Automating microbial taxonomy workflows with PHANTASM: phylogenomic analyses for the taxonomy and systematics of microbes.

The nucleotide sequences of 16S ribosomal RNA (rRNA) genes have been used to inform the taxonomic placement of prokaryotes for several decades. Whole-genome approaches can better resolve evolutionary relationships of organisms, but these analyses often require computational proficiencies that are uncommon among microbiologists. PHANTASM is a new tool capable of automating these workflows. This tool was designed to work for a wide range of prokaryotes and is the first example of an automated reconciliation of NCBI's Taxonomy database with that of the List of Prokaryotic names with Standing in Nomenclature (LPSN). In this study, we describe the workflow of PHANTASM and provide several examples of results generated by it. The source code is freely-available on GitHub. In order to facilitate the ease-of-access for researchers, PHANTASM is also available as a Docker image. While other tools exist to facilitate starting points for these analyses, PHANTASM provides users with a greater degree of control and produces outputs that can be used to make publication-quality figures.

xenoGI 3: using the DTLOR model to reconstruct the evolution of gene families in clades of microbes.

To understand genome evolution in a group of microbes, we need to know the timing of events such as duplications, deletions and horizontal transfers. A common approach is to perform a gene-tree / species-tree reconciliation. While a number of software packages perform this type of analysis, none are geared toward a complete reconstruction for all families in an entire clade. Here we describe an update to the xenoGI software package which allows users to perform such an analysis using the newly developed DTLOR (duplication-transfer-loss-origin-rearrangement) reconciliation model starting from genome sequences as input.

xenoGI: reconstructing the history of genomic island insertions in clades of closely related bacteria.

BACKGROUND: Genomic islands play an important role in microbial genome evolution, providing a mechanism for strains to adapt to new ecological conditions. A variety of computational methods, both genome-composition based and comparative, have been developed to identify them. Some of these methods are explicitly designed to work in single strains, while others make use of multiple strains. In general, existing methods do not identify islands in the context of the phylogeny in which they evolved. Even multiple strain approaches are best suited to identifying genomic islands that are present in one strain but absent in others. They do not automatically recognize islands which are shared between some strains in the clade or determine the branch on which these islands inserted within the phylogenetic tree. RESULTS: We have developed a software package, xenoGI, that identifies genomic islands and maps their origin within a clade of closely related bacteria, determining which branch they inserted on. It takes as input a set of sequenced genomes and a tree specifying their phylogenetic relationships. Making heavy use of synteny information, the package builds gene families in a species-tree-aware way, and then attempts to combine into islands those families whose members are adjacent and whose most recent common ancestor is shared. The package provides a variety of text-based analysis functions, as well as the ability to export genomic islands into formats suitable for viewing in a genome browser. We demonstrate the capabilities of the package with several examples from enteric bacteria, including an examination of the evolution of the acid fitness island in the genus Escherichia. In addition we use output from simulations and a set of known genomic islands from the literature to show that xenoGI can accurately identify genomic islands and place them on a phylogenetic tree. CONCLUSIONS: xenoGI is an effective tool for studying the history of genomic island insertions in a clade of microbes. It identifies genomic islands, and determines which branch they inserted on within the phylogenetic tree for the clade. Such information is valuable because it helps us understand the adaptive path that has produced living species.

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12.

The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS ("sensitive" genes), and genes whose expression changes very little with the production of a little RpoS ("insensitive"). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS.IMPORTANCE The sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses.

The dynamic DNA methylation cycle from egg to sperm in the honey bee Apis mellifera.

In honey bees (Apis mellifera), the epigenetic mark of DNA methylation is central to the developmental regulation of caste differentiation, but may also be involved in additional biological functions. In this study, we examine the whole genome methylation profiles of three stages of the haploid honey bee genome: unfertilised eggs, the adult drones that develop from these eggs and the sperm produced by these drones. These methylomes reveal distinct patterns of methylation. Eggs and sperm show 381 genes with significantly different CpG methylation patterns, with the vast majority being more methylated in eggs. Adult drones show greatly reduced levels of methylation across the genome when compared with both gamete samples. This suggests a dynamic cycle of methylation loss and gain through the development of the drone and during spermatogenesis. Although fluxes in methylation during embryogenesis may account for some of the differentially methylated sites, the distinct methylation patterns at some genes suggest parent-specific epigenetic marking in the gametes. Extensive germ line methylation of some genes possibly explains the lower-than-expected frequency of CpG sites in these genes. We discuss the potential developmental and evolutionary implications of methylation in eggs and sperm in this eusocial insect species.

