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Open Access Publications from the University of California

Scholarly Works (25 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2021)

A wide range of specialized (or “secondary”) metabolites are produced by bacteria in naturalecosystems with varying functions, including antibiotics, siderophores, signalling molecules, and antifungals. These specialized metabolites are produced using operonic sets of genes that work in concert, known as biosynthetic gene clusters. In this work, genome-resolved metagenomic approaches were applied to understand the distribution and ecology of biosynthetic genes and the bacteria that possess them. Bacterial genomes were assembled and binned from deeply sequenced metagenomes from a California grassland meadow soil and a permanently wet vernal pool soil, and clusters of biosynthetic genes were identified in each genome. Genomes were reconstructed for novel species that belong to rarely cultivated but ubiquitous soil phyla, including the Acidobacteria, Verrucomicrobia, and the candidate phylum Rokubacteria. Bacteria from the grassland meadow soil were shown to have unexpectedly large numbers of biosynthetic gene clusters. In particular, two novel lineages of Acidobacteria were identified that possessed an unusual genomic capacity for specialized metabolite biosynthesis - up to 15% of their genomes were predicted to be dedicated to the production of nonribosomal peptides and polyketides. Sampling a second study site of soils from a vernal pool, Another three species were obtained from one of these uncultivated lineages — the candidate genus Angelobacter ​ ​ — which also possessed a large genomic repertoire of diverse biosynthetic genes. By mining public soil metagenomes, additional high quality draft genomes from this candidate genus were also analyzed, confirming that species in this genus are widespread across soil environments. It was therefore established that Angelobacter spp. ​with a substantial capacity for specialized metabolite biosynthesis are widespread in soils with a range of moisture contents and vegetation types. Transcriptional activity of nonribosomal peptide synthetase and polyketide synthase genes of abundant organisms from the grassland soil was tracked over time using 120 metatranscriptomic samples from soil microcosms. For several bacterial species within the samples, unsupervised clustering of genes by co-expression across samples identified modules of biosynthetic genes that were tightly co-expressed with genes involved in transcriptional regulation, environmental sensing, and secretion. For some vernal pool samples where ​ Angelobacter ​ were the most abundant microbial community members, metatranscriptomics demonstrated clear transcriptional activity ​ in situ ​ . Transcription of many ​ Angelobacter ​ biosynthetic genes was detected, extending findings from the grassland soil microcosms. Genetic variation in soil bacteria and their biosynthetic genes was investigated using population genomics methods that leverage genetic variation within sequencing reads that map to genomes from metagenomes. Metagenomic methods to track genetic variation within populations in a spatial context were applied to study the most abundant bacterial species across the grassland meadow study site. Genetic variation specifically within biosynthetic genes was elevated, indicating that there can be substantial allelic diversity in the biosynthetic genes of an abundant species in a local soil ecosystem. For about half of the bacterial populations studied, strong genetic population structure associated with spatial scale was observed. Genomes and gene variants were more genetically similar if they were from the same meadow plot. Simultaneously, while genetic gradients were observed across the meadow, within sample genetic diversity was also found to be high. Genomic signatures of recombination and gene-specific selection were also identified, indicating that ongoing selection and recombination may shape genetic divergence of populations on local spatial scales in soils. While biosynthetic gene clusters can be outlined and annotated with confidence in microbial genomes, prediction of the function of the metabolites produced for novel gene clusters is often an unsolved problem. Colocalized transporter genes associated with biosynthetic gene clusters may help predict metabolite function, due to their intimate association with the metabolite(s) they are transporting. This hypothesis was tested and benchmarked on a dataset of characterized biosynthetic gene clusters. In particular, a strong specificity of transporter genes for siderophore export and re-uptake was quantified as a signal of siderophore production. Using this specific genomic signal, putative siderophore BGCs were annotated across bacterial genomes recovered from soil, as well as from better characterized microbes from the adult and premature infant microbiomes. Surprisingly few genomes from soil bacteria contained transporter genes associated with siderophore biosynthesis. While 23% of microbial genomes from premature infant microbiomes possess at least one siderophore-like biosynthetic gene cluster, only 3% of those from adult gut microbiomes do. In sum, this thesis presented a metagenomic perspective on specialized metabolisms, contributed to discovery of novel species, examined evolutionary processes, and improved genomic functional predictions. The strength of this approach lies in its ability to investigate microbes in ​ in situ community contexts and detect ecological trends among the uncultivated microbial majority.

