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Person-centered care and user involvement have gained focus in the public welfare agenda, as well as in the health sector. However, it has become clear that there is a bias in favor of resourceful citizens in the organisational user involvement. This bias can enhance disparities in health and social care. In this review, reasons that may underlie the bias in organisational user involvement are elucidated, and examples of ways to create more inclusive user involvement are given., (Published under Open Access CC-BY-NC-BD 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/.)

Mutualistic bacteria and mitochondria have small genomes that harbor host-essential genes. A major question is why a distinct bacterial or mitochondrial genome is needed to encode these functions. The dual location of genes demand two sets of information processing systems, coordination of gene expression and elaborate transport systems. A simpler solution would be to harbor all genes in a single genome. Functional gene transfers to the host nuclear genome is uncommon in mutualistic bacteria and lost gene functions are rather rescued by co-symbiotic bacteria. Recent findings suggest that the mitochondrial genome is retained to avoid conflicting signals between protein targeting pathways in the cell. However, if the selective pressure for oxygenic respiration is lost, the mitochondrial genome will start to deteriorate and soon be lost., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Gut-associated microbiota of ants include Rhizobiales bacteria with affiliation to the genus Bartonella. These bacteria may enable the ants to fix atmospheric nitrogen, but no genomes have been sequenced yet to test the hypothesis. Sequence reads from a member of the Rhizobiales were identified in the data collected in a genome project of the ant Harpegnathos saltator. We present an analysis of the closed 1.86 Mb genome of the ant-associated bacterium, for which we suggest the species name Candidatus Tokpelaia hoelldoblerii. A phylogenetic analysis reveals a relationship to Bartonella and Brucella, which infect mammals. Novel gene acquisitions include a gene for a putative extracellular protein of more than 6,000 amino acids secreted by the type I secretion system, which may be involved in attachment to the gut epithelium. No genes for nitrogen fixation could be identified, but genes for a multi-subunit urease protein complex are present in the genome. The urease genes are also present in Brucella, which has a fecal-oral transmission pathway, but not in Bartonella, which use blood-borne transmission pathways. We hypothesize that the gain and loss of the urease function is related to transmission strategies and lifestyle changes in the host-associated members of the Rhizobiales.

Most free-living planktonic cells are streamlined and in spite of their limitations in functional flexibility, their vast populations have radiated into a wide range of aquatic habitats. Here we compared the metabolic potential of subgroups in the Alphaproteobacteria lineage SAR11 adapted to marine and freshwater habitats. Our results suggest that the successful leap from marine to freshwaters in SAR11 was accompanied by a loss of several carbon degradation pathways and a rewiring of the central metabolism. Examples for these are C1 and methylated compounds degradation pathways, the Entner-Doudouroff pathway, the glyoxylate shunt and anapleuretic carbon fixation being absent from the freshwater genomes. Evolutionary reconstructions further suggest that the metabolic modules making up these important freshwater metabolic traits were already present in the gene pool of ancestral marine SAR11 populations. The loss of the glyoxylate shunt had already occurred in the common ancestor of the freshwater subgroup and its closest marine relatives, suggesting that the adaptation to freshwater was a gradual process. Furthermore, our results indicate rapid evolution of TRAP transporters in the freshwater clade involved in the uptake of low molecular weight carboxylic acids. We propose that such gradual tuning of metabolic pathways and transporters toward locally available organic substrates is linked to the formation of subgroups within the SAR11 clade and that this process was critical for the freshwater clade to find and fix an adaptive phenotype.

The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of 'Candidatus Arcanobacter lacustris', a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial 'Candidatus Arcanobacter lacustris' genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular.

Mitochondria are energy-producing organelles in eukaryotic cells considered to be of bacterial origin. The mitochondrial genome has evolved under selection for minimization of gene content, yet it is not known why not all mitochondrial genes have been transferred to the nuclear genome. Here, we predict that hydrophobic membrane proteins encoded by the mitochondrial genomes would be recognized by the signal recognition particle and targeted to the endoplasmic reticulum if they were nuclear-encoded and translated in the cytoplasm. Expression of the mitochondrially encoded proteins Cytochrome oxidase subunit 1, Apocytochrome b, and ATP synthase subunit 6 in the cytoplasm of HeLa cells confirms export to the endoplasmic reticulum. To examine the extent to which the mitochondrial proteome is driven by selective constraints within the eukaryotic cell, we investigated the occurrence of mitochondrial protein domains in bacteria and eukaryotes. The accessory protein domains of the oxidative phosphorylation system are unique to mitochondria, indicating the evolution of new protein folds. Most of the identified domains in the accessory proteins of the ribosome are also found in eukaryotic proteins of other functions and locations. Overall, one-third of the protein domains identified in mitochondrial proteins are only rarely found in bacteria. We conclude that the mitochondrial genome has been maintained to ensure the correct localization of highly hydrophobic membrane proteins. Taken together, the results suggest that selective constraints on the eukaryotic cell have played a major role in modulating the evolution of the mitochondrial genome and proteome.

