Paul Carini

Interests

Research

Environmental Microbiology -physiology, cellular evolution, cellular survival mechanisms, and nutrient cycling

Teaching

Environmental Microbiology -physiology, cellular evolution, cellular survival mechanisms, and nutrient cycling

Courses

Scholarly Contributions

Journals/Publications

• Kridler, M. R., Howe, A., Legins, J. A., Guerrero, C., Bartelme, R. P., Taylor, B., & Carini, P. (2025). High-quality PacBio draft genome sequences of 17 free-living Bradyrhizobium and four related Nitrobacteraceae strains isolated from arid soils in the Santa Catalina Mountains of Southern Arizona. Access Microbiology, 7(Issue 2). doi:10.1099/acmi.0.000884.v3
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  Non-symbiotic Bradyrhizobium are among the most abundant and ubiquitous microbes in bulk soils globally. Despite this, most available genomic resources for Bradyrhizobium are derived from plant- associated strains. We present high- quality draft genomes for 17 Bradyrhizobium and four Nitrobacteraceae cultures isolated from bulk semiarid soils in Arizona, USA. The genome sizes range from 5.99 to 10.4 Mbp. Phylogenomic analysis of the 21 genomes indicates they fall into four clades. Two of the clades are nested within the Bradyrhizobium genus. The other two clades were associated with Nitrobacteraceae outgroups basal to Bradyrhizobium. All genomes lack genes coding for molybdenum or vanadium nitrogenases, and nod genes that code for proteins involved in nodulation, suggesting these isolates are free- living, non- symbiotic and do not fix dinitrogen gas. These genomes offer new resources for investigating free- living Bradyrhizobium lineages.
• Snoeyenbos-West, O. L., Guerrero, C. R., Valencia, M., & Carini, P. (2025). Erratum: Correction for Snoeyenbos-West et al., "Cultivating efficiency: high-throughput growth analysis of anaerobic bacteria in compact microplate readers" (Microbiology spectrum (2024) 12 5 DOI: 10.1128/spectrum.03650-23.). Microbiology spectrum, 13(Issue 4). doi:10.1128/spectrum.03322-24
• Barnum, T. P., Crits-Christoph, A., Molla, M. M., Carini, P., Lee, H. H., & Ostrov, N. (2024). Predicting microbial growth conditions from amino acid composition. BioRxiv. doi:10.1101/2024.03.22.586313
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  The ability to grow a microbe in the laboratory enables reproducible study and engineering of its genetics. Unfortunately, the majority of microbes in the tree of life remain uncultivated because of the effort required to identify culturing conditions. Predictions of viable growth conditions to guide experimental testing would be highly desirable. While carbon and energy sources can be computationally predicted with annotated genes, it is harder to predict other requirements for growth such as oxygen, temperature, salinity, and pH. Here, we developed genome-based computational models capable of predicting oxygen tolerance (92% balanced accuracy), optimum temperature (R2=0.73), salinity (R2=0.81) and pH (R2=0.48) for novel taxonomic microbial families without requiring functional gene annotations. Using growth conditions and genome sequences of 15,596 bacteria and archaea, we found that amino acid frequencies are predictive of growth requirements. As little as two amino acids can predict oxygen tolerance with 88% balanced accuracy. Using cellular localization of proteins to compute amino acid frequencies improved prediction of pH (R2 increase of 0.36). Because these models do not rely on the presence or absence of specific genes, they can be applied to incomplete genomes, requiring as little as 10% completeness. We applied our models to predict growth requirements for all 85,205 species of sequenced bacteria and archaea and found that uncultivated species are enriched in thermophiles, anaerobes, and acidophiles. Finally, we applied our models to 3,349 environmental samples with metagenome-assembled genomes and showed that individual microbes within a community have differing growth requirements. This work guides identification of growth constraints for laboratory cultivation of diverse microbes.
• Carini, P. (2024). Predicting microbial growth conditions from amino acid composition. BioRxiv. doi:https://doi.org/10.1101/2024.03.22.586313
• Carini, P. (2024). Draft genome sequences of Arthrobacter sp. AZCC_0090 and Mycobacterium sp. AZCC_0083 isolated from oligotrophic subsurface forest soil in the Santa Catalina mountains of Southern Arizona. Microbiology Resource Announcements, 13(3). doi:10.1128/mra.01089-23
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  ABSTRACT Here, we present the genomes of two soil actinobacteria: Arthrobacter sp. strain AZCC_0090 and Mycobacterium sp. strain AZCC_0083, isolated from oligotrophic subsurface soils in Southern Arizona, USA.
