Alex Mira

Deletional bias and the evolution of bacterial genomes

Although bacteria increase their DNA content through horizontal transfer and gene duplication, their genomes remain small and, in particular, lack nonfunctional sequences. This pattern is most readily explained by a pervasive bias towards higher numbers of deletions than insertions. When selection is not strong enough to maintain them, genes are lost in large deletions or inactivated and subsequently eroded. Gene inactivation and loss are particularly apparent in obligate parasites and symbionts, in which dramatic reductions in genome size can result not from selection to lose DNA, but from decreased selection to maintain gene functionality. Here we discuss the evidence showing that deletional bias is a major force that shapes bacterial genomes.

Explaining microbial population genomics through phage predation

The remarkable differences that have been detected by metagenomics in the genomes of strains of the same bacterial species are difficult to reconcile with the widely accepted paradigm that periodic selection within bacterial populations will regularly purge genomic diversity by clonal replacement. We have found that many of the genes that differ between strains affect regions that are potential phage recognition targets. We therefore propose the constant-diversity dynamics model, in which the diversity of prokaryotic populations is preserved by phage predation. We provide supporting evidence for this model from metagenomics, mathematical analysis and computer simulations. Periodic selection and phage predation dynamics are not mutually exclusive; we compare their predictions to shed light on the ecological circumstances under which each type of dynamics could predominate.

The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery

BACKGROUND Breast milk is recognized as the most important postpartum element in metabolic and immunologic programming of health of neonates. The factors influencing the milk microbiome and the potential impact of microbes on infant health have not yet been uncovered. OBJECTIVE Our objective was to identify pre- and postnatal factors that can potentially influence the bacterial communities inhabiting human milk. DESIGN We characterized the milk microbial community at 3 different time points by pyrosequencing and quantitative polymerase chain reaction in mothers (n = 18) who varied in BMI, weight gain, and mode of delivery. RESULTS We found that the human milk microbiome changes over lactation. Weisella, Leuconostoc, Staphylococcus, Streptococcus, and Lactococcus were predominant in colostrum samples, whereas in 1- and 6-mo milk samples the typical inhabitants of the oral cavity (eg, Veillonella, Leptotrichia, and Prevotella) increased significantly. Milk from obese mothers tended to contain a different and less diverse bacterial community compared with milk from normal-weight mothers. Milk samples from elective but not from nonelective mothers who underwent cesarean delivery contained a different bacterial community than did milk samples from individuals giving birth by vaginal delivery, suggesting that it is not the operation per se but rather the absence of physiological stress or hormonal signals that could influence the microbial transmission process to milk. CONCLUSIONS Our results indicate that milk bacteria are not contaminants and suggest that the milk microbiome is influenced by several factors that significantly skew its composition. Because bacteria present in breast milk are among the very first microbes entering the human body, our data emphasize the necessity to understand the biological role that the milk microbiome could potentially play for human health.

Viral and microbial community dynamics in four aquatic environments.

The species composition and metabolic potential of microbial and viral communities are predictable and stable for most ecosystems. This apparent stability contradicts theoretical models as well as the viral–microbial dynamics observed in simple ecosystems, both of which show Kill-the-Winner behavior causing cycling of the dominant taxa. Microbial and viral metagenomes were obtained from four human-controlled aquatic environments at various time points separated by one day to >1 year. These environments were maintained within narrow geochemical bounds and had characteristic species composition and metabolic potentials at all time points. However, underlying this stability were rapid changes at the fine-grained level of viral genotypes and microbial strains. These results suggest a model wherein functionally redundant microbial and viral taxa are cycling at the level of viral genotypes and virus-sensitive microbial strains. Microbial taxa, viral taxa, and metabolic function persist over time in stable ecosystems and both communities fluctuate in a Kill-the-Winner manner at the level of viral genotypes and microbial strains.

The oral metagenome in health and disease.