Chromatin analysis of occluded genes.

We recently described two opposing states of transcriptional competency. One is termed 'competent' whereby a gene is capable of responding to trans-acting transcription factors of the cell, such that it is active if appropriate transcriptional activators are present, though it can also be silent if activators are absent or repressors are present. The other is termed 'occluded' whereby a gene is silenced by cis-acting, chromatin-based mechanisms in a manner that blocks it from responding to trans-acting factors, such that it is silent even when activators are present in the cellular milieu. We proposed that gene occlusion is a mechanism by which differentiated cells stably maintain their phenotypic identities. Here, we describe chromatin analysis of occluded genes. We found that DNA methylation plays a causal role in maintaining occlusion for a subset of occluded genes. We further examined a variety of other chromatin marks typically associated with transcriptional silencing, including histone variants, covalent histone modifications and chromatin-associated proteins. Surprisingly, we found that although many of these marks are robustly linked to silent genes (which include both occluded genes and genes that are competent but silent), none is linked specifically to occluded genes. Although the observation does not rule out a possible causal role of these chromatin marks in occlusion, it does suggest that these marks might be secondary effect rather than primary cause of the silent state in many genes.

Context dependent substitution biases vary within the human genome.

BACKGROUND: Models of sequence evolution typically assume that different nucleotide positions evolve independently. This assumption is widely appreciated to be an over-simplification. The best known violations involve biases due to adjacent nucleotides. There have also been suggestions that biases exist at larger scales, however this possibility has not been systematically explored. RESULTS: To address this we have developed a method which identifies over- and under-represented substitution patterns and assesses their overall impact on the evolution of genome composition. Our method is designed to account for biases at smaller pattern sizes, removing their effects. We used this method to investigate context bias in the human lineage after the divergence from chimpanzee. We examined bias effects in substitution patterns between 2 and 5 bp long and found significant effects at all sizes. This included some individual three and four base pair patterns with relatively large biases. We also found that bias effects vary across the genome, differing between transposons and non-transposons, between different classes of transposons, and also near and far from genes. CONCLUSIONS: We found that nucleotides beyond the immediately adjacent one are responsible for substantial context effects, and that these biases vary across the genome.

A genome-wide screen for noncoding elements important in primate evolution.

BACKGROUND: A major goal in the study of human evolution is to identify key genetic changes which occurred over the course of primate evolution. According to one school of thought, many such changes are likely to be found in noncoding sequence. An approach to identifying these involves comparing multiple genomes to identify conserved regions with an accelerated substitution rate in a particular lineage. Such acceleration could be the result of positive selection. RESULTS: Here we develop a likelihood ratio test method to identify such regions. We apply it not only to the human terminal lineage, as has been done in previous studies, but also to a number of other branches in the primate tree. We present the top scoring elements, and compare our results with previous studies. We also present resequencing data from one particular element accelerated on the human lineage. These data indicate that the element lies in a region of low polymorphism in humans, consistent with the possibility of a recent selective sweep. They also show that the AT to GC bias for polymorphism in this region differs dramatically from that for substitutions. CONCLUSION: Our results suggest that screens of this type will be helpful in unraveling the complex set of changes which occurred during primate evolution.

The evolution of word composition in metazoan promoter sequence.