• Kridler, M. R., Viney, I. A., Custer, J. M., Schlottman, B., Bartelme, R., & Carini, P. (2024). Draft genome sequences of sp. AZCC_0090 and sp. AZCC_0083 isolated from oligotrophic subsurface forest soil in the Santa Catalina mountains of Southern Arizona. Microbiology resource announcements, 13(3), e0108923.
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  Here, we present the genomes of two soil actinobacteria: sp. strain AZCC_0090 and sp. strain AZCC_0083, isolated from oligotrophic subsurface soils in Southern Arizona, USA.
• Kridler, M., Viney, I., Custer, J., Schlottman, B., Bartelme, R., & Carini, P. (2024). Draft genome sequences of Arthrobacter sp. AZCC_0090 and Mycobacterium sp. AZCC_0083 isolated from oligotrophic subsurface forest soil in the Santa Catalina mountains of Southern Arizona. Microbiology Resource Announcements, 13(3). doi:10.1128/mra.01089-23
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  Here, we present the genomes of two soil actinobacteria: Arthrobacter sp. strain AZCC_0090 and Mycobacterium sp. strain AZCC_0083, isolated from oligotrophic subsurface soils in Southern Arizona, USA.
• Snoeyenbos-West, O. L., Guerrero, C. R., Valencia, M., & Carini, P. (2024). Cultivating efficiency: high-throughput growth analysis of anaerobic bacteria in compact microplate readers. Microbiology spectrum, 12(5), e0365023.
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  Anaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource-intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics. Yet, their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions using DSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grown and DSM 29485 to different doses of the toxin sodium arsenite. The growth of and was sensitive to arsenite doses of 1.3 µM and 0.4 µM, respectively. Complete inhibition of growth was achieved at 38 µM arsenite for and 338 µM in . These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus.
• Snoeyenbos-West, O., , ., Guerrero, C., Valencia, M., & Carini, P. (2024). Cultivating efficiency: high-throughput growth analysis of anaerobic bacteria in compact microplate readers. Microbiology Spectrum, 12(5). doi:10.1128/spectrum.03650-23
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  Anaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource-intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics. Yet, their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions using Escherichia coli DSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grown E. coli and Clostridium bolteae DSM 29485 to different doses of the toxin sodium arsenite. The growth of E. coli and C. bolteae was sensitive to arsenite doses of 1.3 µM and 0.4 µM, respectively. Complete inhibition of growth was achieved at 38 µM arsenite for C. bolteae and 338 µM in E. coli. These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus. IMPORTANCE We present a workflow that facilitates the production and analysis of growth curves for anaerobic microbes using small-footprint microplate readers and an R script. This workflow is a cost and space-effective solution to most high-throughput solutions for collecting growth data from anaerobic microbes. This technology can be used for applications where high throughput would advance discovery, including microbial isolation, bioprospecting, co-culturing, host-microbe interactions, and drug/ toxin-microbial interactions.
• Snoeyenbos-West, O. L., Guerrero, C. R., Valencia, M., & Carini, P. (2023). Cultivating efficiency: High-throughput growth analysis of anaerobic bacteria in compact microplate readers. bioRxiv : the preprint server for biology.
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  Anaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics but their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions using DSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grown and DSM 29485 to different doses of the toxin sodium arsenite. The growth of and was sensitive to arsenite doses of 1.3 μM and 0.4 μM, respectively. Complete inhibition of growth was achieved at 38 μM arsenite for , and 338 μM in . These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus.
• Carini, P., & Snoeyenbos-West, O. (2022). The Microbial Borg: New Allies Against Climate Change?. GEN Biotechnology, 1(6), 485-486. doi:10.1089/genbio.2022.29067.osw
• Carini, P., & Snoeyenbos-West, O. (2022). The Microbial Borg: New Allies Against Climate Change?. GEN Biotechnology. doi:10.1089/genbio.2022.29067.osw
• Carini, P. (2021). Hazardous gases sustain microbes underfoot.. Nature microbiology, 6(2), 145-146. doi:10.1038/s41564-020-00855-y
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  Most soil microorganisms can use the trace gases carbon monoxide, hydrogen and methane — and potentially other inorganic compounds — to supplement their cellular energetic needs.
