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Recovering and annotating bacterial genomes from metagenomes involves a series of complex computational tools that are often difficult to use for researches without a specialistic bioinformatic background. In this chapter we review all the steps that lead from raw reads to a collection of quality-controlled, functionally annotated bacterial genomes and propose a working protocol using state-of-the-art, open source software tools.

Wolbachia is an iconic example of a successful intracellular bacterium. Despite its importance as a manipulator of invertebrate biology, its evolutionary dynamics have been poorly studied from a genomic viewpoint. To expand the number of Wolbachia genomes, we screen over 30,000 publicly available shotgun DNA sequencing samples from 500 hosts. By assembling over 1000 Wolbachia genomes, we provide a substantial increase in host representation. Our phylogenies based on both core-genome and gene content provide a robust reference for future studies, support new strains in model organisms, and reveal recent horizontal transfers amongst distantly related hosts. We find various instances of gene function gains and losses in different super-groups and in cytoplasmic incompatibility inducing strains. Our Wolbachia-host co-phylogenies indicate that horizontal transmission is widespread at the host intraspecific level and that there is no support for a general Wolbachia-mitochondrial synchronous divergence.

Streptococcus agalactiae (Group B Streptococcus, GBS) causes life-threatening infections in newborns and adults with chronic medical conditions. Serotype IV strains are emerging both among carriers and as cause of invasive disease and recent studies revealed two main Sequence Types (STs), ST-452 and ST-459 assigned to Clonal Complexes CC23 and CC1, respectively. Whole genome sequencing of 70 type IV GBS and subsequent phylogenetic analysis elucidated the localization of type IV isolates in a SNP-based phylogenetic tree and suggested that ST-452 could have originated through genetic recombination. SNPs density analysis of the core genome confirmed that the founder strain of this lineage originated from a single large horizontal gene transfer event between CC23 and the hypervirulent CC17. Indeed, ST-452 genomes are composed by two parts that are nearly identical to corresponding regions in ST-24 (CC23) and ST-291 (CC17). Chromosome mapping of the major GBS virulence factors showed that ST-452 strains have an intermediate yet unique profile among CC23 and CC17 strains. We described unreported large recombination events, involving the cps IV operon and resulting in the expansion of serotype IV to CC23. This work sheds further light on the evolution of GBS providing new insights on the recent emergence of serotype IV.

One of the main hurdles for the development of an effective and broadly protective vaccine against nonencapsulated isolates of Haemophilus influenzae (NTHi) lies in the genetic diversity of the species, which renders extremely difficult the identification of cross-protective candidate antigens. To assess whether a population structure of NTHi could be defined, we performed genome sequencing of a collection of diverse clinical isolates representative of both carriage and disease and of the diversity of the natural population. Analysis of the distribution of polymorphic sites in the core genome and of the composition of the accessory genome defined distinct evolutionary clades and supported a predominantly clonal evolution of NTHi, with the majority of genetic information transmitted vertically within lineages. A correlation between the population structure and the presence of selected surface-associated proteins and lipooligosaccharide structure, known to contribute to virulence, was found. This high-resolution, genome-based population structure of NTHi provides the foundation to obtain a better understanding, of NTHi adaptation to the host as well as its commensal and virulence behavior, that could facilitate intervention strategies against disease caused by this important human pathogen.

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.

Advances in high-throughput DNA sequencing technologies have determined an explosion in the number of sequenced bacterial genomes. Comparative sequence analysis frequently reveals evidences of homologous recombination occurring with different mechanisms and rates in different species, but the large-scale use of computational methods to identify recombination events is hampered by their high computational costs. Here, we propose a new method to identify recombination events in large datasets of whole genome sequences. Using a filtering procedure of the gene conservation profiles of a test genome against a panel of strains, this algorithm identifies sets of contiguous genes acquired by homologous recombination. The locations of the recombination breakpoints are determined using a statistical test that is able to account for the differences in the natural rate of evolution between different genes. The algorithm was tested on a dataset of 75 genomes of Staphylococcus aureus and 50 genomes comprising different streptococcal species, and was able to detect intra-species recombination events in S. aureus and in Streptococcus pneumoniae. Furthermore, we found evidences of an inter-species exchange of genetic material between S. pneumoniae and Streptococcus mitis, a closely related commensal species that colonizes the same ecological niche. The method has been implemented in an R package, Reco, which is freely available from supplementary material, and provides a rapid screening tool to investigate recombination on a genome-wide scale from sequence data.