The field of molecular evolution provides many examples of the principle that molecular differences between species contain information about evolutionary history. One surprising case can be found in the frequency of short words in DNA: more closely related species have more similar word compositions. Interest in this has often focused on its utility in deducing phylogenetic relationships. However, it is also of interest because of the opportunity it provides for studying the evolution of genome function. Word-frequency differences between species change too slowly to be purely the result of random mutational drift. Rather, their slow pattern of change reflects the direct or indirect action of purifying selection and the presence of functional constraints. Many such constraints are likely to exist, and an important challenge is to distinguish them. Here we develop a method to do so by isolating the effects acting at different word sizes. We apply our method to 2-, 4-, and 8-base-pair (bp) words across several classes of noncoding sequence. Our major result is that similarities in 8-bp word frequencies scale with evolutionary time for regions immediately upstream of genes. This association is present although weaker in intronic sequence, but cannot be detected in intergenic sequence using our method. In contrast, 2-bp and 4-bp word frequencies scale with time in all classes of noncoding sequence. These results suggest that different genomic processes are involved at different word sizes. The pattern in 2-bp and 4-bp words may be due to evolutionary changes in processes such as DNA replication and repair, as has been suggested before. The pattern in 8-bp words may reflect evolutionary changes in gene-regulatory machinery, such as changes in the frequencies of transcription-factor binding sites, or in the affinity of transcription factors for particular sequences.

Selective constraint on noncoding regions of hominid genomes.

An important challenge for human evolutionary biology is to understand the genetic basis of human-chimpanzee differences. One influential idea holds that such differences depend, to a large extent, on adaptive changes in gene expression. An important step in assessing this hypothesis involves gaining a better understanding of selective constraint on noncoding regions of hominid genomes. In noncoding sequence, functional elements are frequently small and can be separated by large nonfunctional regions. For this reason, constraint in hominid genomes is likely to be patchy. Here we use conservation in more distantly related mammals and amniotes as a way of identifying small sequence windows that are likely to be functional. We find that putatively functional noncoding elements defined in this manner are subject to significant selective constraint in hominids.

Three-dimensional structure and evolution of primate primary visual cortex.

In this study, three-dimensional reconstructions of primate primary visual cortex (V1) were used to address questions about its evolution. The three-dimensional shape of V1 in anthropoids is significantly longer and narrower than in strepsirrhines. This difference is an effect of clade and is not due to differences in activity pattern or V1 size. New measurements of V1 volume were also provided in order to reassess V1 size differences between strepsirrhines and anthropoids. It was found that for a given lateral geniculate nucleus (LGN) volume, anthropoids have a significantly larger V1 than strepsirrhines do. This is important since LGN is the principal source of V1's input. Finally, independent contrasts analysis was used to examine the scaling of V1 relative to LGN, the rest of cortex, and the rest of the brain. It was confirmed that V1 scales with positive allometry relative to LGN. A number of possible explanations for scaling are discussed. V1 scaling may have to do with the tendency of large brains to be more compartmentalized than small brains, or V1 scaling might reflect the geometry of information representation.

High-resolution computed tomography study of the cranium of a fossil anthropoid primate, Parapithecus grangeri: new insights into the evolutionary history of primate sensory systems.

Extant anthropoids have large brains, small olfactory bulbs, and high-acuity vision compared with other primates. The relative timing of the evolution of these characteristics may have important implications for brain evolution. Here computed tomography is used to examine the cranium of a fossil anthropoid, Parapithecus grangeri. It is found that P. grangeri had a relatively small brain compared with living primates. In addition, it had an olfactory bulb in the middle of the range for living primates. Methods for relating optic foramen area and other cranial measurements to acuity are discussed. Multiple regression is used to estimate retinal ganglion cell number in P. grangeri. Given currently available comparative data, P. grangeri seems to have had retinal ganglion cell counts intermediate for living primates, overlapping with the upper end of the range for strepsirrhines and possibly with the lower end for anthropoids.

Whole-genome DNA methylation profile of the jewel wasp (Nasonia vitripennis).

