The recovery and assembly of genome sequences from samples containing communities of organisms pose several challenges. Because it is rarely possible to disassociate the resident organisms prior to sequencing, a major obstacle is the assignment of sequences to a single genome that can be fully assembled. This chapter delineates many of the decisions, methodologies, and approaches that can lead to the generation of complete or nearly complete microbial genome sequences from heterogeneous samples-that is, the procedures that allow us to turn metagenomes into genomes.

Explaining the diversity of gene repertoires has been a major problem in modern evolutionary biology. In eukaryotes, this diversity is believed to result mainly from gene duplication and loss, but in prokaryotes, lateral gene transfer (LGT) can also contribute substantially to genome contents. To determine the histories of gene inventories, we conducted an exhaustive analysis of gene phylogenies for all gene families in a widely sampled group, the gamma-Proteobacteria. We show that, although these bacterial genomes display striking differences in gene repertoires, most gene families having representatives in several species have congruent histories. Other than the few vast multigene families, gene duplication has contributed relatively little to the contents of these genomes; instead, LGT, over time, provides most of the diversity in genomic repertoires. Most such acquired genes are lost, but the majority of those that persist in genomes are transmitted strictly vertically. Although our analyses are limited to the gamma-Proteobacteria, these results resolve a long-standing paradox-i.e., the ability to make robust phylogenetic inferences in light of substantial LGT.

Wild great apes harbor clades of gut bacteria that are restricted to each host species. Previous research shows the evolutionary relationships among several host-restricted clades mirror those of great-ape species. However, processes such as geographic separation, host-shift speciation, and host-filtering based on diet or gut physiology can generate host-restricted bacterial clades and mimic patterns of co-diversification across host species. To gain insight into the distribution of host-restricted taxa, we examine captive great apes living under conditions where sharing of bacterial strains is readily possible. Here, we show that increased sampling of wild and captive apes identifies additional host-restricted lineages whose relationships are not concordant with the host phylogeny. Moreover, the gut microbiomes of captive apes converge through the displacement of strains that are restricted to their wild conspecifics by human-restricted strains. We demonstrate that host-restricted and co-diversifying bacterial strains in wild apes lack persistence and fidelity in captive environments.

Microbial communities serve as reservoirs of antibiotic resistance genes (ARGs) and facilitate the dissemination of these genes to bacteria that infect humans. Relatively little is known about the taxonomic distribution of bacteria harboring ARGs in these reservoirs and the avenues of transmission due to the technical hurdles associated with characterizing the contents of complex microbial populations and the assignment of genes to particular genomes. Focusing on the array of tetracycline resistance (Tcr) genes in the primary and secondary phases of wastewater treatment, 17 of the 22 assayed Tcr genes were detected in at least one sample. We then applied emulsion, paired isolation, and concatenation PCR (epicPCR) to link tetracycline resistance genes to specific bacterial hosts. Whereas Tcr genes tend to vary in their distributions among bacterial taxa according to their modes of action, there were numerous instances in which a particular Tcr gene was associated with a host that was distantly related to all other bacteria bearing the same gene, including several hosts not previously identified. Tcr genes are far less host-restricted than previously assumed, indicating that complex microbial communities serve as settings where ARGs are spread among divergent bacterial phyla.

The numerous classes of repeats often impede the assembly of genome sequences from the short reads provided by new sequencing technologies. We demonstrate a simple and rapid means to ascertain the repeat structure and total size of a bacterial or archaeal genome without the need for assembly by directly analyzing the abundances of distinct k-mers among reads.

Multiple factors over the lifetime of an individual, including diet, geography, and physiologic state, will influence the microbial communities within the primate gut. To determine the source of variation in the composition of the microbiota within and among species, we investigated the distal gut microbial communities harbored by great apes, as present in fecal samples recovered within their native ranges. We found that the branching order of host-species phylogenies based on the composition of these microbial communities is completely congruent with the known relationships of the hosts. Although the gut is initially and continuously seeded by bacteria that are acquired from external sources, we establish that over evolutionary timescales, the composition of the gut microbiota among great ape species is phylogenetically conserved and has diverged in a manner consistent with vertical inheritance.