• Emerson, D., Yarza, P., Whitman, W. B., Wang, F., Wagner, M., Venter, S. N., Vandamme, P., Tiedje, J. M., Thrash, J. C., Sutcliffe, I. C., Stott, M. B., Stewart, F. J., Steenkamp, E. T., Steen, A. D., Spang, A., Smits, T. H., Santos, P. E., Rossello-mora, R., Rios, M. A., , Reysenbach, A., et al. (2021). Author Correction: Roadmap for naming uncultivated Archaea and Bacteria.. Nature microbiology, 6(1), 136. doi:10.1038/s41564-020-00827-2
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  In the version of this Consensus Statement originally published, Pablo Yarza was mistakenly not included in the author list. Also, in Supplementary Table 1, Alexander Jaffe was missing from the list of endorsees. These errors have now been corrected and the updated Supplementary Table 1 is available online.
• Orsi, W. D., Magritsch, T., Vargas, S., Coskun, Ö. K., Vuillemin, A., Höhna, S., Wörheide, G., D'Hondt, S., Shapiro, B. J., & Carini, P. (2021). Genome Evolution in Bacteria Isolated from Million-Year-Old Subseafloor Sediment. mBio, 12(4), e0115021.
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  Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes have been unknown. Here, we show that the genomes of bacterial populations cultured from million-year-old subseafloor sediments evolve in clonal populations by point mutation, with a relatively low rate of homologous recombination and elevated numbers of pseudogenes. Ratios of nonsynonymous to synonymous substitutions correlate with the accumulation of pseudogenes, consistent with a role for genetic drift in the subseafloor strains but not in type strains of isolated from the surface world. Consistent with this, pangenome analysis reveals that the subseafloor bacterial genomes have a significantly lower number of singleton genes than the type strains, indicating a reduction in recent gene acquisitions. Numerous insertion-deletion events and pseudogenes were present in a flagellar operon of the subseafloor bacteria, indicating that motility is nonessential in these million-year-old subseafloor sediments. This genomic evolution in subseafloor clonal populations coincided with a phenotypic difference: all subseafloor isolates have a lower rate of growth under laboratory conditions than the Thalassospira xiamenensis type strain. Our findings demonstrate that the long-term physical isolation of , in the absence of recombination, has resulted in clonal populations whereby reduced access to novel genetic material from neighbors has resulted in the fixation of new mutations that accumulate in genomes over millions of years. The nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor are unknown. Subseafloor populations have ultralow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting that the genome evolution of these bacteria has been dominated by genetic drift. Thus, reduced recombination, stemming from long-term physical isolation, resulted in small clonal populations of that have accumulated mutations in their genomes over millions of years.
• Orsi, W. D., Magritsch, T., Vargas, S., Coskun, Ö. K., Vuillemin, A., Höhna, S., Wörheide, G., D’Hondt, S., Shapiro, B. J., & Carinih, P. (2021). Genome Evolution in Bacteria Isolated from Million-Year-Old Subseafloor Sediment. mBio, 12(Issue 4). doi:10.1128/mbio.01150-21
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  Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes have been unknown. Here, we show that the genomes of Thalassospira bacterial populations cultured from million-year-old subseafloor sediments evolve in clonal populations by point mutation, with a relatively low rate of homologous recombination and elevated numbers of pseudogenes. Ratios of nonsynonymous to synonymous substitutions correlate with the accumulation of pseudogenes, consistent with a role for genetic drift in the subseafloor strains but not in type strains of Thalassospira isolated from the surface world. Consistent with this, pangenome analysis reveals that the subseafloor bacterial genomes have a significantly lower number of singleton genes than the type strains, indicating a reduction in recent gene acquisitions. Numerous insertion-deletion events and pseudogenes were present in a flagellar operon of the subseafloor bacteria, indicating that motility is nonessential in these million-year-old subseafloor sediments. This genomic evolution in subseafloor clonal populations coincided with a phenotypic difference: all subseafloor isolates have a lower rate of growth under laboratory conditions than the Thalassospira xiamenensis type strain. Our findings demonstrate that the long-term physical isolation of Thalassospira, in the absence of recombination, has resulted in clonal populations whereby reduced access to novel genetic material from neighbors has resulted in the fixation of new mutations that accumulate in genomes over millions of years. IMPORTANCE The nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor are unknown. Subseafloor populations have ultralow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus Thalassospira isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting that the genome evolution of these bacteria has been dominated by genetic drift. Thus, reduced recombination, stemming from long-term physical isolation, resulted in small clonal populations of Thalassospira that have accumulated mutations in their genomes over millions of years.
