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1. INTRODUCTION Evolutionary history of species is now inferred from the evolutionary histories of their genomes. Genomes can be viewed as a collection of genes and whole genome evolution is [...], The tree representation as a model for organismal evolution has been in use since before Darwin. However, with the recent unprecedented access to biomolecular data, it has been discovered that, especially in the microbial world, individual genes making up the genome of an organism give rise to different and sometimes conflicting evolutionary tree topologies. This discovery calls into question the notion of a single evolutionary tree for an organism and gives rise to the notion of an evolutionary consensus tree based on the evolutionary patterns of the majority of genes in a genome embedded in a network of gene histories. Here, we discuss an approach to the analysis of genomic data of multiple genomes using bipartition spectral analysis and unsupervised learning. An interesting observation is that genes within genomes that have evolutionary tree topologies, which are in substantial conflict with the evolutionary consensus tree of an organism, point to possible horizontal gene transfer events which often delineate significant evolutionary events.

Gene transfer agents (GTAs) are enigmatic elements that resemble small viruses and are known to be produced during nutritional stress by some bacteria and archaea. The production of GTAs is regulated by quorum sensing, under which a small fraction of the population acts as GTA producers, while the rest becomes GTA recipients. In contrast to canonical viruses, GTAs cannot propagate themselves because they package pieces of the producing cell's genome. In alphaproteobacteria, GTAs are mostly vertically inherited and reside in their hosts' genomes for hundreds of millions of years. While GTAs' ability to transfer genetic material within a population and their long-term preservation suggest an increased fitness of GTA-producing microbes, the associated benefits and type of selection that maintains GTAs are poorly understood. By comparing rates of evolutionary change in GTA genes to the rates in gene families abundantly present across 293 alphaproteobacterial genomes, we detected 59 gene families that likely co-evolve with GTA genes. These gene families are predominantly involved in stress response, DNA repair, and biofilm formation. We hypothesize that biofilm formation enables the physical proximity of GTA-producing cells, limiting GTA-derived benefits only to a group of closely related cells. We further conjecture that the population structure of biofilm-forming sub-populations ensures that the trait of GTA production is maintained despite the inevitable rise of "cheating" genotypes. Because release of GTA particles kills the producing cell, maintenance of GTAs is an exciting example of social evolution in a microbial population.IMPORTANCEGene transfer agents (GTAs) are viruses domesticated by some archaea and bacteria as vehicles for carrying pieces of the host genome. Produced under certain environmental conditions, GTA particles can deliver DNA to neighboring, closely related cells. The function of GTAs remains uncertain. While making GTAs is suicidal for a cell, GTA-encoding genes are widespread in genomes of alphaproteobacteria. Such GTA persistence implies functional benefits but raises questions about how selection maintains this lethal trait. By showing that GTA genes co-evolve with genes involved in stress response, DNA repair, and biofilm formation, we provide support for the hypothesis that GTAs facilitate DNA exchange during the stress conditions and present a model for how GTAs persist in biofilm-forming bacterial populations despite being lethal., Competing Interests: The authors declare no conflict of interest.

The placement of a nonhyperthermophilic order Mesoaciditogales as the earliest branching clade within the Thermotogota phylum challenges the prevailing hypothesis that the last common ancestor of Thermotogota was a hyperthermophile. Yet, given the long branch leading to the only two Mesoaciditogales described to date, the phylogenetic position of the order may be due to the long branch attraction artifact. By testing various models and applying data recoding in phylogenetic reconstructions, we observed that early branching of Mesoaciditogales within Thermotogota is strongly supported by the conserved marker genes assumed to be vertically inherited. However, based on the taxonomic content of 1,181 gene families and a phylogenetic analysis of 721 gene family trees, we also found that a substantial number of Mesoaciditogales genes are more closely related to species from the order Petrotogales. These genes contribute to coenzyme transport and metabolism, fatty acid biosynthesis, genes known to respond to heat and cold stressors, and include many genes of unknown functions. The Petrotogales comprise moderately thermophilic and mesophilic species with similar temperature tolerances to that of Mesoaciditogales. Our findings hint at extensive horizontal gene transfer (HGT) between, or parallel independent gene gains by, the two ecologically similar lineages and suggest that the exchanged genes may be important for adaptation to comparable temperature niches., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Many Archaea produce membrane-spanning lipids that enable life in extreme environments. These isoprenoid glycerol dibiphytanyl glycerol tetraethers (GDGTs) may contain up to eight cyclopentyl and one cyclohexyl ring, where higher degrees of cyclization are associated with more acidic, hotter or energy-limited conditions. Recently, the genes encoding GDGT ring synthases, grsAB, were identified in two Sulfolobaceae; however, the distribution and abundance of grs homologs across environments inhabited by these and related organisms remain a mystery. To address this, we examined the distribution of grs homologs in relation to environmental temperature and pH, from thermal springs across Earth, where sequences derive from metagenomes, metatranscriptomes, single-cell and cultivar genomes. The abundance of grs homologs shows a strong negative correlation to pH, but a weak positive correlation to temperature. Archaeal genomes and metagenome-assembled genomes (MAGs) that carry two or more grs copies are more abundant in low pH springs. We also find grs in 12 archaeal classes, with the most representatives in Thermoproteia, followed by MAGs of the uncultured Korarchaeia, Bathyarchaeia and Hadarchaeia, while several Nitrososphaeria encodes >3 copies. Our findings highlight the key role of grs-catalysed lipid cyclization in archaeal diversification across hot and acidic environments., (© 2023 Applied Microbiology International and John Wiley & Sons Ltd.)