    Cover page: Ecology and evolution of specialized metabolism in uncultivated bacteria
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2018)
    In soil ecosystems, microorganisms produce diverse secondary metabolites such as antibiotics, antifungals and siderophores that mediate communication, competition and interactions with other organisms and the environment1,2. Most known antibiotics are derived from a few culturable microbial taxa 3 , and the biosynthetic potential of the vast majority of bacteria in soil has rarely been investigated 4 . Here we reconstruct hundreds of near-complete genomes from grassland soil metagenomes and identify microorganisms from previously understudied phyla that encode diverse polyketide and nonribosomal peptide biosynthetic gene clusters that are divergent from well-studied clusters. These biosynthetic loci are encoded by newly identified members of the Acidobacteria, Verrucomicobia and Gemmatimonadetes, and the candidate phylum Rokubacteria. Bacteria from these groups are highly abundant in soils5-7, but have not previously been genomically linked to secondary metabolite production with confidence. In particular, large numbers of biosynthetic genes were characterized in newly identified members of the Acidobacteria, which is the most abundant bacterial phylum across soil biomes 5 . We identify two acidobacterial genomes from divergent lineages, each of which encodes an unusually large repertoire of biosynthetic genes with up to fifteen large polyketide and nonribosomal peptide biosynthetic loci per genome. To track gene expression of genes encoding polyketide synthases and nonribosomal peptide synthetases in the soil ecosystem that we studied, we sampled 120 time points in a microcosm manipulation experiment and, using metatranscriptomics, found that gene clusters were differentially co-expressed in response to environmental perturbations. Transcriptional co-expression networks for specific organisms associated biosynthetic genes with two-component systems, transcriptional activation, putative antimicrobial resistance and iron regulation, linking metabolite biosynthesis to processes of environmental sensing and ecological competition. We conclude that the biosynthetic potential of abundant and phylogenetically diverse soil microorganisms has previously been underestimated. These organisms may represent a source of natural products that can address needs for new antibiotics and other pharmaceutical compounds.
      Cover page: Novel soil bacteria possess diverse genes for secondary metabolite biosynthesis
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2022)
      The relative importance of separation by distance and by environment to population genetic diversity can be conveniently tested in river networks, where these two drivers are often independently distributed over space. To evaluate the importance of dispersal and environmental conditions in shaping microbial population structures, we performed genome-resolved metagenomic analyses of benthic Microcoleus-dominated cyanobacterial mats collected in the Eel and Russian River networks (California, USA). The 64 Microcoleus genomes were clustered into three species that shared >96.5% average nucleotide identity (ANI). Most mats were dominated by one strain, but minor alleles within mats were often shared, even over large spatial distances (>300 km). Within the most common Microcoleus species, the ANI between the dominant strains within mats decreased with increasing spatial separation. However, over shorter spatial distances (tens of kilometres), mats from different subwatersheds had lower ANI than mats from the same subwatershed, suggesting that at shorter spatial distances environmental differences between subwatersheds in factors like canopy cover, conductivity, and mean annual temperature decreases ANI. Since mats in smaller creeks had similar levels of nucleotide diversity (π) as mats in larger downstream subwatersheds, within-mat genetic diversity does not appear to depend on the downstream accumulation of upstream-derived strains. The four-gamete test and sequence length bias suggest recombination occurs between almost all strains within each species, even between populations separated by large distances or living in different habitats. Overall, our results show that, despite some isolation by distance and environmental conditions, sufficient gene-flow occurs among cyanobacterial strains to prevent either driver from producing distinctive population structures across the watershed.