• Bartelme, R. P., Custer, J. M., Dupont, C. L., Espinoza, J. L., Torralba, M., Khalili, B., & Carini, P. (2020). Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation. mSphere, 5(1).
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  The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted cells extracted from soil into one of two custom-designed defined growth media that differed by 100-fold in the concentration of amino acids and organic carbon. Across the two media, we isolated a total of 133 pure cultures, all of which were classified as or The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of We isolated cultures that were representative of the most abundant phylotype in the soil microbial community ( spp.) and representatives of five of the top 10 most abundant phylotypes, including spp., spp., and several other phylogenetically divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and that the concentration of substrates in culture medium influences the culturability of specific microbial lineages. Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities-the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.
• Bartelme, R. P., Custer, J. M., Dupont, C. L., Espinoza, J. L., Torralba, M., Khalili, B., & Carini, P. (2020). Influence of substrate concentration on the culturability of heterotrophic soil microbes isolated by high-throughput dilution-to-extinction cultivation. mSphere, 5(Issue 1). doi:10.1128/msphere.0024-20
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  The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-toextinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted cells extracted from soil into one of two custom-designed defined growth media that differed by 100-fold in the concentration of amino acids and organic carbon. Across the two media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria. The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria. We isolated cultures that were representative of the most abundant phylotype in the soil microbial community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and that the concentration of substrates in culture medium influences the culturability of specific microbial lineages. IMPORTANCE Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities-the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.
• Carini, P. (2020). Genome evolution in bacteria isolated from million-year-old subseafloor sediment. mbio. doi:10.1101/2020.12.19.423498
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  Abstract Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes has been unknown. Here we show that the genomes of Thalassospira bacterial populations cultured from million-year-old subseafloor sediments evolve by point mutation, with a relatively low rate of homologous recombination and a high frequency of pseudogenes. Ratios of synonymous to non-synonymous mutation rates correlate with the accumulation of pseudogenes, consistent with a dominant role for genetic drift in the subseafloor strains, but not in type strains of Thalassospira isolated from the surface world. Our findings demonstrate that the long term physical isolation of these bacteria, in the absence of recombination, has resulted in clonal populations that evolve consistent with ‘Mullers Ratchet’, whereby reduced access to novel genetic material from neighbors has resulted in fixation of new mutations that accumulate in genomes over millions of years. Significance statement The nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor is unknown. Subseafloor populations have ultra-slow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus Thalassospira isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting the genome evolution of these bacteria has been dominated by genetic drift, whereby under long-term physical isolation in small population sizes, and in the absence of homologous recombination, newly acquired mutations accumulate in the genomes of clonal populations over millions of years.
• Carini, P. (2020). Microbial Methane from Methylphosphonate Isotopically Records Source. Geophysical Research Letters.
• Carini, P., Delgado-Baquerizo, M., Hinckley, E. L., Holland-Moritz, H., Brewer, T. E., Rue, G., Vanderburgh, C., McKnight, D., & Fierer, N. (2020). Effects of spatial variability and relic DNA removal on the detection of temporal dynamics in soil microbial communities. mBio, 11(Issue 1). doi:10.1128/mbio.02776-19
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  Few studies have comprehensively investigated the temporal variability in soil microbial communities despite widespread recognition that the belowground environment is dynamic. In part, this stems from the challenges associated with the high degree of spatial heterogeneity in soil microbial communities and because the presence of relic DNA (DNA from dead cells or secreted extracellular DNA) may dampen temporal signals. Here, we disentangle the relationships among spatial, temporal, and relic DNA effects on prokaryotic and fungal communities in soils collected from contrasting hillslopes in Colorado, USA. We intensively sampled plots on each hillslope over 6 months to discriminate between temporal variability, intraplot spatial heterogeneity, and relic DNA effects on the soil prokaryotic and fungal communities. We show that the intraplot spatial variability in microbial community composition was strong and independent of relic DNA effects and that these spatial patterns persisted throughout the study. When controlling for intraplot spatial variability, we identified significant temporal variability in both plots over the 6-month study. These microbial communities were more dissimilar over time after relic DNA was removed, suggesting that relic DNA hinders the detection of important temporal dynamics in belowground microbial communities. We identified microbial taxa that exhibited shared temporal responses and show that these responses were often predictable from temporal changes in soil conditions. Our findings highlight approaches that can be used to better characterize temporal shifts in soil microbial communities, information that is critical for predicting the environmental preferences of individual soil microbial taxa and identifying linkages between soil microbial community composition and belowground processes. IMPORTANCE Nearly all microbial communities are dynamic in time. Understanding how temporal dynamics in microbial community structure affect soil biogeochemis-try and fertility are key to being able to predict the responses of the soil microbiome to environmental perturbations. Here, we explain the effects of soil spatial structure and relic DNA on the determination of microbial community fluctuations over time. We found that intensive spatial sampling was required to identify temporal effects in microbial communities because of the high degree of spatial heterogeneity in soil and that DNA from nonliving sources masks important temporal patterns. We identified groups of microbes with shared temporal responses and show that these patterns were predictable from changes in soil characteristics. These results provide insight into the environmental preferences and temporal relationships between individual microbial taxa and highlight the importance of considering relic DNA when trying to detect temporal dynamics in belowground communities.