The oral cavity of humans is inhabited by hundreds of bacterial species and some of them have a key role in the development of oral diseases, mainly dental caries and periodontitis. We describe for the first time the metagenome of the human oral cavity under health and diseased conditions, with a focus on supragingival dental plaque and cavities. Direct pyrosequencing of eight samples with different oral-health status produced 1 Gbp of sequence without the biases imposed by PCR or cloning. These data show that cavities are not dominated by Streptococcus mutans (the species originally identified as the ethiological agent of dental caries) but are in fact a complex community formed by tens of bacterial species, in agreement with the view that caries is a polymicrobial disease. The analysis of the reads indicated that the oral cavity is functionally a different environment from the gut, with many functional categories enriched in one of the two environments and depleted in the other. Individuals who had never suffered from dental caries showed an over-representation of several functional categories, like genes for antimicrobial peptides and quorum sensing. In addition, they did not have mutans streptococci but displayed high recruitment of other species. Several isolates belonging to these dominant bacteria in healthy individuals were cultured and shown to inhibit the growth of cariogenic bacteria, suggesting the use of these commensal bacterial strains as probiotics to promote oral health and prevent dental caries.

The process of genome shrinkage in the obligate symbiont Buchnera aphidicola

BackgroundVery small genomes have evolved repeatedly in eubacterial lineages that have adopted obligate associations with eukaryotic hosts. Complete genome sequences have revealed that small genomes retain very different gene sets, raising the question of how final genome content is determined. To examine the process of genome reduction, the tiny genome of the endosymbiont Buchnera aphidicola was compared to the larger ancestral genome, reconstructed on the basis of the phylogenetic distribution of gene orthologs among fully sequenced relatives of Escherichia coli and Buchnera.ResultsThe reconstructed ancestral genome contained 2,425 open reading frames (ORFs). The Buchnera genome, containing 564 ORFs, consists of 153 fragments of 1-34 genes that are syntenic with reconstructed ancestral regions. On the basis of this reconstruction, 503 genes were eliminated within syntenic fragments, and 1,403 genes were lost from the gaps between syntenic fragments, probably in connection with genome rearrangements. Lost regions are sometimes large, and often span functionally unrelated genes. In addition, individual genes and regulatory regions have been lost or eroded. For the categories of DNA repair genes and rRNA genes, most lost loci fall in regions between syntenic fragments. This history of gene loss is reflected in the sequences of intergenic spacers at positions where genes were once present.ConclusionsThe most plausible interpretation of this reconstruction is that Buchnera lost many genes through the fixation of large deletions soon after the acquisition of an obligate endosymbiotic lifestyle. An implication is that final genome composition may be partly the chance outcome of initial deletions and that neighboring genes influence the likelihood of loss of particular genes and pathways.

Comparative genomics of two ecotypes of the marine planktonic copiotroph Alteromonas macleodii suggests alternative lifestyles associated with different kinds of particulate organic matter

Alteromonas macleodii is a common marine heterotrophic γ-proteobacterium. Isolates from this microbe cluster by molecular analysis into two major genotypic groups or ecotypes, one found in temperate latitudes in the upper water column and another that is for the most part found in the deep water column of the Mediterranean. Here, we describe the genome of one strain of the ‘deep ecotype’ (AltDE) isolated from 1000 m in the Eastern Mediterranean and compare this genome with that of the type strain ATCC 27126, a representative of the global ‘surface’ ecotype. The genomes are substantially different with DNA sequence similarity values that are borderline for microbes belonging to the same species, and a large differential gene content, mainly found in islands larger than 20 kbp, that also recruit poorly to the Global Ocean Sampling project (GOS). These genomic differences indicate that AltDE is probably better suited to microaerophilic conditions and for the degradation of recalcitrant compounds such as urea. These, together with other features, and the distribution of this genotypic group, indicate that this microbe colonizes relatively large particles that sink rapidly to meso and bathypelagic depths. The genome of ATCC 27126 on the other hand has more potential for regulation (two component systems) and degrades more sugars and amino acids, which is consistent with a more transient particle attachment, as would be expected for lineages specialized in colonizing smaller particulate organic matter with much slower sinking rates. The genomic data are also consistent with a picture of incipient speciation driven by niche specialization.