Pseudogenes are usually considered to be completely neutral sequences whose evolution is shaped by random mutations and chance events. It is possible, however, for disrupted genes to generate products that are deleterious due either to the energetic costs of their transcription and translation or to the formation of toxic proteins. We found that after their initial formation, the youngest pseudogenes in Salmonella genomes have a very high likelihood of being removed by deletional processes and are eliminated too rapidly to be governed by a strictly neutral model of stochastic loss. Those few highly degraded pseudogenes that have persisted in Salmonella genomes correspond to genes with low expression levels and low connectivity in gene networks, such that their inactivation and any initial deleterious effects associated with their inactivation are buffered. Although pseudogenes have long been considered the paradigm of neutral evolution, the distribution of pseudogenes among Salmonella strains indicates that removal of many of these apparently functionless regions is attributable to positive selection.

Knowledge of population-level processes is essential to understanding the efficacy of selection operating within a species. However, attempts at estimating effective population sizes (Ne) are particularly challenging in bacteria due to their extremely large census populations sizes, varying rates of recombination and arbitrary species boundaries.

Gokushoviruses are single-stranded, circular DNA bacteriophages found in metagenomic datasets from diverse ecosystems worldwide, including human gut microbiomes. Despite their ubiquity and abundance, little is known about their biology or host range: Isolates are exceedingly rare, known only from three obligate intracellular bacterial genera. By synthesizing circularized phage genomes from prophages embedded in diverse enteric bacteria, we produced gokushoviruses in an experimentally tractable model system, allowing us to investigate their features and biology. We demonstrate that virions can reliably infect and lysogenize hosts by hijacking a conserved chromosome-dimer resolution system. Sequence motifs required for lysogeny are detectable in other metagenomically defined gokushoviruses; however, we show that even partial motifs enable phages to persist cytoplasmically without leading to collapse of their host culture. This ability to employ multiple, disparate survival strategies is likely key to the long-term persistence and global distribution of Gokushovirinae.

Microviruses encompass an astonishing array of small, single-stranded DNA phages that, due to the surge in metagenomic surveys, are now known to be prevalent in most environments. Current taxonomy concedes the considerable diversity within this lineage to a single family (the Microviridae), which has rendered it difficult to adequately and accurately assess the amount of variation that actually exists within this group. We amassed and curated the largest collection of microviral genomes to date and, through a combination of protein-sharing networks and phylogenetic analysis, discovered at least three meaningful taxonomic levels between the current ranks of family and genus. When considering more than 13,000 microviral genomes from recognized lineages and as-yet-unclassified microviruses in metagenomic samples, microviral diversity is better understood by elevating microviruses to the level of an order that consists of three suborders and at least 19 putative families, each with their respective subfamilies. These revisions enable fine-scale assessment of microviral dynamics: for example, in the human gut, there are considerable differences in the abundances of microviral families both between urban and rural populations and in individuals over time. In addition, our analysis of genome contents and gene exchange shows that microviral families carry no recognizable accessory metabolic genes and rarely, if ever, engage in horizontal gene transfer across microviral families or with their bacterial hosts. These insights bring microviral taxonomy in line with current developments in the taxonomy of other phages and increase the understanding of microvirus biology. IMPORTANCE Microviruses are the most abundant single-stranded DNA phages on the planet and an important component of the human gut virome. And yet, productive research into their biology is hampered by the inadequacies of current taxonomic ordering: microviruses are lumped into a single family and treated as a monolithic group, thereby obscuring the extent of their diversity and resulting in little comparative research. Our investigations into the diversity of microviruses define numerous groups, most lacking any isolated representatives, and point toward high-value targets for future research. To expedite microvirus discovery and comparison, we developed a pipeline that enables the fast and facile sorting of novel microvirus genomes into well-defined taxonomic groups. These improvements provide new insights into the biology of microviruses and emphasize fundamental differences between these miniature phages and their large, double-stranded DNA phage competitors.