• Carini, P., Delgado-Baquerizo, M., Hinckley, E. S., Holland-Moritz, H., Brewer, T. E., Rue, G., Vanderburgh, C., McKnight, D., & Fierer, N. (2020). Effects of Spatial Variability and Relic DNA Removal on the Detection of Temporal Dynamics in Soil Microbial Communities. mBio, 11(1).
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  Few studies have comprehensively investigated the temporal variability in soil microbial communities despite widespread recognition that the belowground environment is dynamic. In part, this stems from the challenges associated with the high degree of spatial heterogeneity in soil microbial communities and because the presence of relic DNA (DNA from dead cells or secreted extracellular DNA) may dampen temporal signals. Here, we disentangle the relationships among spatial, temporal, and relic DNA effects on prokaryotic and fungal communities in soils collected from contrasting hillslopes in Colorado, USA. We intensively sampled plots on each hillslope over 6 months to discriminate between temporal variability, intraplot spatial heterogeneity, and relic DNA effects on the soil prokaryotic and fungal communities. We show that the intraplot spatial variability in microbial community composition was strong and independent of relic DNA effects and that these spatial patterns persisted throughout the study. When controlling for intraplot spatial variability, we identified significant temporal variability in both plots over the 6-month study. These microbial communities were more dissimilar over time after relic DNA was removed, suggesting that relic DNA hinders the detection of important temporal dynamics in belowground microbial communities. We identified microbial taxa that exhibited shared temporal responses and show that these responses were often predictable from temporal changes in soil conditions. Our findings highlight approaches that can be used to better characterize temporal shifts in soil microbial communities, information that is critical for predicting the environmental preferences of individual soil microbial taxa and identifying linkages between soil microbial community composition and belowground processes. Nearly all microbial communities are dynamic in time. Understanding how temporal dynamics in microbial community structure affect soil biogeochemistry and fertility are key to being able to predict the responses of the soil microbiome to environmental perturbations. Here, we explain the effects of soil spatial structure and relic DNA on the determination of microbial community fluctuations over time. We found that intensive spatial sampling was required to identify temporal effects in microbial communities because of the high degree of spatial heterogeneity in soil and that DNA from nonliving sources masks important temporal patterns. We identified groups of microbes with shared temporal responses and show that these patterns were predictable from changes in soil characteristics. These results provide insight into the environmental preferences and temporal relationships between individual microbial taxa and highlight the importance of considering relic DNA when trying to detect temporal dynamics in belowground communities.
• Murray, A. E., Freudenstein, J., Gribaldo, S., Hatzenpichler, R., Hugenholtz, P., Kämpfer, P., Konstantinidis, K. T., Lane, C. E., Papke, R. T., Parks, D. H., Rossello-Mora, R., Stott, M. B., Sutcliffe, I. C., Thrash, J. C., Venter, S. N., Whitman, W. B., Acinas, S. G., Amann, R. I., Anantharaman, K., , Armengaud, J., et al. (2020). Author Correction: Roadmap for naming uncultivated Archaea and Bacteria. Nature microbiology.
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  An amendment to this paper has been published and can be accessed via a link at the top of the paper.