The genetic variability in bacterial species is much larger than in other kingdoms of life. The gene content between pairs of isolates can diverge by as much as 30% in species like Escherichia coli or Streptococcus pneumoniae. This unexpected finding led to the introduction of the concept of the pan-genome, the set of genes that can be found in a given bacterial species. The genome of any isolate is thus composed by a "core genome" shared by all strains and characteristic of the species, and a "dispensable genome" that accounts for many of the phenotypic differences between strains. The pan-genome is usually much larger than the genome of any single isolate and, given the ability of many bacteria to exchange genetic material with the environment, constitutes a reservoir that could enhance their ability to survive in a mutating environment. To understand the evolution of the pan-genome of an important pathogen and its interactions with the commensal microbial flora, we have analyzed the genomes of 44 strains of Streptococcus pneumoniae, one of the most important causes of microbial diseases in humans. Despite evidence of extensive homologous recombination, the S. pneumoniae phylogenetic tree reconstructed from polymorphisms in the core genome identified major groups of genetically related strains. With the exception of serotype 1, the tree correlated poorly with capsular serotype, geographical site of isolation and disease outcome. The distribution of dispensable genes was consistent with phylogeny, although horizontal gene transfer events attenuated this correlation in the case of ancient lineages. Homologous recombination, involving short stretches of DNA, was the dominant evolutionary process of the core genome of S. pneumoniae. Genetic exchange with related species sharing the same ecological niche was the main mechanism of evolution of S. pneumonia; and S. mitis was the main reservoir of genetic diversity of S. pneumoniae. The pan-genome of S. pneumoniae increased logarithmically with the number of strains and linearly with the variability of the sample, suggesting that acquired genes accumulate proportionately to the age of clones., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Background: Streptococcus pneumoniae is one of the most important causes of microbial diseases in humans. The genomes of 44 diverse strains of S. pneumoniae were analyzed and compared with strains of non-pathogenic streptococci of the Mitis group., Results: Despite evidence of extensive recombination, the S. pneumoniae phylogenetic tree revealed six major lineages. With the exception of serotype 1, the tree correlated poorly with capsular serotype, geographical site of isolation and disease outcome. The distribution of dispensable genes--genes present in more than one strain but not in all strains--was consistent with phylogeny, although horizontal gene transfer events attenuated this correlation in the case of ancient lineages. Homologous recombination, involving short stretches of DNA, was the dominant evolutionary process of the core genome of S. pneumoniae. Genetic exchange occurred both within and across the borders of the species, and S. mitis was the main reservoir of genetic diversity of S. pneumoniae. The pan-genome size of S. pneumoniae increased logarithmically with the number of strains and linearly with the number of polymorphic sites of the sampled genomes, suggesting that acquired genes accumulate proportionately to the age of clones. Most genes associated with pathogenicity were shared by all S. pneumoniae strains, but were also present in S. mitis, S. oralis and S. infantis, indicating that these genes are not sufficient to determine virulence., Conclusions: Genetic exchange with related species sharing the same ecological niche is the main mechanism of evolution of S. pneumoniae. The open pan-genome guarantees the species a quick and economical response to diverse environments.

Genomics has revolutionized the way in which novel vaccine candidates are identified for the development of efficacious vaccines. Reverse vaccinology, whereby all candidates of interest are identified by analysis of a pathogen's genome, enables characterization of many candidates simultaneously. It accelerates the initial steps of vaccine development and greatly increases the chances of obtaining reliable candidates or cocktails thereof. The availability of one or two genome sequences for any given pathogen provides access to strain-specific vaccine candidates but often fails to identify candidates that would confer general protection. The analysis of multiple genomes of group B Streptococcus revealed tremendous diversity and identified candidates that are not shared by all the strains sequenced, but provide general protection when combined.

The advent of whole-genome sequencing of bacteria and advances in bioinformatics have revolutionized the study of bacterial pathogenesis, enabling the targeting of possible vaccine candidates starting from genomic information. Nowadays, the availability of hundreds of bacterial genomes enables identification of the genetic differences across several genomes from the same species. The unexpected degree of intra-species diversity suggests that a single genome sequence is not entirely representative and does not offer a complete picture of the genetic variability of a species. The practical consequence is that, in many cases, a universal vaccine is possible only by including a combination of antigens and this combination must take into account the pathogen population structure.

A decade after the beginning of the genomic era, the question of how genomics can describe a bacterial species has not been fully addressed. Experimental data have shown that in some species new genes are discovered even after sequencing the genomes of several strains. Mathematical modeling predicts that new genes will be discovered even after sequencing hundreds of genomes per species. Therefore, a bacterial species can be described by its pan-genome, which is composed of a "core genome" containing genes present in all strains, and a "dispensable genome" containing genes present in two or more strains and genes unique to single strains. Given that the number of unique genes is vast, the pan-genome of a bacterial species might be orders of magnitude larger than any single genome.

The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae, the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for approximately 80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.