Delineation of species is fundamental to organizing and understanding biological diversity. The most widely applied criterion for distinguishing species is the Biological Species Concept (BSC), which defines species as groups of interbreeding individuals that remain reproductively isolated from other such groups. The BSC has broad appeal; however, many organisms, most notably asexual lineages, cannot be classified according to the BSC. Despite their exclusively asexual mode of reproduction, Bacteria and Archaea can transfer and exchange genes though homologous recombination. Here we show that barriers to homologous gene exchange define biological species in prokaryotes with the same efficacy as in sexual eukaryotes. By analyzing the impact of recombination on the polymorphisms in thousands of genome sequences, we find that over half of named bacterial species undergo continuous recombination among sequenced constituents, indicative of true biological species. However, nearly a quarter of named bacterial species show sharp discontinuities and comprise multiple biological species. These interruptions of gene flow are not a simple function of genome identity, indicating that bacterial speciation does not uniformly proceed by the gradual divergence of genome sequences. The same genomic approach based on recombinant polymorphisms retrieves known species boundaries in sexually reproducing eukaryotes. Thus, a single biological species definition based on gene flow, once thought to be limited only to sexually reproducing organisms, is applicable to all cellular lifeforms.

Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing functionality genome-wide. Applying such an approach, we assayed all transcripts in two closely related bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, and demonstrate that, although the levels of genome-wide antisense transcription are similarly high in both bacteria, only a small fraction of asRNAs are shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. These findings indicate that despite the genome-wide transcription of asRNAs, many of these transcripts are likely nonfunctional.

For both historical and technical reasons, 16S ribosomal RNA has been the most common molecular marker used to analyze the contents of microbial communities. However, its slow rate of evolution hinders the resolution of closely related bacteria--individual 16S-phylotypes, particularly when clustered at 97% sequence identity, conceal vast amounts of species- and strain-level variation. Protein-coding genes, which evolve more quickly, are useful for differentiating among more recently diverged lineages, but their application is complicated by difficulties in designing low-redundancy primers that amplify homologous regions from distantly related taxa. Given the now-common practice of multiplexing hundreds of samples, adopting new genes usually entails the synthesis of large sets of barcoded primers. To circumvent problems associated with use of protein-coding genes to survey microbial communities, we develop an approach--termed phyloTAGs--that offers an automatic solution for primer design and can be easily adapted to target different taxonomic groups and/or different protein-coding regions. We applied this method to analyze diversity within the gorilla gut microbiome and recovered hundreds of strains that went undetected after deep-sequencing of 16S rDNA amplicons. PhyloTAGs provides a powerful way to recover the fine-level diversity within microbial communities and to study stability and dynamics of bacterial populations.

Bacterial species, and even strains within species, can vary greatly in their gene contents and metabolic capabilities. We examine the evolution of this diversity by assessing the distribution and ancestry of each gene in 13 sequenced isolates of Escherichia coli and Shigella. We focus on the emergence and demise of two specific classes of genes, ORFans (genes with no homologs in present databases) and HOPs (genes with distant homologs), since these genes, in contrast to most conserved ancestral sequences, are known to be a major source of the novel features in each strain. We find that the rates of gain and loss of these genes vary greatly among strains as well as through time, and that ORFans and HOPs show very different behavior with respect to their emergence and demise. Although HOPs, which mostly represent gene acquisitions from other bacteria, originate more frequently, ORFans are much more likely to persist. This difference suggests that many adaptive traits are conferred by completely novel genes that do not originate in other bacterial genomes. With respect to the demise of these acquired genes, we find that strains of Shigella lose genes, both by disruption events and by complete removal, at accelerated rates.

The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.

Analyses of genome reduction in obligate bacterial symbionts typically focus on the removal and retention of protein-coding regions, which are subject to ongoing inactivation and deletion. However, these same forces operate on intergenic spacers (IGSs) and affect their contents, maintenance, and rates of evolution. IGSs comprise both non-coding, non-functional regions, including decaying pseudogenes at varying stages of recognizability, as well as functional elements, such as genes for sRNAs and regulatory control elements. The genomes of Buchnera and other small genome symbionts display biased nucleotide compositions and high rates of sequence evolution and contain few recognizable regulatory elements. However, IGS lengths are highly correlated across divergent Buchnera genomes, suggesting the presence of functional elements. To identify functional regions within the IGSs, we sequenced two Buchnera genomes (from aphid species Uroleucon ambrosiae and Acyrthosiphon kondoi) and applied a phylogenetic footprinting approach to alignments of orthologous IGSs from a total of eight Buchnera genomes corresponding to six aphid species. Inclusion of these new genomes allowed comparative analyses at intermediate levels of divergence, enabling the detection of both conserved elements and previously unrecognized pseudogenes. Analyses of these genomes revealed that 232 of 336 IGS alignments over 50 nucleotides in length displayed substantial sequence conservation. Conserved alignment blocks within these IGSs encompassed 88 Shine-Dalgarno sequences, 55 transcriptional terminators, 5 Sigma-32 binding sites, and 12 novel small RNAs. Although pseudogene formation, and thus IGS formation, are ongoing processes in these genomes, a large proportion of intergenic spacers contain functional sequences.