Oceanotoga sp. strain T3B was isolated from an estuarine sinkhole in the Bahamas. Here, we report its complete genome, which is currently the only sequenced genome from the genus Oceanotoga . The genome sequence provides new data for the genus Oceanotoga .

Gene transfer agents (GTAs) are virus-like elements that are encoded by some bacterial and archaeal genomes. The production of GTAs can be induced by carbon depletion and results in host lysis and the release of virus-like particles that contain mostly random fragments of the host DNA. The remaining members of a GTA-producing population act as GTA recipients by producing proteins needed for GTA-mediated DNA acquisition. Here, we detected a codon usage bias toward codons with more readily available tRNAs in the RcGTA-like GTA genes of alphaproteobacterial genomes. Such bias likely improves the translational efficacy during GTA gene expression. While the strength of codon usage bias fluctuates substantially among individual GTA genes and across taxonomic groups, it is especially pronounced in Sphingomonadales , whose members are known to inhabit nutrient-depleted environments. By screening genomes for gene families with trends in codon usage biases similar to those in GTA genes, we found a gene that likely encodes head completion protein in some GTAs where it appeared missing, and 13 genes previously not implicated in the GTA life cycle. The latter genes are involved in various molecular processes, including the homologous recombination and transport of scarce organic matter. Our findings provide insights into the role of selection for translational efficiency in the evolution of GTA genes and outline genes that are potentially involved in the previously hypothesized integration of GTA-delivered DNA into the host genome. IMPORTANCE Horizontal gene transfer (HGT) is a fundamental process that drives evolution of microorganisms. HGT can result in a rapid dissemination of beneficial genes within and among microbial communities and can be achieved via multiple mechanisms. One peculiar HGT mechanism involves viruses "domesticated" by some bacteria and archaea (their hosts). These so-called gene transfer agents (GTAs) are encoded in hosts' genomes, produced under starvation conditions, and cannot propagate themselves as viruses. We show that GTA genes are under selection to improve the efficiency of their translation when the host activates GTA production. The selection is especially pronounced in bacteria that occupy nutrient-depleted environments. Intriguingly, several genes involved in incorporation of DNA into a genome are under similar selection pressure, suggesting that they may facilitate the integration of GTA-delivered DNA into the host genome. Our findings underscore the potential importance of GTAs as a mechanism of HGT under nutrient-limited conditions, which are widespread in microbial habitats.

Morphological and genetic features strongly suggest that gene transfer agents (GTAs) are caudoviricete-derived entities that have evolved in concert with cellular genomes to such a degree that they should not be considered viruses. Indeed, GTA particles resemble caudoviricete virions, but, in contrast to caudoviricetes (or any viruses), GTAs can encapsidate at best only part of their own genomes, are induced solely in small subpopulations of prokaryotic host cells, and are transmitted vertically as part of cellular genomes during replication and division. Therefore, the lifecycles of GTAs are analogous to virus-derived entities found in the parasitoid wasps, which have recently been recognized as non-virus entities and therefore reclassified as viriforms. We evaluated three distinct, independently exapted GTA groups, for which the genetic basis for GTA particle production has been established. Based on the evidence, we outline a classification scheme for these viriforms., (© The Author(s) 2022. Published by Oxford University Press.)

Phylogenomic analyses of bacteria from the phylum Thermotogota have shown extensive lateral gene transfer with distantly related organisms, particularly with Firmicutes. One likely mechanism of such DNA transfer is viruses. However, to date, only three temperate viruses have been characterized in this phylum, all infecting bacteria from the Marinitoga genus. Here we report 17 proviruses integrated into genomes of bacteria belonging to eight Thermotogota genera and induce viral particle production from one of the proviruses. All except an incomplete provirus from Mesotoga fall into two groups based on sequence similarity, gene synteny and taxonomic classification. Proviruses of Group 1 are found in the genera Geotoga, Kosmotoga, Marinitoga, Thermosipho and Mesoaciditoga and are similar to the previously characterized Marinitoga viruses, while proviruses from Group 2 are distantly related to the Group 1 proviruses, have different genome organization and are found in Petrotoga and Defluviitoga. Genes carried by both groups are closely related to Firmicutes and Firmicutes (pro)viruses in phylogenetic analyses. Moreover, one of the groups show evidence of recent gene exchange and may be capable of infecting cells from both phyla. We hypothesize that viruses are responsible for a large portion of the observed gene flow between Firmicutes and Thermotogota., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

All environments including hypersaline ones harbor measurable concentrations of dissolved extracellular DNA (eDNA) that can be utilized by microbes as a nutrient. However, it remains poorly understood which eDNA components are used, and who in a community utilizes it. For this study, we incubated a saltern microbial community with combinations of carbon, nitrogen, phosphorus, and DNA, and tracked the community response in each microcosm treatment via 16S rRNA and rpoB gene sequencing. We show that microbial communities used DNA only as a phosphorus source, and provision of other sources of carbon and nitrogen was needed to exhibit a substantial growth. The taxonomic composition of eDNA in the water column changed with the availability of inorganic phosphorus or supplied DNA, hinting at preferential uptake of eDNA from specific organismal sources. Especially favored for growth was eDNA from the most abundant taxa, suggesting some haloarchaea prefer eDNA from closely related taxa. The preferential eDNA consumption and differential growth under various nutrient availability regimes were associated with substantial shifts in the taxonomic composition and diversity of microcosm communities. Therefore, we conjecture that in salterns the microbial community assembly is driven by the available resources, including eDNA., (© 2021. The Author(s).)