Lactobacillus kunkeei is the most abundant bacterial species in the honey crop and food products of honeybees. The 16 S rRNA genes of strains isolated from different bee species are nearly identical in sequence and therefore inadequate as markers for studies of coevolutionary patterns. Here, we have compared the 1.5 Mb genomes of ten L. kunkeei strains isolated from all recognized Apis species and another two strains from Meliponini species. A gene flux analysis, including previously sequenced Lactobacillus species as outgroups, indicated the influence of reductive evolution. The genome architecture is unique in that vertically inherited core genes are located near the terminus of replication, whereas genes for secreted proteins and putative host-adaptive traits are located near the origin of replication. We suggest that these features have resulted from a genome-wide loss of genes, with integrations of novel genes mostly occurring in regions flanking the origin of replication. The phylogenetic analyses showed that the bacterial topology was incongruent with the host topology, and that strains of the same microcluster have recombined frequently across the host species barriers, arguing against codiversification. Multiple genotypes were recovered in the individual hosts and transfers of mobile elements could be demonstrated for strains isolated from the same host species. Unlike other bacteria with small genomes, short generation times and multiple rRNA operons suggest that L. kunkeei evolves under selection for rapid growth in its natural growth habitat. The results provide an extended framework for reductive genome evolution and functional genome organization in bacteria., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera., Results: Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations., Conclusions: The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Background: Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome., Results: Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F)., Conclusions: This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.

Little is known about the diversity and structuring of freshwater microbial communities beyond the patterns revealed by tracing their distribution in the landscape with common taxonomic markers such as the ribosomal RNA. To address this gap in knowledge, metagenomes from temperate lakes were compared to selected marine metagenomes. Taxonomic analyses of rRNA genes in these freshwater metagenomes confirm the previously reported dominance of a limited subset of uncultured lineages of freshwater bacteria, whereas Archaea were rare. Diversification into marine and freshwater microbial lineages was also reflected in phylogenies of functional genes, and there were also significant differences in functional beta-diversity. The pathways and functions that accounted for these differences are involved in osmoregulation, active transport, carbohydrate and amino acid metabolism. Moreover, predicted genes orthologous to active transporters and recalcitrant organic matter degradation were more common in microbial genomes from oligotrophic versus eutrophic lakes. This comparative metagenomic analysis allowed us to formulate a general hypothesis that oceanic- compared with freshwater-dwelling microorganisms, invest more in metabolism of amino acids and that strategies of carbohydrate metabolism differ significantly between marine and freshwater microbial communities., (© 2013 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs (mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

SAR11 is one of the most abundant bacterioplanktons in the upper surface waters of the oceans. In a recent issue of The ISME Journal, Thrash and colleagues present the genomes of four single SAR11 cells isolated from the deep oceans that are enriched in genes for membrane biosynthetic functions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt- and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet., Results: We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules., Conclusions: We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host.

The multiple displacement amplification method has revolutionized genomic studies of uncultured bacteria, where the extraction of pure DNA in sufficient quantity for next-generation sequencing is challenging. However, the method is problematic in that it amplifies the target DNA unevenly, induces the formation of chimeric reads and also amplifies contaminating DNA. Here, we have tested the reproducibility of the multiple displacement amplification method using serial dilutions of extracted genomic DNA and intact cells from the cultured endosymbiont Bartonella australis. The amplified DNA was sequenced with the Illumina sequencing technology, and the results were compared to sequence data obtained from unamplified DNA in this study as well as from a previously published genome project. We show that artifacts such as the extent of the amplification bias, the percentage of chimeric reads and the relative fraction of contaminating DNA increase dramatically for the smallest amounts of template DNA. The pattern of read coverage was reproducibly obtained for samples with higher amounts of template DNA, suggesting that the bias is non-random and genome-specific. A re-analysis of previously published sequence data obtained after amplification from clonal endosymbiont populations confirmed these predictions. We conclude that many of the artifacts associated with the use of the multiple displacement amplification method can be alleviated or much reduced by using multiple cells as the template for the amplification. These findings should be particularly useful for researchers studying the genomes of endosymbionts and other uncultured bacteria, for which a small clonal population of cells can be isolated.

SAR11 is a globally abundant group of Alphaproteobacteria in the oceans that is taxonomically not well defined. It has been suggested SAR11 should be classified into the novel order Pelagibacterales. Features such as conservation of gene content and synteny have been taken as evidence that also the divergent member HIMB59 should be included in the order. However, this proposition is controversial since phylogenetic analyses have questioned the monophyly of this grouping. Here, we performed phylogenetic analyses and reinvestigated the genomic similarity of SAR11 and HIMB59. Our phylogenetic analysis confirmed that HIMB59 is not a sister group to the other SAR11 strains. By placing the comparison in the context of the evolution of the Alphaproteobacteria, we found that none of the measures of genomic similarity supports a clustering of HIMB59 and SAR11 to the exclusion of other Alphaproteobacteria. First, pairwise sequence similarity measures for the SAR11 and HIMB59 genomes were within the range observed for unrelated pairs of Alphaproteobacteria. Second, pairwise comparisons of gene contents revealed a higher similarity of SAR11 to several other alphaproteobacterial genomes than to HIMB59. Third, the SAR11 genomes are not more similar in gene order to the HIMB59 genome than what they are to several other alphaproteobacterial genomes. Finally, in contrast to earlier reports, we observed no sequence similarity between the hypervariable region HVR2 in the SAR11 genomes and the region located at the corresponding position in the HIMB59 genome. Based on these observations, we conclude that the alphaproteobacterium HIMB59 is not monophyletic with the SAR11 strains and that genome streamlining has evolved multiple times independently in Alphaproteobacteria adapted to the upper surface waters of the oceans.