        Cover page: Microcoleus (Cyanobacteria) form watershed‐wide populations without strong gradients in population structure
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2020)
        Soil microbial diversity is often studied from the perspective of community composition, but less is known about genetic heterogeneity within species. The relative impacts of clonal interference, gene-specific selection, and recombination in many abundant but rarely cultivated soil microbes remain unknown. Here we track genome-wide population genetic variation for 19 highly abundant bacterial species sampled from across a grassland meadow. Genomic inferences about population structure are made using the millions of sequencing reads that are assembled de novo into consensus genomes from metagenomes, as each read pair describes a short genomic sequence from a cell in each population. Genomic nucleotide identity of assembled genomes was significantly associated with local geography for over half of the populations studied, and for a majority of populations within-sample nucleotide diversity could often be as high as meadow-wide nucleotide diversity. Genes involved in metabolite biosynthesis and extracellular transport were characterized by elevated nucleotide diversity in multiple species. Microbial populations displayed varying degrees of homologous recombination and recombinant variants were often detected at 7-36% of loci genome-wide. Within multiple populations we identified genes with unusually high spatial differentiation of alleles, fewer recombinant events, elevated ratios of nonsynonymous to synonymous variants, and lower nucleotide diversity, suggesting recent selective sweeps for gene variants. Taken together, these results indicate that recombination and gene-specific selection commonly shape genetic variation in several understudied soil bacterial lineages.
          Cover page: Soil bacterial populations are shaped by recombination and gene-specific selection across a grassland meadowCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2021)
          Biosynthetic gene clusters (BGCs) are operonic sets of microbial genes that synthesize specialized metabolites with diverse functions, including siderophores and antibiotics, which often require export to the extracellular environment. For this reason, genes for transport across cellular membranes are essential for the production of specialized metabolites and are often genomically colocalized with BGCs. Here, we conducted a comprehensive computational analysis of transporters associated with characterized BGCs. In addition to known exporters, in BGCs we found many importer-specific transmembrane domains that co-occur with substrate binding proteins possibly for uptake of siderophores or metabolic precursors. Machine learning models using transporter gene frequencies were predictive of known siderophore activity, molecular weights, and a measure of lipophilicity (log P) for corresponding BGC-synthesized metabolites. Transporter genes associated with BGCs were often equally or more predictive of metabolite features than biosynthetic genes. Given the importance of siderophores as pathogenicity factors, we used transporters specific for siderophore BGCs to identify both known and uncharacterized siderophore-like BGCs in genomes from metagenomes from the infant and adult gut microbiome. We find that 23% of microbial genomes from premature infant guts have siderophore-like BGCs, but only 3% of those assembled from adult gut microbiomes do. Although siderophore-like BGCs from the infant gut are predominantly associated with Enterobacteriaceae and Staphylococcus, siderophore-like BGCs can be identified from taxa in the adult gut microbiome that have rarely been recognized for siderophore production. Taken together, these results show that consideration of BGC-associated transporter genes can inform predictions of specialized metabolite structure and function.
            Cover page: Transporter genes in biosynthetic gene clusters predict metabolite characteristics and siderophore activityCreative Commons 'BY-NC' version 4.0 license
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2021)
            Coexisting microbial cells of the same species often exhibit genetic variation that can affect phenotypes ranging from nutrient preference to pathogenicity. Here we present inStrain, a program that uses metagenomic paired reads to profile intra-population genetic diversity (microdiversity) across whole genomes and compares microbial populations in a microdiversity-aware manner, greatly increasing the accuracy of genomic comparisons when benchmarked against existing methods. We use inStrain to profile >1,000 fecal metagenomes from newborn premature infants and find that siblings share significantly more strains than unrelated infants, although identical twins share no more strains than fraternal siblings. Infants born by cesarean section harbor Klebsiella with significantly higher nucleotide diversity than infants delivered vaginally, potentially reflecting acquisition from hospital rather than maternal microbiomes. Genomic loci that show diversity in individual infants include variants found between other infants, possibly reflecting inoculation from diverse hospital-associated sources. inStrain can be applied to any metagenomic dataset for microdiversity analysis and rigorous strain comparison.
              Cover page: inStrain profiles population microdiversity from metagenomic data and sensitively detects shared microbial strains
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2020)
              Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination.IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.