Gene transfer agents (GTAs) are virus-like particles encoded and produced by many bacteria and archaea. Unlike viruses, GTAs package fragments of the host genome instead of the genes that encode the components of the GTA itself. As a result of this non-specific DNA packaging, GTAs can transfer genes within bacterial and archaeal communities. GTAs clearly evolved from viruses and are thought to have been maintained in prokaryotic genomes due to the advantages associated with their DNA transfer capacity. The most-studied GTA is produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), which packages random portions of the host genome at a lower DNA density than usually observed in tailed bacterial viruses. How the DNA packaging properties of RcGTA evolved from those of the ancestral virus remains unknown. To address this question, we reconstructed the evolutionary history of the large subunit of the terminase (TerL), a highly conserved enzyme used by viruses and GTAs to package DNA. We found that RcGTA-like TerLs grouped within viruses that employ the headful packaging strategy. Because distinct mechanisms of viral DNA packaging correspond to differences in the TerL amino acid sequence, our finding suggests that RcGTA evolved from a headful packaging virus. Headful packaging is the least sequence-specific mode of DNA packaging, which would facilitate the switch from packaging of the viral genome to packaging random pieces of the host genome during GTA evolution., (© The Author(s) 2021. Published by Oxford University Press.)

Gene transfer agents (GTAs) are virus-like elements integrated into bacterial genomes, particularly, those of Alphaproteobacteria The GTAs can be induced under conditions of nutritional stress, incorporate random fragments of bacterial DNA into miniphage particles, lyse the host cells, and infect neighboring bacteria, thus enhancing horizontal gene transfer. We show that GTA genes evolve under conditions of pronounced positive selection for the reduction of the energy cost of protein production as shown by comparison of the amino acid compositions with those of both homologous viral genes and host genes. The energy saving in GTA genes is comparable to or even more pronounced than that in the genes encoding the most abundant, essential bacterial proteins. In cases in which viruses acquire genes from GTAs, the bias in amino acid composition disappears in the course of evolution, showing that reduction of the energy cost of protein production is an important factor of evolution of GTAs but not bacterial viruses. These findings strongly suggest that GTAs represent bacterial adaptations rather than selfish, virus-like elements. Because GTA production kills the host cell and does not propagate the GTA genome, it appears likely that the GTAs are retained in the course of evolution via kin or group selection. Therefore, we hypothesize that GTAs facilitate the survival of bacterial populations under energy-limiting conditions through the spread of metabolic and transport capabilities via horizontal gene transfer and increases in nutrient availability resulting from the altruistic suicide of GTA-producing cells. IMPORTANCE Kin selection and group selection remain controversial topics in evolutionary biology. We argue that these types of selection are likely to operate in bacterial populations by showing that bacterial gene transfer agents (GTAs), but not related viruses, evolve under conditions of positive selection for the reduction of the energy cost of GTA particle production. We hypothesize that GTAs are dedicated devices mediating the survival of bacteria under conditions of nutrient limitation. The benefits conferred by GTAs under nutritional stress conditions appear to include horizontal dissemination of genes that could provide bacteria with enhanced capabilities for nutrient utilization and increases of nutrient availability occurring through the lysis of GTA-producing bacteria., (Copyright © 2020 Kogay et al.)