The sequencing of highly virulent Escherichia coli O104:H4 strains isolated during the outbreak of bloody diarrhea and hemolytic uremic syndrome in Europe in 2011 revealed a genome that contained a Shiga toxin encoding prophage and a plasmid encoding enteroaggregative fimbriae. Here, we present the draft genome sequence of a strain isolated in Sweden from a patient who had travelled to Tunisia in 2010 (E112/10) and was found to differ from the outbreak strains by only 38 SNPs in non-repetitive regions, 16 of which were mapped to the branch to the outbreak strain. We identified putatively adaptive mutations in genes for transporters, outer surface proteins and enzymes involved in the metabolism of carbohydrates. A comparative analysis with other historical strains showed that E112/10 contained Shiga toxin prophage genes of the same genotype as the outbreak strain, while these genes have been replaced by a different genotype in two otherwise very closely related strains isolated in the Republic of Georgia in 2009. We also present the genome sequences of two enteroaggregative E. coli strains affiliated with phylogroup A (C43/90 and C48/93) that contain the agg genes for the AAF/I-type fimbriae characteristic of the outbreak population. Interestingly, C43/90 also contained a tet/mer antibiotic resistance island that was nearly identical in sequence to that of the outbreak strain, while the corresponding island in the Georgian strains was most similar to E. coli strains of other serotypes. We conclude that the pan-genome of the outbreak population is shared with strains of the A phylogroup and that its evolutionary history is littered with gene replacement events, including most recently independent acquisitions of antibiotic resistance genes in the outbreak strains and its nearest neighbors. The results are summarized in a refined evolutionary model for the emergence of the O104:H4 outbreak population.

Background: The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized., Principal Findings: Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences., Conclusions/significance: Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

The importance of host-specialization to speciation processes in obligate host-associated bacteria is well known, as is also the ability of recombination to generate cohesion in bacterial populations. However, whether divergent strains of highly recombining intracellular bacteria, such as Wolbachia, can maintain their genetic distinctness when infecting the same host is not known. We first developed a protocol for the genome sequencing of uncultivable endosymbionts. Using this method, we have sequenced the complete genomes of the Wolbachia strains wHa and wNo, which occur as natural double infections in Drosophila simulans populations on the Seychelles and in New Caledonia. Taxonomically, wHa belong to supergroup A and wNo to supergroup B. A comparative genomics study including additional strains supported the supergroup classification scheme and revealed 24 and 33 group-specific genes, putatively involved in host-adaptation processes. Recombination frequencies were high for strains of the same supergroup despite different host-preference patterns, leading to genomic cohesion. The inferred recombination fragments for strains of different supergroups were of short sizes, and the genomes of the co-infecting Wolbachia strains wHa and wNo were not more similar to each other and did not share more genes than other A- and B-group strains that infect different hosts. We conclude that Wolbachia strains of supergroup A and B represent genetically distinct clades, and that strains of different supergroups can co-exist in the same arthropod host without converging into the same species. This suggests that the supergroups are irreversibly separated and that barriers other than host-specialization are able to maintain distinct clades in recombining endosymbiont populations. Acquiring a good knowledge of the barriers to genetic exchange in Wolbachia will advance our understanding of how endosymbiont communities are constructed from vertically and horizontally transmitted genes., Competing Interests: The authors have declared that no competing interests exist.

Gene transfer agents (GTAs) randomly transfer short fragments of a bacterial genome. A novel putative GTA was recently discovered in the mouse-infecting bacterium Bartonella grahamii. Although GTAs are widespread in phylogenetically diverse bacteria, their role in evolution is largely unknown. Here, we present a comparative analysis of 16 Bartonella genomes ranging from 1.4 to 2.6 Mb in size, including six novel genomes from Bartonella isolated from a cow, two moose, two dogs, and a kangaroo. A phylogenetic tree inferred from 428 orthologous core genes indicates that the deadly human pathogen B. bacilliformis is related to the ruminant-adapted clade, rather than being the earliest diverging species in the genus as previously thought. A gene flux analysis identified 12 genes for a GTA and a phage-derived origin of replication as the most conserved innovations. These are located in a region of a few hundred kb that also contains 8 insertions of gene clusters for type III, IV, and V secretion systems, and genes for putatively secreted molecules such as cholera-like toxins. The phylogenies indicate a recent transfer of seven genes in the virB gene cluster for a type IV secretion system from a cat-adapted B. henselae to a dog-adapted B. vinsonii strain. We show that the B. henselae GTA is functional and can transfer genes in vitro. We suggest that the maintenance of the GTA is driven by selection to increase the likelihood of horizontal gene transfer and argue that this process is beneficial at the population level, by facilitating adaptive evolution of the host-adaptation systems and thereby expansion of the host range size. The process counters gene loss and forces all cells to contribute to the production of the GTA and the secreted molecules. The results advance our understanding of the role that GTAs play for the evolution of bacterial genomes., Competing Interests: The authors have declared that no competing interests exist.