• Murray, A. E., Freudenstein, J., Gribaldo, S., Hatzenpichler, R., Hugenholtz, P., Kämpfer, P., Konstantinidis, K. T., Lane, C. E., Papke, R. T., Parks, D. H., Rossello-Mora, R., Stott, M. B., Sutcliffe, I. C., Thrash, J. C., Venter, S. N., Whitman, W. B., Acinas, S. G., Amann, R. I., Anantharaman, K., , Armengaud, J., et al. (2020). Roadmap for naming uncultivated Archaea and Bacteria. Nature microbiology, 5(8), 987-994.
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  The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.
• Taenzer, L., Carini, P. C., Masterson, A. M., Bourque, B., Gaube, J. H., & Leavitt, W. D. (2020). Microbial Methane From Methylphosphonate Isotopically Records Source. Geophysical Research Letters, 47(Issue 1). doi:10.1029/2019gl085872
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  Methane is a potent greenhouse gas commonly supersaturated in the oxic surfaces waters of oceans and lakes, yet canonical microbial methanogens are obligate anaerobes. One proposed methane production pathway involves microbial degradation of methylphosphonate (MPn), which can proceed in the presence of oxygen. Directly tracing dissolved methane to its source in oxic waters, however, remains a challenge. To address this knowledge gap, we quantified the carbon isotopic fractionation between substrate MPn and product methane (1.3‰) in lab experiments, which was 1 to 2 orders of magnitude smaller than canonical pathways of microbial methanogenesis (20 to 100‰). Together, these results indicated that microbial catabolism of MPn is a source of methane in surface oceans and lake waters, but to differentiate sources of MPn in nature a further accounting of all sources is necessary. Methane from this pathway must be considered in constraining the marine carbon cycle and methane budget.
• Carini, P. (2019). A "Cultural" Renaissance: Genomics Breathes New Life into an Old Craft. MSYSTEMS, 4(3).
• Carini, P. (2019). The influence of substrate concentration on the culturability of heterotrophic soil microbes isolated by high-throughput dilution-to-extinction cultivation. msphere. doi:10.1101/726661
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  Abstract The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted extracted cells into one of two custom-designed defined growth media that differed only by a 100-fold difference in the concentration of amino acids and organic carbon. Across both media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria. The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria. We isolated cultures that were representative of the most abundant phylotype in the soil microbial community ( Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically-divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic-acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and the concentration of substrates in culture medium influences the culturability of specific microbial lineages. Importance Isolating environmental microbes and studying their physiology under controlled conditions is an essential aspect of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities—the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.
• Steen, A. D., Crits-Christoph, A., Carini, P., DeAngelis, K. M., Fierer, N., Lloyd, K. G., & Cameron Thrash, J. (2019). High proportions of bacteria and archaea across most biomes remain uncultured. ISME Journal, 13(Issue 12). doi:10.1038/s41396-019-0484-y
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  A recent paper by Martiny argues that “high proportions” of bacteria in diverse Earth environments have been cultured. Here we reanalyze a portion of the data in that paper, and argue that the conclusion is based on several technical errors, most notably a calculation of sequence similarity that does not account for sequence gaps, and the reliance on 16S rRNA gene amplicons that are known to be biased towards cultured organisms. We further argue that the paper is also based on a conceptual error: namely, that sequence similarity cannot be used to infer “culturability” because one cannot infer physiology from 16S rRNA gene sequences. Combined with other recent, more reliable studies, the evidence supports the conclusion that most bacterial and archaeal taxa remain uncultured.
• Steen, A. D., Crits-Christoph, A., Carini, P., DeAngelis, K. M., Fierer, N., Lloyd, K. G., & Thrash, J. C. (2019). High proportions of bacteria and archaea across most biomes remain uncultured. ISME JOURNAL, 13(12), 3126-3130.
• Becker, K. W., Collins, J. R., Durham, B. P., Groussman, R. D., White, A. E., Fredricks, H. F., Ossolinski, J. E., Repeta, D. J., Carini, P., Armbrust, E. V., & Van Mooy, B. A. (2018). Daily changes in phytoplankton lipidomes reveal mechanisms of energy storage in the open ocean. Nature Communications, 9(Issue 1). doi:10.1038/s41467-018-07346-z
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  Sunlight is the dominant control on phytoplankton biosynthetic activity, and darkness deprives them of their primary external energy source. Changes in the biochemical composition of phytoplankton communities over diel light cycles and attendant consequences for carbon and energy flux in environments remain poorly elucidated. Here we use lipidomic data from the North Pacific subtropical gyre to show that biosynthesis of energy-rich triacylglycerols (TAGs) by eukaryotic nanophytoplankton during the day and their subsequent consumption at night drives a large and previously uncharacterized daily carbon cycle. Diel oscillations in TAG concentration comprise 23 ± 11% of primary production by eukaryotic nanophytoplankton representing a global flux of about 2.4 Pg C yr−1. Metatranscriptomic analyses of genes required for TAG biosynthesis indicate that haptophytes and dinoflagellates are active members in TAG production. Estimates suggest that these organisms could contain as much as 40% more calories at sunset than at sunrise due to TAG production.