Plants must rearrange the network of complex carbohydrates in their cell walls during normal growth and development. To accomplish this, all plants depend on proteins called expansins that nonenzymatically loosen noncovalent bonding between cellulose microfibrils. Surprisingly, expansin genes have more recently been found in some bacteria and microbial eukaryotes, where their biological functions are largely unknown. Here, we reconstruct a comprehensive phylogeny of microbial expansin genes. We find these genes in all eukaryotic microorganisms that have structural cell wall cellulose, suggesting expansins evolved in ancient marine microorganisms long before the evolution of land plants. We also find expansins in an unexpectedly high diversity of bacteria and fungi that do not have cellulosic cell walls. These bacteria and fungi inhabit varied ecological contexts, mirroring the diversity of terrestrial and aquatic niches where plant and/or algal cellulosic cell walls are present. The microbial expansin phylogeny shows evidence of multiple horizontal gene transfer events within and between bacterial and eukaryotic microbial lineages, which may in part underlie their unusually broad phylogenetic distribution. Overall, expansins are unexpectedly widespread in bacteria and eukaryotes, and the contribution of these genes to microbial ecological interactions with plants and algae has probbaly been underappreciated., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Many of the sequenced bacterial and archaeal genomes encode regions of viral provenance. Yet, not all of these regions encode bona fide viruses. Gene transfer agents (GTAs) are thought to be former viruses that are now maintained in genomes of some bacteria and archaea and are hypothesized to enable exchange of DNA within bacterial populations. In Alphaproteobacteria, genes homologous to the "head-tail" gene cluster that encodes structural components of the Rhodobacter capsulatus GTA (RcGTA) are found in many taxa, even if they are only distantly related to Rhodobacter capsulatus. Yet, in most genomes available in GenBank RcGTA-like genes have annotations of typical viral proteins, and therefore are not easily distinguished from their viral homologs without additional analyses. Here, we report a "support vector machine" classifier that quickly and accurately distinguishes RcGTA-like genes from their viral homologs by capturing the differences in the amino acid composition of the encoded proteins. Our open-source classifier is implemented in Python and can be used to scan homologs of the RcGTA genes in newly sequenced genomes. The classifier can also be trained to identify other types of GTAs, or even to detect other elements of viral ancestry. Using the classifier trained on a manually curated set of homologous viruses and GTAs, we detected RcGTA-like "head-tail" gene clusters in 57.5% of the 1,423 examined alphaproteobacterial genomes. We also demonstrated that more than half of the in silico prophage predictions are instead likely to be GTAs, suggesting that in many alphaproteobacterial genomes the RcGTA-like elements remain unrecognized., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Cyanobacteria of the Prochlorococcus and marine Synechococcus genera are the most abundant photosynthetic microbes in the ocean. Intriguingly, the genomes of these bacteria are strongly divergent even within each genus, both in gene content and at the amino acid level of the encoded proteins. One striking exception to this is a 62-amino-acid protein, termed P rochlorococcus / S ynechococcush yper- c onserved p rotein (PSHCP). PSHCP is not only found in all sequenced Prochlorococcus and marine Synechococcus genomes, but it is also nearly 100% identical in its amino acid sequence across all sampled genomes. Such universal distribution and sequence conservation suggest an essential cellular role of PSHCP in these bacteria. However, its function is unknown. Here, we used NMR spectroscopy to determine its structure, finding that 53 of the 62 amino acids in PSHCP form a Tudor domain, whereas the remainder of the protein is disordered. NMR titration experiments revealed that PSHCP has only a weak affinity for DNA, but an 18.5-fold higher affinity for tRNA, hinting at an involvement of PSHCP in translation. Isothermal titration calorimetry experiments further revealed that PSHCP also binds single-stranded, double-stranded, and hairpin RNAs. These results provide the first insight into the structure and function of PSHCP, suggesting that PSHCP appears to be an RNA-binding protein that can recognize a broad array of RNA molecules., (© 2019 Bauer et al.)

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits, an economically important phytopathogen affecting an economically important phytopathogen affecting few cultivated Cucurbitaceae few cultivated Cucurbitaceae host plant species in temperate eastern North America. However, essentially nothing is known about E. tracheiphila population structure or genetic diversity. To address this shortcoming, a representative collection of 88 E. tracheiphila isolates was gathered from throughout its geographic range, and their genomes were sequenced. Phylogenomic analysis revealed three genetic clusters with distinct hrp T3SS virulence gene repertoires, host plant association patterns, and geographic distributions. Low genetic heterogeneity within each cluster suggests a recent population bottleneck followed by population expansion. We showed that in the field and greenhouse, cucumber ( Cucumis sativus ), which was introduced to North America by early Spanish conquistadors, is the most susceptible host plant species and the only species susceptible to isolates from all three lineages. The establishment of large agricultural populations of highly susceptible C. sativus in temperate eastern North America may have facilitated the original emergence of E. tracheiphila into cucurbit agroecosystems, and this introduced plant species may now be acting as a highly susceptible reservoir host. Our findings have broad implications for agricultural sustainability by drawing attention to how worldwide crop plant movement, agricultural intensification, and locally unique environments may affect the emergence, evolution, and epidemic persistence of virulent microbial pathogens. IMPORTANCE Erwinia tracheiphila is a virulent phytopathogen that infects two genera of cucurbit crop plants, Cucurbita spp. (pumpkin and squash) and Cucumis spp. (muskmelon and cucumber). One of the unusual ecological traits of this pathogen is that it is limited to temperate eastern North America. Here, we complete the first large-scale sequencing of an E. tracheiphila isolate collection. From phylogenomic, comparative genomic, and empirical analyses, we find that introduced Cucumis spp. crop plants are driving the diversification of E. tracheiphila into multiple lineages. Together, the results from this study show that locally unique biotic (plant population) and abiotic (climate) conditions can drive the evolutionary trajectories of locally endemic pathogens in unexpected ways., (Copyright © 2018 Shapiro et al.)

