A fundamental question in microbial ecology relates to community structure, and how this varies across environment types. It is widely believed that some environments, such as those at very low pH, host simple communities based on the low number of taxa, possibly due to the extreme environmental conditions. However, most analyses of species richness have relied on methods that provide relatively low ribosomal RNA (rRNA) sampling depth. Here we used community transcriptomics to analyze the microbial diversity of natural acid mine drainage biofilms from the Richmond Mine at Iron Mountain, California. Our analyses target deep pools of rRNA gene transcripts recovered from both natural and laboratory-grown biofilms across varying developmental stages. In all, 91.8% of the ∼ 254 million Illumina reads mapped to rRNA genes represented in the SILVA database. Up to 159 different taxa, including Bacteria, Archaea and Eukaryotes, were identified. Diversity measures, ordination and hierarchical clustering separate environmental from laboratory-grown biofilms. In part, this is due to the much larger number of rare members in the environmental biofilms. Although Leptospirillum bacteria generally dominate biofilms, we detect a wide variety of other Nitrospira organisms present at very low abundance. Bacteria from the Chloroflexi phylum were also detected. The results indicate that the primary characteristic that has enabled prior extensive cultivation-independent 'omic' analyses is not simplicity but rather the high dominance by a few taxa. We conclude that a much larger variety of organisms than previously thought have adapted to this extreme environment, although only few are selected for at any one time.

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth ∼20×). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types (∼94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination.

The vast majority of bacterial diversity lies within phylum-level lineages called "candidate phyla," which lack isolated representatives and are poorly understood. These bacteria are surprisingly abundant in the oral cavity of marine mammals. We employed a genome-resolved metagenomic approach to recover and characterize genomes and functional potential from microbes in the oral gingival sulcus of two bottlenose dolphins (Tursiops truncatus). We detected organisms from 24 known bacterial phyla and one archaeal phylum. We also recovered genomes from two deep-branching, previously uncharacterized phylum-level lineages (here named "Candidatus Delphibacteria" and "Candidatus Fertabacteria"). The Delphibacteria lineage is found in both managed and wild dolphins; its metabolic profile suggests a capacity for denitrification and a possible role in dolphin health. We uncovered a rich diversity of predicted Cas9 proteins, including the two longest predicted Cas9 proteins to date. Notably, we identified the first type II CRISPR-Cas systems encoded by members of the Candidate Phyla Radiation. Using their spacer sequences, we subsequently identified and assembled a complete Saccharibacteria phage genome. These findings underscore the immense microbial diversity and functional potential that await discovery in previously unexplored environments.

Recent studies suggest that the microbiome has an impact on gestational health and outcome. However, characterization of the pregnancy-associated microbiome has largely relied on 16S rRNA gene amplicon-based surveys. Here, we describe an assembly-driven, metagenomics-based, longitudinal study of the vaginal, gut, and oral microbiomes in 292 samples from 10 subjects sampled every three weeks throughout pregnancy. Nonhuman sequences in the amount of 1.53 Gb were assembled into scaffolds, and functional genes were predicted for gene- and pathway-based analyses. Vaginal assemblies were binned into 97 draft quality genomes. Redundancy analysis (RDA) of microbial community composition at all three body sites revealed gestational age to be a significant source of variation in patterns of gene abundance. In addition, health complications were associated with variation in community functional gene composition in the mouth and gut. The diversity of Lactobacillus iners-dominated communities in the vagina, unlike most other vaginal community types, significantly increased with gestational age. The genomes of co-occurring Gardnerella vaginalis strains with predicted distinct functions were recovered in samples from two subjects. In seven subjects, gut samples contained strains of the same Lactobacillus species that dominated the vaginal community of that same subject and not other Lactobacillus species; however, these within-host strains were divergent. CRISPR spacer analysis suggested shared phage and plasmid populations across body sites and individuals. This work underscores the dynamic behavior of the microbiome during pregnancy and suggests the potential importance of understanding the sources of this behavior for fetal development and gestational outcome.