Ankyrin repeat domain-encoding genes are common in the eukaryotic and viral domains of life, but they are rare in bacteria, the exception being a few obligate or facultative intracellular Proteobacteria species. Despite having a reduced genome, the arthropod strains of the alphaproteobacterium Wolbachia contain an unusually high number of ankyrin repeat domain-encoding genes ranging from 23 in wMel to 60 in wPip strain. This group of genes has attracted considerable attention for their astonishing large number as well as for the fact that ankyrin proteins are known to participate in protein-protein interactions, suggesting that they play a critical role in the molecular mechanism that determines host-Wolbachia symbiotic interactions. We present a comparative evolutionary analysis of the wMel-related ankyrin repeat domain-encoding genes present in different Drosophila-Wolbachia associations. Our results show that the ankyrin repeat domain-encoding genes change in size by expansion and contraction mediated by short directly repeated sequences. We provide examples of intra-genic recombination events and show that these genes are likely to be horizontally transferred between strains with the aid of bacteriophages. These results confirm previous findings that the Wolbachia genomes are evolutionary mosaics and illustrate the potential that these bacteria have to generate diversity in proteins potentially involved in the symbiotic interactions.

Background: Rates of recombination vary by three orders of magnitude in bacteria but the reasons for this variation is unclear. We performed a genome-wide study of recombination rate variation among genes in the intracellular bacterium Bartonella henselae, which has among the lowest estimated ratio of recombination relative to mutation in prokaryotes., Results: The 1.9 Mb genomes of B. henselae strains IC11, UGA10 and Houston-1 genomes showed only minor gene content variation. Nucleotide sequence divergence levels were less than 1% and the relative rate of recombination to mutation was estimated to 1.1 for the genome overall. Four to eight segments per genome presented significantly enhanced divergences, the most pronounced of which were the virB and trw gene clusters for type IV secretion systems that play essential roles in the infection process. Consistently, multiple recombination events were identified inside these gene clusters. High recombination frequencies were also observed for a gene putatively involved in iron metabolism. A phylogenetic study of this gene in 80 strains of Bartonella quintana, B. henselae and B. grahamii indicated different population structures for each species and revealed horizontal gene transfers across Bartonella species with different host preferences., Conclusions: Our analysis has shown little novel gene acquisition in B. henselae, indicative of a closed pan-genome, but higher recombination frequencies within the population than previously estimated. We propose that the dramatically increased fixation rate for recombination events at gene clusters for type IV secretion systems is driven by selection for sequence variability.

The SAR11 clade, here represented by Candidatus Pelagibacter ubique, is the most successful group of bacteria in the upper surface waters of the oceans. In contrast to previous studies that have associated the 1.3 Mb genome of Ca. Pelagibacter ubique with the less than 1.5 Mb genomes of the Rickettsiales, our phylogenetic analysis suggests that Ca. Pelagibacter ubique is most closely related to soil and aquatic Alphaproteobacteria with large genomes. This implies that the SAR11 clade and the Rickettsiales have undergone genome reduction independently. A gene flux analysis of 46 representative alphaproteobacterial genomes indicates the loss of more than 800 genes in each of Ca. Pelagibacter ubique and the Rickettsiales. Consistent with their different phylogenetic affiliations, the pattern of gene loss differs with a higher loss of genes for repair and recombination processes in Ca. Pelagibacter ubique as compared with a more extensive loss of genes for biosynthetic functions in the Rickettsiales. Some of the lost genes in Ca. Pelagibacter ubique, such as mutLS, recFN, and ruvABC, are conserved in all other alphaproteobacterial genomes including the small genomes of the Rickettsiales. The mismatch repair genes mutLS are absent from all currently sequenced SAR11 genomes and also underrepresented in the global ocean metagenome data set. We hypothesize that the unique loss of genes involved in repair and recombination processes in Ca. Pelagibacter ubique has been driven by selection and that this helps explain many of the characteristics of the SAR11 population, such as the streamlined genomes, the long branch lengths, the high recombination frequencies, and the extensive sequence divergence within the population.