Several bacterial and archaeal lineages produce nanostructures that morphologically resemble small tailed viruses, but, unlike most viruses, contain apparently random pieces of the host genome. Since these elements can deliver the packaged DNA to other cells, they were dubbed gene transfer agents (GTAs). Because many genes involved in GTA production have viral homologs, it has been hypothesized that the GTA ancestor was a virus. Whether GTAs represent an atypical virus, a defective virus, or a virus co-opted by the prokaryotes for some function, remains to be elucidated. To evaluate these possibilities, we examined the distribution and evolutionary histories of genes that encode a GTA in the α-proteobacterium Rhodobacter capsulatus (RcGTA). We report that although homologs of many individual RcGTA genes are abundant across bacteria and their viruses, RcGTA-like genomes are mainly found in one subclade of α-proteobacteria. When compared with the viral homologs, genes of the RcGTA-like genomes evolve significantly slower, and do not have higher %A+T nucleotides than their host chromosomes. Moreover, they appear to reside in stable regions of the bacterial chromosomes that are generally conserved across taxonomic orders. These findings argue against RcGTA being an atypical or a defective virus. Our phylogenetic analyses suggest that RcGTA ancestor likely originated in the lineage that gave rise to contemporary α-proteobacterial orders Rhizobiales , Rhodobacterales , Caulobacterales , Parvularculales , and Sphingomonadales , and since that time the RcGTA-like element has co-evolved with its host chromosomes. Such evolutionary history is compatible with maintenance of these elements by bacteria due to some selective advantage. As for many other prokaryotic traits, horizontal gene transfer played a substantial role in the evolution of RcGTA-like elements, not only in shaping its genome components within the orders, but also in occasional dissemination of RcGTA-like regions across the orders and even to different bacterial phyla.

Temperature is one of the defining parameters of an ecological niche. Most organisms thrive within a temperature range that rarely exceeds ~30 °C, but the deep subsurface bacterium Kosmotoga olearia can grow over a temperature range of 59 °C (20-79 °C). To identify genes correlated with this flexible phenotype, we compared transcriptomes of K. olearia cultures grown at its optimal 65 °C to those at 30, 40, and 77 °C. The temperature treatments affected expression of 573 of 2224 K. olearia genes. Notably, this transcriptional response elicits re-modeling of the cellular membrane and changes in metabolism, with increased expression of genes involved in energy and carbohydrate metabolism at high temperatures and up-regulation of amino acid metabolism at lower temperatures. At sub-optimal temperatures, many transcriptional changes were similar to those observed in mesophilic bacteria at physiologically low temperatures, including up-regulation of typical cold stress genes and ribosomal proteins. Comparative genomic analysis of additional Thermotogae genomes indicates that one of K. olearia's strategies for low-temperature growth is increased copy number of some typical cold response genes through duplication and/or lateral acquisition. At 77 °C one-third of the up-regulated genes are of hypothetical function, indicating that many features of high-temperature growth are unknown.

Whether prokaryotes (Bacteria and Archaea) are naturally organized into phenotypically and genetically cohesive units comparable to animal or plant species remains contested, frustrating attempts to estimate how many such units there might be, or to identify the ecological roles they play. Analyses of gene sequences in various closely related prokaryotic groups reveal that sequence diversity is typically organized into distinct clusters, and processes such as periodic selection and extensive recombination are understood to be drivers of cluster formation ("speciation"). However, observed patterns are rarely compared with those obtainable with simple null models of diversification under stochastic lineage birth and death and random genetic drift. Via a combination of simulations and analyses of core and phylogenetic marker genes, we show that patterns of diversity for the genera Escherichia , Neisseria , and Borrelia are generally indistinguishable from patterns arising under a null model. We suggest that caution should thus be taken in interpreting observed clustering as a result of selective evolutionary forces. Unknown forces do, however, appear to play a role in Helicobacter pylori , and some individual genes in all groups fail to conform to the null model. Taken together, we recommend the presented birth-death model as a null hypothesis in prokaryotic speciation studies. It is only when the real data are statistically different from the expectations under the null model that some speciation process should be invoked., Competing Interests: The authors declare no conflict of interest.

Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell's genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures "repurposed" by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA "genome" consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA "genome" loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA "genome" is presumably a result of selection on the host organism to retain GTA functionality., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Modern industrial agriculture depends on high-density cultivation of genetically similar crop plants, creating favorable conditions for the emergence of novel pathogens with increased fitness in managed compared with ecologically intact settings. Here, we present the genome sequence of six strains of the cucurbit bacterial wilt pathogen Erwinia tracheiphila (Enterobacteriaceae) isolated from infected squash plants in New York, Pennsylvania, Kentucky, and Michigan. These genomes exhibit a high proportion of recent horizontal gene acquisitions, invasion and remarkable amplification of mobile genetic elements, and pseudogenization of approximately 20% of the coding sequences. These genome attributes indicate that E. tracheiphila recently emerged as a host-restricted pathogen. Furthermore, chromosomal rearrangements associated with phage and transposable element proliferation contribute to substantial differences in gene content and genetic architecture between the six E. tracheiphila strains and other Erwinia species. Together, these data lead us to hypothesize that E. tracheiphila has undergone recent evolution through both genome decay (pseudogenization) and genome expansion (horizontal gene transfer and mobile element amplification). Despite evidence of dramatic genomic changes, the six strains are genetically monomorphic, suggesting a recent population bottleneck and emergence into E. tracheiphila's current ecological niche., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Thermophiles are extremophiles that grow optimally at temperatures >45 °C. To survive and maintain function of their biological molecules, they have a suite of characteristics not found in organisms that grow at moderate temperature (mesophiles). At the cellular level, thermophiles have mechanisms for maintaining their membranes, nucleic acids, and other cellular structures. At the protein level, each of their proteins remains stable and retains activity at temperatures that would denature their mesophilic homologs. Conversely, cellular structures and proteins from thermophiles may not function optimally at moderate temperatures. These differences between thermophiles and mesophiles presumably present a barrier for evolutionary transitioning between the 2 lifestyles. Therefore, studying closely related thermophiles and mesophiles can help us determine how such lifestyle transitions may happen. The bacterial phylum Thermotogae contains hyperthermophiles, thermophiles, mesophiles, and organisms with temperature ranges wide enough to span both thermophilic and mesophilic temperatures. Genomic, proteomic, and physiological differences noted between other bacterial thermophiles and mesophiles are evident within the Thermotogae. We argue that the Thermotogae is an ideal group of organisms for understanding of the response to fluctuating temperature and of long-term evolutionary adaptation to a different growth temperature range.

