot springs (16), and many other oxic habitats (15). In addition, Nitrospira members are the key NOB in most WWTPs (14, 15). Nitrospira are notoriously difficult to culture under laboratory conditions and, hence, despite their ecological and biotechnological importance, little is known about their ecophysiology. Interestingly, not all members of this genus are restricted to nitrite as their sole source of energy and reductant. Some Nitrospira from marine ecosystems or activated sludge can use simple organic substrates, such as pyruvate, formate, and glycerol, for carbon assimilation and probably also as energy sources in addition to CO₂ and nitrite (mixotrophy) (15, 19, 21, 22). Nitrospira moscoviensis even grows by aerobic hydrogen oxidation as an alternative lifestyle outside the N cycle (23). Furthermore, this organism can reduce nitrate with H₂ as an electron donor, but under these conditions, growth was not detected (24).So far, only one study analyzed a fully sequenced Nitrospira genome, which was obtained from N. defluvii (25). This Nitrospira lineage I member had been enriched from a WWTP (26). Here, we analyzed the genome of N. moscovienis representing Nitrospira lineage II, which is the Nitrospira clade most widely distributed in both natural and engineered ecosystems (15). This newly sequenced Nitrospira genome revealed surprising metabolic features that were experimentally confirmed. These findings change the current perception on the interdependence of nitrifiers and demonstrate an unexpected ecophysiological versatility of Nitrospira with contributions to N-cycling processes other than nitrite oxidation.     

Rate,spectrum, and evolutionary dynamics of spontaneous epimutations

Stochastic changes in cytosine methylation are a source of heritable epigenetic and phenotypic diversity in plants. Using the model plant Arabidopsis thaliana, we derive robust estimates of the rate at which methylation is spontaneously gained (forward epimutation) or lost (backward epimutation) at individual cytosines and construct a comprehensive picture of the epimutation landscape in this species. We demonstrate that the dynamic interplay between forward and backward epimutations is modulated by genomic context and show that subtle contextual differences have profoundly shaped patterns of methylation diversity in A. thaliana natural populations over evolutionary timescales. Theoretical arguments indicate that the epimutation rates reported here are high enough to rapidly uncouple genetic from epigenetic variation, but low enough for new epialleles to sustain long-term selection responses. Our results provide new insights into methylome evolution and its population-level consequences.Plant genomes make extensive use of cytosine methylation to control the expression of transposable elements (TEs) and genes (1). Despite its tight regulation, methylation losses or gains at individual cytosines or clusters of cytosines can emerge spontaneously, in an event termed “epimutation” (2, 3). Many examples of segregating epimutations have been documented in experimental and wild populations of plants and in some cases contribute to heritable variation in phenotypes independently of DNA sequence variation (4, 5). These observations have led to much speculation about the role of DNA methylation in plant evolution (6–8), and its potential in breeding programs (9). In the model plant Arabidopsis thaliana, spontaneous methylation changes at CG dinucleotides accumulate in a rapid but nonlinear fashion over generations (2, 3, 10), thus pointing to high forward–backward epimutation rates (11). Precise estimates of these rates are necessary to be able to quantify the long-term dynamics of epigenetic variation under laboratory or natural conditions, and to understand the molecular mechanisms that drive methylome evolution (12–14). Here we combine theoretical modeling with high-resolution methylome analysis of multiple independent A. thaliana mutation accumulation (MA) lines (15), including measurements of methylation changes in continuous generations, to obtain robust estimates of forward and backward epimutation rates.     