The significance of introgression as an evolutionary force shaping natural populations is well established, especially in animal and plant systems. However, the abundance and size of introgression tracts, and to what degree interspecific gene flow is the result of adaptive processes, are largely unknown. In this study, we present medium coverage genomic data from species of the filamentous ascomycete Neurospora, and we use comparative genomics to investigate the introgression landscape at the genomic level in this model genus. We revealed one large introgression tract in each of the three investigated phylogenetic lineages of Neurospora tetrasperma (sizes of 5.6 Mbp, 5.2 Mbp, and 4.1 Mbp, respectively). The tract is located on the chromosome containing the locus conferring sexual identity, the mating-type (mat) chromosome. The region of introgression is confined to the region of suppressed recombination and is found on one of the two mat chromosomes (mat a). We used Bayesian concordance analyses to exclude incomplete lineage sorting as the cause for the observed pattern, and multilocus genealogies from additional species of Neurospora show that the introgression likely originates from two closely related, freely recombining, heterothallic species (N. hispaniola and N. crassa/N. perkinsii). Finally, we investigated patterns of molecular evolution of the mat chromosome in Neurospora, and we show that introgression is correlated with reduced level of molecular degeneration, consistent with a shorter time of recombination suppression. The chromosome specific (mat) and allele specific (mat a) introgression reported herein comprise the largest introgression tracts reported to date from natural populations. Furthermore, our data contradicts theoretical predictions that introgression should be less likely on sex-determining chromosomes. Taken together, the data presented herein advance our general understanding of introgression as a force shaping eukaryotic genomes., Competing Interests: The authors have declared that no competing interests exist.

Background: According to the endosymbiont hypothesis, the mitochondrial system for aerobic respiration was derived from an ancestral Alphaproteobacterium. Phylogenetic studies indicate that the mitochondrial ancestor is most closely related to the Rickettsiales. Recently, it was suggested that Candidatus Pelagibacter ubique, a member of the SAR11 clade that is highly abundant in the oceans, is a sister taxon to the mitochondrial-Rickettsiales clade. The availability of ocean metagenome data substantially increases the sampling of Alphaproteobacteria inhabiting the oxygen-containing waters of the oceans that likely resemble the originating environment of mitochondria., Methodology/principal Findings: We present a phylogenetic study of the origin of mitochondria that incorporates metagenome data from the Global Ocean Sampling (GOS) expedition. We identify mitochondrially related sequences in the GOS dataset that represent a rare group of Alphaproteobacteria, designated OMAC (Oceanic Mitochondria Affiliated Clade) as the closest free-living relatives to mitochondria in the oceans. In addition, our analyses reject the hypothesis that the mitochondrial system for aerobic respiration is affiliated with that of the SAR11 clade., Conclusions/significance: Our results allude to the existence of an alphaproteobacterial clade in the oxygen-rich surface waters of the oceans that represents the closest free-living relative to mitochondria identified thus far. In addition, our findings underscore the importance of expanding the taxonomic diversity in phylogenetic analyses beyond that represented by cultivated bacteria to study the origin of mitochondria.

Unlabelled: The amount of gene and genome data obtained by next-generation sequencing technologies generates a need for comparative visualization tools. Complementing existing software for comparison and exploration of genomics data, genoPlotR automatically creates publication-grade linear maps of gene and genomes, in a highly automatic, flexible and reproducible way., Availability: genoPlotR is a platform-independent R package, available with full source code under a GPL2 license at R-Forge: http://genoplotr.r-forge.r-project.org/.

Host-adapted bacteria include mutualists and pathogens of animals, plants and insects. Their study is therefore important for biotechnology, biodiversity and human health. The recent rapid expansion in bacterial genome data has provided insights into the adaptive, diversifying and reductive evolutionary processes that occur during host adaptation. The results have challenged many pre-existing concepts built from studies of laboratory bacterial strains. Furthermore, recent studies have revealed genetic changes associated with transitions from parasitism to mutualism and opened new research avenues to understand the functional reshaping of bacteria as they adapt to growth in the cytoplasm of a eukaryotic host.

Here, we report the first comprehensive study of Bartonella henselae gene expression during infection of human endothelial cells. Expression of the main cluster of upregulated genes, comprising the VirB type IV secretion system and its secreted protein substrates, is shown to be under the positive control of the transcriptional regulator BatR. We demonstrate binding of BatR to the promoters of the virB operon and a substrate-encoding gene and provide biochemical evidence that BatR and BatS constitute a functional two-component regulatory system. Moreover, in contrast to the acid-inducible (pH 5.5) homologs ChvG/ChvI of Agrobacterium tumefaciens, BatR/BatS are optimally activated at the physiological pH of blood (pH 7.4). By conservation analysis of the BatR regulon, we show that BatR/BatS are uniquely adapted to upregulate a genus-specific virulence regulon during hemotropic infection in mammals. Thus, we propose that BatR/BatS two-component system homologs represent vertically inherited pH sensors that control the expression of horizontally transmitted gene sets critical for the diverse host-associated life styles of the alphaproteobacteria.

Bartonella is a genus of vector-borne bacteria that infect the red blood cells of mammals, and includes several human-specific and zoonotic pathogens. Bartonella grahamii has a wide host range and is one of the most prevalent Bartonella species in wild rodents. We studied the population structure, genome content and genome plasticity of a collection of 26 B. grahamii isolates from 11 species of wild rodents in seven countries. We found strong geographic patterns, high recombination frequencies and large variations in genome size in B. grahamii compared with previously analysed cat- and human-associated Bartonella species. The extent of sequence divergence in B. grahamii populations was markedly lower in Europe and North America than in Asia, and several recombination events were predicted between the Asian strains. We discuss environmental and demographic factors that may underlie the observed differences.

