Archaeal and Bacterial Diversity and Distribution Patterns in Mediterranean-Climate Vernal Pools of Mexico and the Western USA.

Biogeographic patterns in microorganisms are poorly understood, despite the importance of microbial communities for a range of ecosystem processes. Our knowledge of microbial ecology and biogeography is particularly deficient in rare and threatened ecosystems. We tested for three ecological patterns in microbial community composition within ephemeral wetlands-vernal pools-located across Baja California (Mexico) and California (USA): (1) habitat filtering; (2) a latitudinal diversity gradient; and (3) distance decay in community composition. Paired water and soil samples were collected along a latitudinal transect of vernal pools, and bacterial and archaeal communities were characterized using 16S rDNA sequencing. We identified two main microbial communities, with one community present in the soil matrix that included archaeal and bacterial soil taxa, and another community present in the overlying water that was dominated by common freshwater bacterial taxa. Aquatic microbial communities were more diverse in the north, and displayed a significant but inverted latitudinal diversity pattern. Aquatic communities also exhibited a significant distance-decay pattern, with geographic proximity, and precipitation explaining part of the community variation. Collectively these results indicate greater sensitivity to spatial and environmental variation in vernal pool aquatic microbial communities than in soil microbial communities. We conclude that vernal pool aquatic microbial communities can display distribution patterns similar to those exhibited by larger organisms, but differ in some key aspects, such as the latitudinal gradient in diversity.

Influences of Climate on Phyllosphere Endophytic Bacterial Communities of Wild Poplar.

Plant-associated microbial communities play a central role in the plant response to biotic and abiotic stimuli, improving plant fitness under challenging growing conditions. Many studies have focused on the characterization of changes in abundance and composition of root-associated microbial communities as a consequence of the plant response to abiotic factors such as altered soil nutrients and drought. However, changes in composition in response to abiotic factors are still poorly understood concerning the endophytic community associated to the phyllosphere, the above-ground plant tissues. In the present study, we applied high-throughput 16S rDNA gene sequencing of the phyllosphere endophytic bacterial communities colonizing wild Populus trichocarpa (black cottonwood) plants growing in native, nutrient-limited environments characterized by hot-dry (xeric) riparian zones (Yakima River, WA), riparian zones with mid hot-dry (Tieton and Teanaway Rivers, WA) and moist (mesic) climates (Snoqualmie, Skykomish and Skagit Rivers, WA). From sequencing data, 587 Amplicon Sequence Variants (ASV) were identified. Surprisingly, our data show that a core microbiome could be found in phyllosphere-associated endophytic communities in trees growing on opposite sides of the Cascades Mountain Range. Considering only taxa appearing in at least 90% of all samples within each climatic zone, the core microbiome was dominated only by two ASVs affiliated Pseudomonadaceae and two ASVs of the Enterobacteriaceae family. Alpha-diversity measures indicated that plants colonizing hot-dry environments showed a lower diversity than those from mid hot-dry and moist climates. Beta-diversity measures showed that bacterial composition was significantly different across sampling sites. Accordingly, we found that specific ASV affiliated to Pseudomonadaceae and Enterobacteriaceae were significantly more abundant in the phyllosphere endophytic community colonizing plants adapted to the xeric environment. In summary, this study highlights that sampling site is the major driver of variation and that only a few ASV showed a distribution that significantly correlated to climate variables.

Molecular host mimicry and manipulation in bacterial symbionts.

It is common among intracellular bacterial pathogens to use eukaryotic-like proteins that mimic and manipulate host cellular processes to promote colonization and intracellular survival. Eukaryotic-like proteins are bacterial proteins with domains that are rare in bacteria, and known to function in the context of a eukaryotic cell. Such proteins can originate through horizontal gene transfer from eukaryotes or, in the case of simple repeat proteins, through convergent evolution. Recent studies of microbiomes associated with several eukaryotic hosts suggest that similar molecular strategies are deployed by cooperative bacteria that interact closely with eukaryotic cells. Some mimics, like ankyrin repeats, leucine rich repeats and tetratricopeptide repeats are shared across diverse symbiotic systems ranging from amoebae to plants, and may have originated early, or evolved independently in multiple systems. Others, like plant-mimicking domains in members of the plant microbiome are likely to be more recent innovations resulting from horizontal gene transfer from the host, or from microbial eukaryotes occupying the same host. Host protein mimics have only been described in a limited set of symbiotic systems, but are likely to be more widespread. Systematic searches for eukaryote-like proteins in symbiont genomes could lead to the discovery of novel mechanisms underlying host-symbiont interactions.

Subalpine conifers in different geographical locations host highly similar foliar bacterial endophyte communities.