Colonization of the gastrointestinal tract (GIT) of human infants with a suitable microbial community is essential for numerous aspects of health, but the progression of events by which this microbiota becomes established is poorly understood. Here, we investigate two previously unexplored areas of microbiota development in infants: the deployment of functional capabilities at the community level and the population genetics of its most abundant genera. To assess the progression of the infant microbiota toward an adult-like state and to evaluate the contribution of maternal GIT bacteria to the infant gut, we compare the infant's microbiota with that of the mother at 1 and 11 months after delivery. These comparisons reveal that the infant's microbiota rapidly acquires and maintains the range of gene functions present in the mother, without replicating the phylogenetic composition of her microbiota. Microdiversity analyses for Bacteroides and Bifidobacterium, two of the main microbiota constituents, reveal that by 11 months, the phylotypes detected in the infant are distinct from those in the mother, although the maternal Bacteroides phylotypes were transiently present at 1 month of age. The configuration of genetic variants within these genera reveals populations far from equilibrium and likely to be undergoing rapid growth, consistent with recent population turnovers. Such compositional turnovers and the associated loss of maternal phylotypes should limit the potential for long-term coadaptation between specific bacterial and host genotypes.

Because bacteria do not have a robust fossil record, attempts to infer the timing of events in their evolutionary history requires comparisons of molecular sequences. This use of molecular clocks is based on the assumptions that substitution rates for homologous genes or sites are fairly constant through time and across taxa. Violation of these conditions can lead to erroneous inferences and result in estimates that are off by orders of magnitude. In this study, we examine the consistency of substitution rates among a set of conserved genes in diverse bacterial lineages, and address the questions regarding the validity of molecular dating.

Analysis of many bacterial genomes is impeded by the inability to separate individual species from complex mixtures of cells or to propagate cells in pure culture. This problem is an obstacle to the study of many bacterial symbionts that live intracellularly in insects and other animals. To recover bacterial DNA from complex samples, we devised a method that facilitates the cloning of DNA fragments of distinctive G+C contents in order to generate shotgun DNA libraries enriched in inserts having a specific base composition. DNA preparations are first treated with a restriction enzyme having a common cleavage site in a particular genome and then shotgun cloned following size-fractionation. This method was applied to whole bacteriomes of the psyllid, Pachypsylla venusta, which harbors the bacterial symbiont Candidatus Carsonella ruddii. The resulting libraries were highly enriched in bacterial sequences. Through the use of alternate enzymes and partial digests, this technique can be adapted to yield virtually pure DNA libraries for individual bacterial species.

Pseudogenes are now known to be a regular feature of bacterial genomes and are found in particularly high numbers within the genomes of recently emerged bacterial pathogens. As most pseudogenes are recognized by sequence alignments, we use newly available genomic sequences to identify the pseudogenes in 11 genomes from 4 bacterial genera, each of which contains at least 1 human pathogen. The numbers of pseudogenes range from 27 in Staphylococcus aureus MW2 to 337 in Yersinia pestis CO92 (e.g. 1-8% of the annotated genes in the genome). Most pseudogenes are formed by small frameshifting indels, but because stop codons are A + T-rich, the two low-G + C Gram-positive taxa (Streptococcus and Staphylococcus) have relatively high fractions of pseudogenes generated by nonsense mutations when compared with more G + C-rich genomes. Over half of the pseudogenes are produced from genes whose original functions were annotated as 'hypothetical' or 'unknown'; however, several broadly distributed genes involved in nucleotide processing, repair or replication have become pseudogenes in one of the sequenced Vibrio vulnificus genomes. Although many of our comparisons involved closely related strains with broadly overlapping gene inventories, each genome contains a largely unique set of pseudogenes, suggesting that pseudogenes are formed and eliminated relatively rapidly from most bacterial genomes.