Synthetic genomics is a new field of research in which small DNA pieces are assembled in a series of steps into whole genomes. The highly reduced genomes of host-associated bacteria are now being used as models for de novo synthesis of small genomes in the laboratory. Bacteria with the smallest genomes identified in nature provide nutrients to their hosts, such as amino acids, co-factors and vitamins. Comparative genomics of these bacteria enables predictions to be made about the gene sets required for core cellular functions and the associated metabolic network for the biosynthesis of host-selected compounds. Synthetic biology may ultimately enable researchers to make customized cell-specific organelles for the production and delivery of drugs to humans and domestic animals. Synthetic genomics may also become the method of choice for functional analyses of genes and genomes from bacteria that cannot be cultivated in the laboratory.

Background: Rodents represent a high-risk reservoir for the emergence of new human pathogens. The recent completion of the 2.3 Mb genome of Bartonella grahamii, one of the most prevalent blood-borne bacteria in wild rodents, revealed a higher abundance of genes for host-cell interaction systems than in the genomes of closely related human pathogens. The sequence variability within the global B. grahamii population was recently investigated by multi locus sequence typing, but no study on the variability of putative host-cell interaction systems has been performed., Results: To study the population dynamics of B. grahamii, we analyzed the genomic diversity on a whole-genome scale of 27 B. grahamii strains isolated from four different species of wild rodents in three geographic locations separated by less than 30 km. Even using highly variable spacer regions, only 3 sequence types were identified. This low sequence diversity contrasted with a high variability in genome content. Microarray comparative genome hybridizations identified genes for outer surface proteins, including a repeated region containing the fha gene for filamentous hemaggluttinin and a plasmid that encodes a type IV secretion system, as the most variable. The estimated generation times in liquid culture medium for a subset of strains ranged from 5 to 22 hours, but did not correlate with sequence type or presence/absence patterns of the fha gene or the plasmid., Conclusion: Our study has revealed a geographic microstructure of B. grahamii in wild rodents. Despite near-identity in nucleotide sequence, major differences were observed in gene presence/absence patterns that did not segregate with host species. This suggests that genetically similar strains can infect a range of different hosts.

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella., Competing Interests: The authors have declared that no competing interests exist.

The alpha-proteobacteria represent one of the most diverse bacterial subdivisions, displaying extreme variations in lifestyle, geographical distribution and genome size. Species for which genome data are available have been classified into a species tree based on a conserved set of vertically inherited core genes. By mapping the variation in gene content onto the species tree, genomic changes can be associated with adaptations to specific growth niches. Genes for adaptive traits are mostly located in 'plasticity zones' in the bacterial genome, which also contain mobile elements and are highly variable across strains. By physically separating genes for information processing from genes involved in interactions with the surrounding environment, the rate of evolutionary change can be substantially enhanced for genes underlying adaptation to new growth habitats, possibly explaining the ecological success of the alpha-proteo-bacterial subdivision.

The obligate intracellular bacterium Wolbachia pipientis infects around 20% of all insect species. It is maternally inherited and induces reproductive alterations of insect populations by male killing, feminization, parthenogenesis, or cytoplasmic incompatibility. Here, we present the 1,445,873-bp genome of W. pipientis strain wRi that induces very strong cytoplasmic incompatibility in its natural host Drosophila simulans. A comparison with the previously sequenced genome of W. pipientis strain wMel from Drosophila melanogaster identified 35 breakpoints associated with mobile elements and repeated sequences that are stable in Drosophila lines transinfected with wRi. Additionally, 450 genes with orthologs in wRi and wMel were sequenced from the W. pipientis strain wUni, responsible for the induction of parthenogenesis in the parasitoid wasp Muscidifurax uniraptor. The comparison of these A-group Wolbachia strains uncovered the most highly recombining intracellular bacterial genomes known to date. This was manifested in a 500-fold variation in sequence divergences at synonymous sites, with different genes and gene segments supporting different strain relationships. The substitution-frequency profile resembled that of Neisseria meningitidis, which is characterized by rampant intraspecies recombination, rather than that of Rickettsia, where genes mostly diverge by nucleotide substitutions. The data further revealed diversification of ankyrin repeat genes by short tandem duplications and provided examples of horizontal gene transfer across A- and B-group strains that infect D. simulans. These results suggest that the transmission dynamics of Wolbachia and the opportunity for coinfections have created a freely recombining intracellular bacterial community with mosaic genomes.