Pines in the subalpine environment at Niwot Ridge, CO, have been found to host communities of acetic acid bacteria (AAB) within their needles. The significance and ubiquity of this pattern is not known, but recent evidence of nitrogen (N)-fixing activity in Pinus flexilis (limber pine) foliage calls for a better understanding of the processes that regulate endophytic communities in forest tree canopies. Here, to test if AAB dominate the foliar bacterial microbiota in other subalpine locations, we compared the 16S rRNA community in needles from P. flexilis and P. contorta (lodgepole pine) growing in the Eastern Sierra Nevada, CA, and Niwot Ridge, CO. AAB made up the majority of the bacterial community in both species at both sites. Multiple distinct AAB taxa, resolved at the single nucleotide level, were shared across host species and sites, with dominant OTUs identical or highly similar to database sequences from cold environments, including high altitude air sampled in Colorado, and the endosphere of Arctic plants. Our results suggest strong selection for community composition, potentially amplified by the long lifespan of individual Pinus needles, along with low dispersal constraints on canopy bacteria.

Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer.

Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N2 -fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P. flexilis-AAB association is consistent across years, we re-sampled P. flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N2 , we incubated twigs with (13) N2 -enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using (13) N. We used the acetylene reduction assay to test for nitrogenase activity within P. flexilis twigs four times from June to September. We found evidence for N2 fixation in P. flexilis foliage. N2 diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8-13.6 µg N m(-2)  d(-1) to P. flexilis stands. AAB dominated the P. flexilis needle endophyte community. We propose that foliar endophytes represent a low-cost, evolutionarily stable N2 -fixing strategy for long-lived conifers. This novel source of biological N2 fixation has fundamental implications for understanding forest N budgets.

The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins.

UNLABELLED: Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. IMPORTANCE: All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside all tissues. M. extorquens DSM13060 occupies an unusual niche inside cells of the dividing shoot tissues of a pine and stimulates seedling growth without producing cytokinin, auxin, or other plant hormones commonly synthesized by plant-associated bacteria. Here, we tracked the bacteria using a fluorescent tag and confocal laser scanning microscopy and found that they localize near the nucleus of the plant cell. This prompted us to sequence the genome and identify proteins that may affect host growth by targeting processes in the host cytoplasm and nucleus. We found many novel genes whose products may modulate plant processes from within the plant cell. Our results open up new avenues to better understand how bacteria assist in plant growth, with broad implications for plant science, forestry, and agriculture.

Evolutionary capture of viral and plasmid DNA by yeast nuclear chromosomes.

A 10-kb region of the nuclear genome of the yeast Vanderwaltozyma polyspora contains an unusual cluster of five pseudogenes homologous to five different genes from yeast killer viruses, killer plasmids, the 2microm plasmid, and a Penicillium virus. By further database searches, we show that this phenomenon is not unique to V. polyspora but that about 40% of the sequenced genomes of Saccharomycotina species contain integrated copies of genes from DNA plasmids or RNA viruses. We propose the name NUPAVs (nuclear sequences of plasmid and viral origin) for these objects, by analogy to NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) of organellar origin. Although most of the NUPAVs are pseudogenes, one intact and active gene that was formed in this way is the KHS1 chromosomal killer locus of Saccharomyces cerevisiae. We show that KHS1 is a NUPAV related to M2 killer virus double-stranded RNA. Many NUPAVs are located beside tRNA genes, and some contain sequences from a mixture of different extrachromosomal sources. We propose that NUPAVs are sequences that were captured by the nuclear genome during the repair of double-strand breaks that occurred during evolution and that some of their properties may be explained by repeated breakage at fragile chromosomal sites.

Run-off replication of host-adaptability genes is associated with gene transfer agents in the genome of mouse-infecting Bartonella grahamii.

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.

Diversifying selection and concerted evolution of a type IV secretion system in Bartonella.

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.

Independent sorting-out of thousands of duplicated gene pairs in two yeast species descended from a whole-genome duplication.

Among yeasts that underwent whole-genome duplication (WGD), Kluyveromyces polysporus represents the lineage most distant from Saccharomyces cerevisiae. By sequencing the K. polysporus genome and comparing it with the S. cerevisiae genome using a likelihood model of gene loss, we show that these species diverged very soon after the WGD, when their common ancestor contained >9,000 genes. The two genomes subsequently converged onto similar current sizes (5,600 protein-coding genes each) and independently retained sets of duplicated genes that are strikingly similar. Almost half of their surviving single-copy genes are not orthologs but paralogs formed by WGD, as would be expected if most gene pairs were resolved independently. In addition, by comparing the pattern of gene loss among K. polysporus, S. cerevisiae, and three other yeasts that diverged after the WGD, we show that the patterns of gene loss changed over time. Initially, both members of a duplicate pair were equally likely to be lost, but loss of the same gene copy in independent lineages was increasingly favored at later time points. This trend parallels an increasing restriction of reciprocal gene loss to more slowly evolving gene pairs over time and suggests that, as duplicate genes diverged, one gene copy became favored over the other. The apparent low initial sequence divergence of the gene pairs leads us to propose that the yeast WGD was probably an autopolyploidization.