                Cover page: Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species BoundariesCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2020)
                Bacteria isolated from soils are major sources of specialized metabolites, including antibiotics and other compounds with clinical value that likely shape interactions among microbial community members and impact biogeochemical cycles. Yet, isolated lineages represent a small fraction of all soil bacterial diversity. It remains unclear how the production of specialized metabolites varies across the phylogenetic diversity of bacterial species in soils and whether the genetic potential for production of these metabolites differs with soil depth and vegetation type within a geographic region. We sampled soils and saprolite from three sites in a northern California Critical Zone Observatory with various vegetation and bedrock characteristics and reconstructed 1,334 metagenome-assembled genomes containing diverse biosynthetic gene clusters (BGCs) for secondary metabolite production. We obtained genomes for prolific producers of secondary metabolites, including novel groups within the Actinobacteria, Chloroflexi, and candidate phylum "Candidatus Dormibacteraeota." Surprisingly, one genome of a candidate phyla radiation (CPR) bacterium coded for a ribosomally synthesized linear azole/azoline-containing peptide, a capacity we found in other publicly available CPR bacterial genomes. Overall, bacteria with higher biosynthetic potential were enriched in shallow soils and grassland soils, with patterns of abundance of BGC type varying by taxonomy.IMPORTANCE Microbes produce specialized compounds to compete or communicate with one another and their environment. Some of these compounds, such as antibiotics, are also useful in medicine and biotechnology. Historically, most antibiotics have come from soil bacteria which can be isolated and grown in the lab. Though the vast majority of soil bacteria cannot be isolated, we can extract their genetic information and search it for genes which produce these specialized compounds. These understudied soil bacteria offer a wealth of potential for the discovery of new and important microbial products. Here, we identified the ability to produce these specialized compounds in diverse and novel bacteria in a range of soil environments. This information will be useful to other researchers who wish to isolate certain products. Beyond their use to humans, understanding the distribution and function of microbial products is key to understanding microbial communities and their effects on biogeochemical cycles.
                  Cover page: Bacterial Secondary Metabolite Biosynthetic Potential in Soil Varies with Phylum, Depth, and Vegetation Type.Creative Commons 'BY' version 4.0 license
                  • Article
                  • Peer Reviewed
                  UC Berkeley Previously Published Works (2022)
                  Copper membrane monooxygenases (CuMMOs) play critical roles in the global carbon and nitrogen cycles. Organisms harboring these enzymes perform the first, and rate limiting, step in aerobic oxidation of ammonia, methane, or other simple hydrocarbons. Within archaea, only organisms in the order Nitrososphaerales (Thaumarchaeota) encode CuMMOs, which function exclusively as ammonia monooxygenases. From grassland and hillslope soils and aquifer sediments, we identified 20 genomes from distinct archaeal species encoding divergent CuMMO sequences. These archaea are phylogenetically clustered in a previously unnamed Thermoplasmatota order, herein named the Ca. Angelarchaeales. The CuMMO proteins in Ca. Angelarchaeales are more similar in structure to those in Nitrososphaerales than those of bacteria, and contain all functional residues required for general monooxygenase activity. Ca. Angelarchaeales genomes are significantly enriched in blue copper proteins (BCPs) relative to sibling lineages, including plastocyanin-like electron carriers and divergent nitrite reductase-like (nirK) 2-domain cupredoxin proteins co-located with electron transport machinery. Ca. Angelarchaeales also encode significant capacity for peptide/amino acid uptake and degradation and share numerous electron transport mechanisms with the Nitrososphaerales. Ca. Angelarchaeales are detected at high relative abundance in some of the environments where their genomes originated from. While the exact substrate specificities of the novel CuMMOs identified here have yet to be determined, activity on ammonia is possible given their metabolic and ecological context. The identification of an archaeal CuMMO outside of the Nitrososphaerales significantly expands the known diversity of CuMMO enzymes in archaea and suggests previously unaccounted organisms contribute to critical global nitrogen and/or carbon cycling functions.
                    Cover page: Soils and sediments host Thermoplasmata archaea encoding novel copper membrane monooxygenases (CuMMOs)
                    • Article
                    • Peer Reviewed
                    UC Berkeley Previously Published Works (2020)
                    Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination.IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.
                      Cover page: Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries
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