Among host-dependent bacteria that have evolved by extreme reductive genome evolution, long-term bacterial endosymbionts of insects have the smallest (160-790 kb) and most A + T-rich (>70%) bacterial genomes known to date. These genomes are riddled with poly(A) tracts, and 5-50% of genes contain tracts of 10 As or more. Here, we demonstrate transcriptional slippage at poly(A) tracts within genes of Buchnera aphidicola associated with aphids and Blochmannia pennsylvanicus associated with ants. Several tracts contain single frameshift deletions; these apparent pseudogenes showed patterns of constraint consistent with purifying selection on the encoded proteins. Transcriptional slippage yielded a heterogeneous population of transcripts with variable numbers of As in the tract. Across several frameshifted genes, including B. aphidicola cell wall biosynthesis genes and a B. pennsylvanicus histidine biosynthesis gene, 12-50% of transcripts contained corrected reading frames that could potentially yield full-length proteins. In situ immunostaining confirmed the production of the cell wall biosynthetic enzyme UDP-N-acetylmuramyl pentapeptide synthase encoded by the frameshifted murF gene. Simulation studies indicated an overrepresentation of poly(A) tracts in endosymbiont genomes relative to other A + T-rich bacterial genomes. Polymerase infidelity at poly(A) tracts rescues the functionality of genes with frameshift mutations and, conversely, reduces the efficiency of expression for in-frame genes carrying poly(A) regions. These features of homopolymeric tracts could be exploited to manipulate gene expression in small synthetic genomes.

Berg et al. (Reports, 14 December 2007, p. 1782) reported the discovery of an autotrophic carbon dioxide-fixation pathway in Archaea and implicated a substantial role of this pathway in global carbon cycling based on sequence analysis of Global Ocean Sampling data. We question the validity of the latter claim.

We have studied the evolution of a type IV secretion system (T4SS), in Bartonella, which is thought to have changed function from conjugation to erythrocyte adherence following a recent horizontal gene transfer event. The system, called Trw, is unique among T4SSs in that genes encoding both exo- and intracellular components are located within the same duplicated fragment. This provides an opportunity to study the influence of selection on proteins involved in host-pathogen interactions. We sequenced the trw locus from several strains of Bartonella henselae and investigated its evolutionary history by comparisons to other Bartonella species. Several instances of recombination and gene conversion events where detected in the 2- to 5-fold duplicated gene fragments encompassing trwJIH, explaining the homogenization of the anchoring protein TrwI and the divergence of the minor pilus protein TrwJ. A phylogenetic analysis of the 7- to 8-fold duplicated gene coding for the major pilus protein TrwL displayed 2 distinct clades, likely representing a subfunctionalization event. The analyses of the B. henselae strains also identified a recent horizontal transfer event of almost the complete trwL region. We suggest that the switch in function of the T4SS was mediated by the duplication of the genes encoding pilus components and their diversification by combinatorial sequence shuffling within and among genomes. We suggest that the pilus proteins have evolved by diversifying selection to match a divergent set of erythrocyte surface structures, consistent with the trench warfare coevolutionary model.

Background: Pseudogenes reveal ancestral gene functions. Some obligate intracellular bacteria, such as Mycobacterium leprae and Rickettsia spp., carry substantial fractions of pseudogenes. Until recently, horizontal gene transfers were considered to be rare events in obligate host-associated bacteria., Results: We present a visualization tool that displays the relationships and positions of degraded and partially overlapping gene sequences in multiple genomes. With this tool we explore the origin and deterioration patterns of the Rickettsia pseudogenes and find that variably present genes and pseudogenes tend to have been acquired more recently, are more divergent in sequence, and exhibit a different functional profile compared with genes conserved across all species. Overall, the origin of only one-quarter of the variable genes and pseudogenes can be traced back to the common ancestor of Rickettsia and the outgroup genera Orientia and Wolbachia. These sequences contain only a few disruptive mutations and show a broad functional distribution profile, much like the core genes. The remaining genes and pseudogenes are extensively degraded or solely present in a single species. Their functional profile was heavily biased toward the mobile gene pool and genes for components of the cell wall and the lipopolysaccharide., Conclusion: Reductive evolution of the vertically inherited genomic core accounts for 25% of the predicted genes in the variable segments of the Rickettsia genomes, whereas 75% stems from the flux of the mobile gene pool along with genes for cell surface structures. Thus, most of the variably present genes and pseudogenes in Rickettsia have arisen from recent acquisitions.

Comparative genomics of Rickettsia and Orientia has revealed an exciting interplay between reductive evolutionary forces acting on metabolic genes in all species and proliferation of mobile genetic elements in some species. These contradictory evolutionary forces highlight the influence of chance, adaptation and host-cell exploitation during the evolution of intracellular bacteria.

The Rickettsiales, a genetically diverse group of the alpha-Proteobacteria, include major mammalian pathogens, such as the agents of epidemic typhus, scrub typhus, ehrlichioses and heartwater disease. Sequenced genomes of this bacterial order have provided exciting insights into reductive genome evolution, antigenic variation and host cell manipulation. Recent results suggest that human pathogens emerged relatively late in the evolution of the Rickettsiales. Surprisingly, there is no association between pathogenicity and the acquisition of novel virulence genes. Here, we explore the genomic differences between members of the Rickettsiales and ask what are the changes that enable infectious agents to emerge from seemingly harmless bacteria.