Microbiome Diversity in the Bronchial Tracts of Patients with Chronic Obstructive Pulmonary Disease

ABSTRACT Culture of bacteria from bronchial secretions in respiratory patients has low sensitivity and does not allow for complete assessment of microbial diversity across different bronchial compartments. In addition, a significant number of clinical studies are based on sputum samples, and it is not known to what extent they describe the real diversity of the mucosa. In order to identify previously unrecognized lower airway bacteria and to investigate the complexity and distribution of microbiota in patients with chronic obstructive pulmonary disease (COPD), we performed PCR amplification and pyrosequencing of the 16S rRNA gene in patients not showing signs or symptoms of infection. Four types of respiratory samples (sputum, bronchial aspirate, bronchoalveolar lavage, and bronchial mucosa) were taken from each individual, obtaining on average >1,000 16S rRNA sequences per sample. The total number of genera per patient was >100, showing a high diversity, with Streptococcus, Prevotella, Moraxella, Haemophilus, Acinetobacter, Fusobacterium, and Neisseria being the most commonly identified. Sputum samples showed significantly lower diversity than the other three sample types. Lower-bronchial-tree samples, i.e., bronchoalveolar lavage and bronchial mucosa, showed a very similar bacterial compositions in contrast to sputum and bronchial aspirate samples. Thus, sputum and bronchial aspirate samples are upper bronchial tree samples that are not representative of the lower bronchial mucosa flora, and bronchoalveolar lavage samples showed the results closest to those for the bronchial mucosa. Our data confirm that the bronchial tree is not sterile in COPD patients and support the existence a different microbiota in the upper and lower compartments.

Metagenome of the Mediterranean deep chlorophyll maximum studied by direct and fosmid library 454 pyrosequencing

The deep chlorophyll maximum (DCM) is a zone of maximal photosynthetic activity, generally located toward the base of the photic zone in lakes and oceans. In the tropical waters, this is a permanent feature, but in the Mediterranean and other temperate waters, the DCM is a seasonal phenomenon. The metagenome from a single sample of a mature Mediterranean DCM community has been 454 pyrosequenced both directly and after cloning in fosmids. This study is the first to be carried out at this sequencing depth (ca. 600 Mb combining direct and fosmid sequencing) at any DCM. Our results indicate a microbial community massively dominated by the high-light-adapted Prochlorococcus marinus subsp. pastoris, Synechococcus sp., and the heterotroph Candidatus Pelagibacter. The sequences retrieved were remarkably similar to the existing genome of P. marinus subsp. pastoris with a nucleotide identity over 98%. Besides, we found a large number of cyanophages that could prey on this microbe, although sequence conservation was much lower. The high abundance of phage sequences in the cellular size fraction indicated a remarkably high proportion of cells suffering phage lytic attack. In addition, several fosmids clearly belonging to Group II Euryarchaeota were retrieved and recruited many fragments from the total direct DNA sequencing suggesting that this group might be quite abundant in this habitat. The comparison between the direct and fosmids sequencing revealed a bias in the fosmid libraries against low-GC DNA and specifically against the two most dominant members of the community, Candidatus Pelagibacter and P. marinus subsp. pastoris, thus unexpectedly providing a feasible method to obtain large genomic fragments from other less prevalent members of this community.

Solving the etiology of dental caries

For decades, the sugar-fermenting, acidogenic species Streptococcus mutans has been considered the main causative agent of dental caries and most diagnostic and therapeutic strategies have been targeted toward this microorganism. However, recent DNA- and RNA-based studies from carious lesions have uncovered an extraordinarily diverse ecosystem where S. mutans accounts only a tiny fraction of the bacterial community. This supports the concept that consortia formed by multiple microorganisms act collectively, probably synergistically, to initiate and expand the cavity. Thus, antimicrobial therapies are not expected to be effective in the treatment of caries and other polymicrobial diseases that do not follow classical Kochs postulates.