Prokaryotic viruses play a major role in the microbial ecology and evolution. However, the virosphere associated with deep-sea hydrothermal ecosystems remains largely unexplored. Numerous instances of lateral gene transfer have contributed to the complex and incongruent evolutionary history of Thermotogales, an order well represented in deep-sea hydrothermal vents. The presence of clustered regularly interspaced short palindromic repeats (CRISPR) loci has been reported in all Thermotogales genomes, suggesting that these bacteria have been exposed to viral infections that could have mediated gene exchange. In this study, we isolated and characterized the first virus infecting bacteria from the order Thermotogales, Marinitoga piezophila virus 1 (MPV1). The host, Marinitoga piezophila is a thermophilic, anaerobic and piezophilic bacterium isolated from a deep-sea hydrothermal chimney. MPV1 is a temperate Siphoviridae-like virus with a 43.7 kb genome. Surprisingly, we found that MPV1 virions carry not only the viral DNA but preferentially package a plasmid of 13.3 kb (pMP1) also carried by M. piezophila. This 'ménage à trois' highlights potential relevance of selfish genetic elements in facilitating lateral gene transfer in the deep-sea biosphere., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Oil reservoirs represent a nutrient-rich ecological niche of the deep biosphere. Although most oil reservoirs are occupied by microbial populations, when and how the microbes colonized these environments remains unanswered. To address this question, we compared 11 genomes of Thermotoga maritima-like hyperthermophilic bacteria from two environment types: subsurface oil reservoirs in the North Sea and Japan, and marine sites located in the Kuril Islands, Italy and the Azores. We complemented our genomes with Thermotoga DNA from publicly available subsurface metagenomes from North America and Australia. Our analysis revealed complex non-bifurcating evolutionary history of the isolates' genomes, suggesting high amounts of gene flow across all sampled locations, a conjecture supported by numerous recombination events. Genomes from the same type of environment tend to be more similar, and have exchanged more genes with each other than with geographically close isolates from different types of environments. Hence, Thermotoga populations of oil reservoirs do not appear isolated, a requirement of the 'burial and isolation' hypothesis, under which reservoir bacteria are descendants of the isolated communities buried with sediments that over time became oil reservoirs. Instead, our analysis supports a more complex view, where bacteria from subsurface and marine populations have been continuously migrating into the oil reservoirs and influencing their genetic composition. The Thermotoga spp. in the oil reservoirs in the North Sea and Japan probably entered the reservoirs shortly after they were formed. An Australian oil reservoir, on the other hand, was likely colonized very recently, perhaps during human reservoir development.

Erwinia tracheiphila is one of the most economically important pathogens of cucumbers, melons, squashes, pumpkins, and gourds in the northeastern and midwestern United States, yet its molecular pathology remains uninvestigated. Here, we report the first draft genome sequence of an E. tracheiphila strain isolated from an infected wild gourd (Cucurbita pepo subsp. texana) plant. The genome assembly consists of 7 contigs and includes a putative plasmid and at least 20 phage and prophage elements., (Copyright © 2015 Shapiro et al.)

A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.

All oxygenic photoautotrophs suffer photoinactivation of their Photosystem II complexes, at a rate driven by the instantaneous light level. To maintain photosynthesis, PsbA subunits are proteolytically removed from photoinactivated Photosystem II complexes, primarily by a membrane-bound FtsH protease. Diatoms thrive in environments with fluctuating light, such as coastal regions, in part because they enjoy a low susceptibility to photoinactivation of Photosystem II. In a coastal strain of the diatom Thalassiosira pseudonana growing across a range of light levels, active Photosystem II represents only about 42 % of the total Photosystem II protein, with the remainder attributable to photoinactivated Photosystem II awaiting recycling. The rate constant for removal of PsbA protein increases with growth light, in parallel with an increasing content of the FtsH protease relative to the substrate PsbA. An offshore strain of Thalassiosira pseudonana, originating from a more stable light environment, had a lower content of FtsH and slower rate constants for removal of PsbA. We used this data to generate the first estimates for in vivo proteolytic degradation of photoinactivated PsbA per FtsH6 protease, at ~3.9 × 10(-2) s(-1), which proved consistent across growth lights and across the onshore and offshore strains.