Scrub typhus is caused by the obligate intracellular rickettsia Orientia tsutsugamushi (previously called Rickettsia tsutsugamushi). The bacterium is maternally inherited in trombicuid mites and transmitted to humans by feeding larvae. We report here the 2,127,051-bp genome of the Boryong strain, which represents the most highly repeated bacterial genome sequenced to date. The repeat density of the scrub typhus pathogen is 200-fold higher than that of its close relative Rickettsia prowazekii, the agent of epidemic typhus. A total of 359 tra genes for components of conjugative type IV secretion systems were identified at 79 sites in the genome. Associated with these are >200 genes for signaling and host-cell interaction proteins, such as histidine kinases, ankyrin-repeat proteins, and tetratrico peptide-repeat proteins. Additionally, the O. tsutsugamushi genome contains >400 transposases, 60 phage integrases, and 70 reverse transcriptases. Deletions and rearrangements have yielded unique gene combinations as well as frequent pseudogenization in the tra clusters. A comparative analysis of the tra clusters within the genome and across strains indicates sequence homogenization by gene conversion, whereas complexity, diversity, and pseudogenization are acquired by duplications, deletions, and transposon integrations into the amplified segments. The results suggest intragenomic duplications or multiple integrations of a massively proliferating conjugative transfer system. Diversifying selection on host-cell interaction genes along with repeated population bottlenecks may drive rare genome variants to fixation, thereby short-circuiting selection for low complexity in bacterial genomes.

Genotyping of Chlamydia trachomatis is limited by the low sequence variation in the genome, and no adequate method is available for analysis of the spread of chlamydial infections in the community. We have developed a multilocus sequence typing (MLST) system based on five target regions and compared it with analysis of ompA, the single gene most extensively used for genotyping. Sequence determination of 16 reference strains, comprising all major serotypes, serotypes A to L3, showed that the number of genetic variants in the five separate target regions ranged from 8 to 16. The genetic variation in 47 clinical C. trachomatis isolates of representative serotypes (14 serotype D, 12 serotype E, 11 serotype G, and 10 serotype K strains) was analyzed; and the MLST system detected 32 variants, whereas 12 variants were detected by using ompA analysis. Specimens of the predominant serotype, serotype E, were differentiated into seven genotypes by MLST but into only two by ompA analysis. The MLST system was applied to C. trachomatis specimens from a population of men who have sex with men and was able to differentiate 10 specimens of one predominant ompA genotype G variant into four distinct MLST variants. To conclude, our MLST system can be used to discriminate C. trachomatis strains and can be applied to high-resolution molecular epidemiology.

Many theories favor a fusion of 2 prokaryotic genomes for the origin of the Eukaryotes, but there are disagreements on the origin, timing, and cellular structures of the cells involved. Equally controversial is the source of the nuclear genes for mitochondrial proteins, although the alpha-proteobacterial contribution to the mitochondrial genome is well established. Phylogenetic inferences show that the nuclearly encoded mitochondrial aminoacyl-tRNA synthetases (aaRSs) occupy a position in the tree that is not close to any of the currently sequenced alpha-proteobacterial genomes, despite cohesive and remarkably well-resolved alpha-proteobacterial clades in 12 of the 20 trees. Two or more alpha-proteobacterial clusters were observed in 8 cases, indicative of differential loss of paralogous genes or horizontal gene transfer. Replacement and retargeting events within the nuclear genomes of the Eukaryotes was indicated in 10 trees, 4 of which also show split alpha-proteobacterial groups. A majority of the mitochondrial aaRSs originate from within the bacterial domain, but none specifically from the alpha-Proteobacteria. For some aaRS, the endosymbiotic origin may have been erased by ongoing gene replacements on the bacterial as well as the eukaryotic side. For others that accurately resolve the alpha-proteobacterial divergence patterns, the lack of affiliation with mitochondria is more surprising. We hypothesize that the ancestral eukaryotic gene pool hosted primordial "bacterial-like" genes, to which a limited set of alpha-proteobacterial genes, mostly coding for components of the respiratory chain complexes, were added and selectively maintained.

Cats are the natural host for Bartonella henselae, an opportunistic human pathogen and the agent of cat scratch disease. Here, we have analyzed the natural variation in gene content and genome structure of 38 Bartonella henselae strains isolated from cats and humans by comparative genome hybridizations to microarrays and probe hybridizations to pulsed-field gel electrophoresis (PFGE) blots. The variation in gene content was modest and confined to the prophage and the genomic islands, whereas the PFGE analyses indicated extensive rearrangements across the terminus of replication with breakpoints in areas of the genomic islands. We observed no difference in gene content or structure between feline and human strains. Rather, the results suggest multiple sources of human infection from feline B. henselae strains of diverse genotypes. Additionally, the microarray hybridizations revealed DNA amplification in some strains in the so-called chromosome II-like region. The amplified segments were centered at a position corresponding to a putative phage replication initiation site and increased in size with the duration of cultivation. We hypothesize that the variable gene pool in the B. henselae population plays an important role in the establishment of long-term persistent infection in the natural host by promoting antigenic variation and escape from the host immune response.