The epigenetic mark of DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, has been extensively studied in many mammalian genomes and, although it is commonly found at the promoter regions of genes, it is also involved in a number of different biological functions. In other complex animals, such as social insects, DNA methylation has been determined to be involved in caste differentiation and to occur primarily in gene bodies. The role of methylation in nonsocial insects, however, has not yet been explored thoroughly. Here, we present the whole-genome DNA methylation profile of the nonsocial hymenopteran, the jewel wasp (Nasonia vitripennis). From high-throughput sequencing of bisulfite-converted gDNA extracted from male Nasonia thoraces, we were able to determine which cytosine residues are methylated in the entire genome. We found that an overwhelming majority of methylated sites (99.7%) occur at cytosines followed by a guanine in the 3' direction (CpG sites). Additionally, we found that a majority of methylation in Nasonia occurs within exonic regions of the genome (more than 62%). Overall, methylation is sparse in Nasonia, occurring only at 0.18% of all sites and at 0.63% of CpGs. Our analysis of the Nasonia methylome revealed that in contrast to the methylation profile typically seen in mammals, methylation is sparse and is constrained primarily to exons. This methylation profile is more similar to that of the social hymenopteran species, the honey bee (Apis mellifera). In presenting the Nasonia methylome, we hope to promote future investigation of the regulatory function of DNA methylation in both social and nonsocial hymenoptera.

Modeling the role of negative cooperativity in metabolic regulation and homeostasis.

A significant proportion of enzymes display cooperativity in binding ligand molecules, and such effects have an important impact on metabolic regulation. This is easiest to understand in the case of positive cooperativity. Sharp responses to changes in metabolite concentrations can allow organisms to better respond to environmental changes and maintain metabolic homeostasis. However, despite the fact that negative cooperativity is almost as common as positive, it has been harder to imagine what advantages it provides. Here we use computational models to explore the utility of negative cooperativity in one particular context: that of an inhibitor binding to an enzyme. We identify several factors which may contribute, and show that acting together they can make negative cooperativity advantageous.

Different classes of tissue-specific genes show different levels of noncoding conservation.

We divide tissue-specific genes into two major classes: regulators, defined as genes participating in tissue-specific transcriptional regulation, and effectors, defined as genes involved in rendering the physiological properties of cells. We show that regulators tend to have significantly greater noncoding conservation than effectors. We further show that within the regulator class, tissue-specific transcription factors generally have the greatest noncoding conservation, whereas signal receptors generally have the least noncoding conservation. Using noncoding conservation as a proxy for the complexity of cis-regulatory DNA, we extrapolate that different classes of tissue-specific genes tend to have different levels of cis-regulatory complexity and that greater complexity can be found in genes involved in transcriptional regulation, especially transcription factors.

The scaling of frontal cortex in primates and carnivores.

Size has a profound effect on the structure of the brain. Many brain structures scale allometrically, that is, their relative size changes systematically as a function of brain size. Here we use independent contrasts analysis to examine the scaling of frontal cortex in 43 species of mammals including 25 primates and 15 carnivores. We find evidence for significant differences in scaling between primates and carnivores. Primate frontal cortex hyperscales relative to the rest of neocortex and the rest of the brain. The slope of frontal cortex contrasts on rest of cortex contrasts is 1.18 (95% confidence interval, 1.06-1.30) for primates, which is significantly greater than isometric. It is also significantly greater than the carnivore value of 0.94 (95% confidence interval, 0.82-1.07). This finding supports the idea that there are substantial differences in frontal cortex structure and development between the two groups.

The scaling of white matter to gray matter in cerebellum and neocortex.

It is known that the white matter of neocortex increases disproportionately with brain size. However, relatively few measurements have been made of white matter/gray matter scaling in the cerebellum. We present data on the volumes of white and gray matter in both structures, taken from 45 species of mammals. We find a scaling exponent of 1.13 for cerebellum and 1.28 for neocortex. The 95% confidence intervals for our estimates of these two exponents do not overlap. This difference likely reflects differences in the connectivity and/or micro-structure of white matter in the two regions.

Rapid long lasting learning in a collinear edge-detection task.

We have developed a detection task in which subjects identify a pair of collinear edges in a field of polygons. Five of our six subjects showed significant, rapid learning at this task. Four showed evidence of retention a day and a week later. In several transfer tests, we found that disruption of the distractors produced a significant drop-off in performance. These results are consistent with a model in which collinear targets initially produce a salience signal too weak to be reliably detected over the noise of the distractors. As the experiment proceeds, the visual system learns to dampen the distractor signals, allowing for more reliable detection.