Functional divergence and horizontal transfer of type IV secretion systems.

The type IV secretion system (TFSSs) is a multifunctional family of translocation pathways that mediate the transfer of DNA among bacteria and deliver DNA and proteins to eukaryotic cells during bacterial infections. Horizontal transmission has dominated the evolution of the TFSS, as demonstrated here by a lack of congruence between the tree topology inferred from components of the TFSS and the presumed bacterial species divergence pattern. A parsimony analysis suggests that conjugation represents the ancestral state and that the divergence from conjugation to secretion of effector molecules has occurred independently at multiple sites in the tree. The result shows that the nodes at which functional shifts have occurred coincide with those of horizontal gene transfers among distantly related bacteria. We suggest that it is the transfer between species that paved the way for the divergence of the TFSSs and discuss the general role of horizontal gene transfers for the evolution of novel gene functions.

The louse-borne human pathogen Bartonella quintana is a genomic derivative of the zoonotic agent Bartonella henselae.

We present the complete genomes of two human pathogens, Bartonella quintana (1,581,384 bp) and Bartonella henselae (1,931,047 bp). The two pathogens maintain several similarities in being transmitted by insect vectors, using mammalian reservoirs, infecting similar cell types (endothelial cells and erythrocytes) and causing vasculoproliferative changes in immunocompromised hosts. A primary difference between the two pathogens is their reservoir ecology. Whereas B. quintana is a specialist, using only the human as a reservoir, B. henselae is more promiscuous and is frequently isolated from both cats and humans. Genome comparison elucidated a high degree of overall similarity with major differences being B. henselae specific genomic islands coding for filamentous hemagglutinin, and evidence of extensive genome reduction in B. quintana, reminiscent of that found in Rickettsia prowazekii. Both genomes are reduced versions of chromosome I from the highly related pathogen Brucella melitensis. Flanked by two rRNA operons is a segment with similarity to genes located on chromosome II of B. melitensis, suggesting that it was acquired by integration of megareplicon DNA in a common ancestor of the two Bartonella species. Comparisons of the vector-host ecology of these organisms suggest that the utilization of host-restricted vectors is associated with accelerated rates of genome degradation and may explain why human pathogens transmitted by specialist vectors are outnumbered by zoonotic agents, which use vectors of broad host ranges.

Computational inference of scenarios for alpha-proteobacterial genome evolution.

The alpha-proteobacteria, from which mitochondria are thought to have originated, display a 10-fold genome size variation and provide an excellent model system for studies of genome size evolution in bacteria. Here, we use computational approaches to infer ancestral gene sets and to quantify the flux of genes along the branches of the alpha-proteobacterial species tree. Our study reveals massive gene expansions at branches diversifying plant-associated bacteria and extreme losses at branches separating intracellular bacteria of animals and humans. Alterations in gene numbers have mostly affected functional categories associated with regulation, transport, and small-molecule metabolism, many of which are encoded by paralogous gene families located on auxiliary chromosomes. The results suggest that the alpha-proteobacterial ancestor contained 3,000-5,000 genes and was a free-living, aerobic, and motile bacterium with pili and surface proteins for host cell and environmental interactions. Approximately one third of the ancestral gene set has no homologs among the eukaryotes. More than 40% of the genes without eukaryotic counterparts encode proteins that are conserved among the alpha-proteobacteria but for which no function has yet been identified. These genes that never made it into the eukaryotes but are widely distributed in bacteria may represent bacterial drug targets and should be prime candidates for future functional characterization.

Genome deterioration: loss of repeated sequences and accumulation of junk DNA.

A global survey of microbial genomes reveals a correlation between genome size, repeat content and lifestyle. Free-living bacteria have large genomes with a high content of repeated sequences and self-propagating DNA, such as transposons and bacteriophages. In contrast, obligate intracellular bacteria have small genomes with a low content of repeated sequences and no or few genetic parasites. In extreme cases, such as in the 650 kb-genomes of aphid endosymbionts of the genus Buchnera all repeated sequences above 200bp have been eliminated. We speculate that the initial downsizing of the genomes of obligate symbionts and parasites occurred by homologous recombination at repeated genes, leading to the loss of large blocks of DNA as well as to the consumption of repeated sequences. Further sequence elimination in these small genomes seems primarily to result from the accumulation of short deletions within genic sequences. This process may lead to temporary increases in the genomic content of pseudogenes and 'junk' DNA. We discuss causes and long-term consequences of extreme genome size reductions in obligate intracellular bacteria.