Biogeography of microbial populations remains to be poorly understood, and a novel technique of single cell sorting promises a new level of resolution for microbial diversity studies. Using single cell sorting, we compared saturated NaCl brine environments (32-35 %) of the South Bay Salt Works in Chula Vista in California (USA) and Santa Pola saltern near Alicante (Spain). Although some overlap in community composition was detected, both samples were significantly different and included previously undiscovered 16S rRNA sequences. The community from Chula Vista saltern had a large bacterial fraction, which consisted of diverse Bacteroidetes and Proteobacteria. In contrast, Archaea dominated Santa Pola's community and its bacterial fraction consisted of the previously known Salinibacter lineages. The recently reported group of halophilic Archaea, Nanohaloarchaea, was detected at both sites. We demonstrate that cell sorting is a useful technique for analysis of halophilic microbial communities, and is capable of identifying yet unknown or divergent lineages. Furthermore, we argue that observed differences in community composition reflect restricted dispersal between sites, a likely mechanism for diversification of halophilic microorganisms.

Horizontal gene transfer is important in the evolution of bacterial and archaeal genomes. An interesting genetic exchange process is carried out by diverse phage-like gene transfer agents (GTAs) that are found in a wide range of prokaryotes. Although GTAs resemble phages, they lack the hallmark capabilities that define typical phages, and they package random pieces of the producing cell's genome. In this Review, we discuss the defining characteristics of the GTAs that have been identified to date, along with potential functions for these agents and the possible evolutionary forces that act on the genes involved in their production.

Background: The frequent exchange of genetic material among prokaryotes means that extracting a majority or plurality phylogenetic signal from many gene families, and the identification of gene families that are in significant conflict with the plurality signal is a frequent task in comparative genomics, and especially in phylogenomic analyses. Decomposition of gene trees into embedded quartets (unrooted trees each with four taxa) is a convenient and statistically powerful technique to address this challenging problem. This approach was shown to be useful in several studies of completely sequenced microbial genomes., Results: We present here a web server that takes a collection of gene phylogenies, decomposes them into quartets, generates a Quartet Spectrum, and draws a split network. Users are also provided with various data download options for further analyses. Each gene phylogeny is to be represented by an assessment of phylogenetic information content, such as sets of trees reconstructed from bootstrap replicates or sampled from a posterior distribution. The Quartet Decomposition server is accessible at http://quartets.uga.edu., Conclusions: The Quartet Decomposition server presented here provides a convenient means to perform Quartet Decomposition analyses and will empower users to find statistically supported phylogenetic conflicts.

Prochlorococcus is a genus of marine cyanobacteria characterized by small cell and genome size, an evolutionary trend toward low GC content, the possession of chlorophyll b, and the absence of phycobilisomes. Whereas many shared derived characters define Prochlorococcus as a clade, many genome-based analyses recover them as paraphyletic, with some low-light adapted Prochlorococcus spp. grouping with marine Synechococcus. Here, we use 18 Prochlorococcus and marine Synechococcus genomes to analyze gene flow within and between these taxa. We introduce embedded quartet scatter plots as a tool to screen for genes whose phylogeny agrees or conflicts with the plurality phylogenetic signal, with accepted taxonomy and naming, with GC content, and with the ecological adaptation to high and low light intensities. We find that most gene families support high-light adapted Prochlorococcus spp. as a monophyletic clade and low-light adapted Prochlorococcus sp. as a paraphyletic group. But we also detect 16 gene families that were transferred between high-light adapted and low-light adapted Prochlorococcus sp. and 495 gene families, including 19 ribosomal proteins, that do not cluster designated Prochlorococcus and Synechococcus strains in the expected manner. To explain the observed data, we propose that frequent gene transfer between marine Synechococcus spp. and low-light adapted Prochlorococcus spp. has created a "highway of gene sharing" (Beiko RG, Harlow TJ, Ragan MA. 2005. Highways of gene sharing in prokaryotes. Proc Natl Acad Sci USA. 102:14332-14337) that tends to erode genus boundaries without erasing the Prochlorococcus-specific ecological adaptations.

The notion that all prokaryotes belong to genomically and phenomically cohesive clusters that we might legitimately call "species" is a contentious one. At issue are (1) whether such clusters actually exist; (2) what species definition might most reliably identify them, if they do; and (3) what species concept -- by which is meant a genetic and ecological theory of speciation -- might best explain species existence and rationalize a species definition, if we could agree on one. We review existing theories and some relevant data. We conclude that microbiologists now understand in some detail the various genetic, population, and ecological processes that effect the evolution of prokaryotes. There will be on occasion circumstances under which these, working together, will form groups of related organisms sufficiently like each other that we might all agree to call them "species," but there is no reason that this must always be so. Thus, there is no principled way in which questions about prokaryotic species, such as how many there are, how large their populations are, or how globally they are distributed, can be answered. These questions can, however, be reformulated so that metagenomic methods and thinking will meaningfully address the biological patterns and processes whose understanding is our ultimate target.