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.DNA double-strand breaks (DSBs) are toxic lesions that can be repaired by two major pathways in eukaryotes: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (1). Although HR repairs DSBs in a template-dependent, high-fidelity manner, NHEJ functions to ligate DSB ends together using no or very short (1–4 bp) homology. Recently, a new pathway was identified in eukaryotes, which uses microhomologies (MHs) to repair a DSB and does not require the central proteins used in HR (Rad51, Rad52) or NHEJ (Ku70–Ku80) (2–5). In mammalian cells, this pathway of repair is known as alternative end-joining (Alt-EJ) and is often but not always associated with MHs, whereas in budding yeast, the commensurate pathway, MH-mediated end-joining (MMEJ), will typically use 5–25 bp of MH (6, 7). These pathways are associated with genomic rearrangements, and cancer genomes show evidence of MH-mediated rearrangements (8–12). In addition, eukaryotic genomes contain many dispersed repetitive elements that can lead to genome rearrangements when recombination occurs between them (13–16). Therefore, controlling DSB repair in the human genome, which features a variety of repeats, is especially important given the fact that recombination between repetitive elements has been implicated in genomic instability associated with disease (17–20).The original characterization of Alt-EJ in mammalian cells suggested it did not represent a significant DNA repair pathway and only operated in the absence of functional HR and NHEJ pathways. More recent analyses demonstrate a physiological role of Alt-EJ during DNA repair in the presence of active HR and NHEJ pathways (2, 12, 21, 22). Furthermore, examination of I-SceI–induced translocation junctions in mammalian cells revealed the frequent presence of MHs (23, 24). NHEJ-deficient and p53-null mice develop pro–B-cell lymphomas, and nonreciprocal translocations characterized by small MHs are found at their break point junctions (25–28). Similarly, in human cancers, many translocation break point junctions contain MHs, suggesting a role for Alt-EJ in cancer development (29–31) and resistance to chemotherapy and genetic disease (32–36). Hence, the presence of many short repetitive sequences in the human genome is likely to increase rearrangements mediated by MHs following the creation of a DSB.MMEJ is a distinct DSB repair pathway that operates in the presence of functional NHEJ and HR pathways (10, 37). The genetic requirements of MMEJ are being studied in the model eukaryote Saccharomyces cerevisiae and involve components traditionally considered specific to the NHEJ (Pol λ) and HR (Rad1–Rad10, Rad59, and Mre11–Rad50–Xrs2) pathways (4, 5, 10, 38). Although being clearly independent of the central NHEJ factor Ku70–Ku80 heterodimer (10, 37), the involvement of the key HR factor Rad52 in MMEJ remains uncertain. It has been reported that Rad52 is required for MMEJ repair (4, 10, 38), whereas in another assay system Rad52 suppresses MMEJ repair (37). More recently, it has been proposed that the replication protein A (RPA) regulates pathway choice between HR and MMEJ (37). In addition, several models have been proposed that identify specific pathways that may use MHs for the repair of DNA damage (39–41). Despite current advancements in our understanding of MMEJ, the precise involvement of DNA polymerases in supporting the repair of DSBs using MHs remains poorly understood. DNA polymerase λ (also called Pol4 in budding yeast) and its human homolog Pol λ are considered to be the primary candidates for the DNA polymerases working in NHEJ and MMEJ (4, 5, 42–46). Both genetic and biochemical evidence shows that Pol δ is recruited during HR to extend Rad51-dependent recombination intermediates (47–50). Recent analysis using pol32 mutants (5, 10) implicated the Pol32 subunit of Pol δ in MMEJ. Pol32 and Pol31 were also identified as subunits of the DNA polymerase zeta complex (Pol ζ) (51, 52), but previous analysis showed no effect of rev3 mutants in MMEJ (10). REV3 encodes the catalytic subunit of Pol ζ. However, an involvement of Pol δ had not been demonstrated directly before, and it is possible that Pol32 could act in conjunction with yet another DNA polymerase.Here, we report the development of a series of interchromosomal MMEJ assays in diploid S. cerevisiae to assess the mechanisms underlying the repair of DSBs using varying MHs. We focus on diploid cells, as they represent the natural state of budding yeast, which is a diplontic organism (53). The yeast mating-type switching system represents a mechanism to return haploid yeast as efficiently as possible to diploidy (54). Using a combination of genetic, molecular, and in vivo chromatin immunoprecipitation (ChIP) experiments, we provide compelling evidence for a direct involvement of Pol δ in coordinating with Pol λ in MMEJ in budding yeast.