Since publication of the first Thermotogales genome, Thermotoga maritima strain MSB8, single- and multi-gene analyses have disagreed on the phylogenetic position of this order of Bacteria. Here we present the genome sequences of 4 additional members of the Thermotogales (Tt. petrophila, Tt. lettingae, Thermosipho melanesiensis, and Fervidobacterium nodosum) and a comprehensive comparative analysis including the original T. maritima genome. While ribosomal protein genes strongly place Thermotogales as a sister group to Aquificales, the majority of genes with sufficient phylogenetic signal show affinities to Archaea and Firmicutes, especially Clostridia. Indeed, on the basis of the majority of genes in their genomes (including genes that are also found in Aquificales), Thermotogales should be considered members of the Firmicutes. This result highlights the conflict between the taxonomic goal of assigning every species to a unique position in an inclusive Linnaean hierarchy and the evolutionary goal of understanding phylogenesis in the presence of pervasive horizontal gene transfer (HGT) within prokaryotes. Amino acid compositions of reconstructed ancestral sequences from 423 gene families suggest an origin of this gene pool even more thermophilic than extant members of this order, followed by adaptation to lower growth temperatures within the Thermotogales.

Lateral gene transfers (LGT) (also called horizontal gene transfers) have been a major force shaping the Thermosipho africanus TCF52B genome, whose sequence we describe here. Firmicutes emerge as the principal LGT partner. Twenty-six percent of phylogenetic trees suggest LGT with this group, while 13% of the open reading frames indicate LGT with Archaea.

This chapter discusses the pros and cons of the existing computational methods for the detection of horizontal (or lateral) gene transfer and highlights the genome-wide studies utilizing these methods. The impact of horizontal gene transfer (HGT) on prokaryote genome evolution is discussed.

Proteorhodopsins are light-energy-harvesting transmembrane proteins encoded by genes recently discovered in the surface waters of the world's oceans. Metagenomic data from the Global Ocean Sampling expedition (GOS) recovered 2674 proteorhodopsin-related sequences from 51 aquatic samples. Four of these samples were from non-marine environments, specifically, Lake Gatun within the Panama Canal, Delaware Bay and Chesapeake Bay and the Punta Cormorant Lagoon in Ecuador. Rhodopsins related to but phylogenetically distinct from most sequences designated proteorhodopsins were present at all four of these non-marine sites and comprised three different clades that were almost completely absent from marine samples. Phylogenomic analyses of genes adjacent to those encoding these novel rhodopsins suggest affiliation to the Actinobacteria, and hence we propose to name these divergent, non-marine rhodopsins 'actinorhodopsins'. Actinorhodopsins conserve the acidic amino acid residues critical for proton pumping and their genes lack genomic association with those encoding photo-sensory transducer proteins, thus supporting a putative ion pumping function. The ratio of recA and radA to rhodopsin genes in the different environment types sampled within the GOS indicates that rhodopsins of one type or another are abundant in microbial communities in freshwater, estuarine and lagoon ecosystems, supporting an important role for these photosystems in all aquatic environments influenced by sunlight.

The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of approximately 3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling (Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins.

An ORF (encoding a hyperconserved protein of unknown function) in Prochlorococcus/marine Synechococcus genomes that is 100% conserved on the amino acid level but lacks homologs outside this group is described here. Such conservation of an uncharacterized group-specific gene is unusual, and unexpected given the extensive genomic divergence of this group at other loci. Comparative analyses indicate that the hyperconserved protein, which may be involved in interactions with nucleic acids, is under stabilizing selection and has resided in these genomes since the last common ancestor of the group.

Prokaryotic (bacterial and archaeal) species definitions and the biological concepts that underpin them entail clustering (cohesion) among individuals, in terms of genome content and gene sequence similarity. Homologous recombination can maintain gene sequence similarity within, while permitting divergence between, clusters and is thus the basis for recent efforts to apply the Biological Species Concept in prokaryote systematics and ecology. In this study, we examine isolates of the haloarchaeal genus Halorubrum from two adjacent ponds of different salinities at a Spanish saltern and a natural saline lake in Algeria by using multilocus sequence analysis. We show that, although clusters can be defined by concatenation of multiple marker sequences, barriers to exchange between them are leaky. We suggest that no nonarbitrary way to circumscribe "species" is likely to emerge for this group, or by extension, to apply generally across prokaryotes. Arbitrary criteria might have limited practical use, but still must be agreed upon by the community.

A recent paper, which will surely figure centrally in the debate between evolutionists and Intelligent Design creationists, proposes a (perhaps too simple) scheme for the evolution of bacterial flagella.

The variability in genome content among closely related strains of prokaryotes has been one of the most remarkable discoveries of genomics. One way to approach the description of this so-called pan-genome is to compare one reference strain genome with metagenomic sequences from the environment. We have applied this approach to one extreme aquatic habitat, saturated brines in a solar saltern. The genome of Haloquadratum walsbyi strain DSM 16790 was compared to an environmental metagenome obtained from the exact site of its isolation. This approach revealed that some regions of the strain genome were scarcely represented in the metagenome. Here we have analyzed these genomic islands (GI) in the genome of DSM 16790 and compared them with the complete sequence of some fosmids from the environmental library. Two of the islands, GI 2 and GI 4, overlapped with two large guanine and cytosine (GC)-rich regions that showed evidence of high variability through mobile elements. GI 3 seemed to be a phage or phage-remnant acquired by the reference genome, but not present in most environmental lineages. Most differential gene content was related to small molecule transport and detection, probably reflecting adaptation to different pools of organic nutrients. GI 1 did not possess traces of mobile elements and had normal GC content. This island contained the main cluster of cell envelope glycoproteins and the variability found was different from the other GIs. Rather than containing different genes it consisted of homologs with low similarity. This variation might reflect a phage evasion strategy.