• Becker, K. W., Collins, J. R., Durham, B. P., Groussman, R. D., White, A. E., Fredricks, H. F., Ossolinski, J. E., Repeta, D. J., Carini, P., Armbrust, E. V., & Van, M. (2018). Daily changes in phytoplankton lipidomes reveal mechanisms of energy storage in the open ocean. NATURE COMMUNICATIONS, 9.
• Carini, P. (2018). Unraveling the effects of spatial variability and relic DNA on the temporal dynamics of soil microbial communities. mbio. doi:10.1101/402438
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  Abstract Few studies have comprehensively investigated the temporal variability in soil microbial communities despite widespread recognition that the belowground environment is dynamic. In part, this stems from the challenges associated with the high degree of spatial heterogeneity in soil microbial communities and because the presence of relic DNA (DNA from non-living cells) may dampen temporal signals. Here we disentangle the relationships among spatial, temporal, and relic DNA effects on bacterial, archaeal, and fungal communities in soils collected from contrasting hillslopes in Colorado, USA. We intensively sampled plots on each hillslope over six months to discriminate between temporal variability, intra-plot spatial heterogeneity, and relic DNA effects on the soil prokaryotic and fungal communities. We show that the intra-plot spatial variability in microbial community composition was strong and independent of relic DNA effects with these spatial patterns persisting throughout the study. When controlling for intra-plot spatial variability, we identified significant temporal variability in both plots over the six-month study. These microbial communities were more dissimilar over time after relic DNA was removed, suggesting that relic DNA hinders the detection of important temporal dynamics in belowground microbial communities. We identified microbial taxa that exhibited shared temporal responses and show these responses were often predictable from temporal changes in soil conditions. Our findings highlight approaches that can be used to better characterize temporal shifts in soil microbial communities, information that is critical for predicting the environmental preferences of individual soil microbial taxa and identifying linkages between soil microbial community composition and belowground processes. Importance Nearly all microbial communities are dynamic in time. Understanding how temporal dynamics in microbial community structure affect soil biogeochemistry and fertility are key to being able to predict the responses of the soil microbiome to environmental perturbations. Here we explain the effects of soil spatial structure and relic DNA on the determination of microbial community fluctuations over time. We found that intensive spatial sampling is required to identify temporal effects in microbial communities because of the high degree of spatial heterogeneity in soil and that DNA from non-living microbial cells masks important temporal patterns. We identified groups of microbes that display correlated behavior over time and show that these patterns are predictable from soil characteristics. These results provide insight into the environmental preferences and temporal relationships between individual microbial taxa and highlight the importance of considering relic DNA when trying to detect temporal dynamics in belowground communities.
• Carini, P., Becker, K. W., Collins, J. R., Durham, B. R., Groussman, R. D., White, A. E., Fredricks, H. F., Ossolinski, J. E., Repeta, D. J., Armbrust, E. V., & Van Mooy, B. A. (2018). Daily changes in phytoplankton lipidomes reveal mechanisms of energy storage in the open ocean.. Nature Communications. doi:https://doi.org/10.1038/s41467-018-07346-z
• Carini, P., Dupont, C. L., & Santoro, A. E. (2018). Patterns of thaumarchaeal gene expression in culture and diverse marine environments. ENVIRONMENTAL MICROBIOLOGY, 20(6), 2112-2124.
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  Preprint of this paper is availible here: https://www.biorxiv.org/content/early/2018/03/19/175141
• Carini, P., Dupont, C. L., & Santoro, A. E. (2018). Patterns of thaumarchaeal gene expression in culture and diverse marine environments. Environmental Microbiology, 20(Issue 6). doi:10.1111/1462-2920.14107
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  Thaumarchaea are ubiquitous in marine habitats where they participate in carbon and nitrogen cycling. Although metatranscriptomes suggest thaumarchaea are active microbes in marine waters, we understand little about how thaumarchaeal gene expression patterns relate to substrate utilization and activity. Here, we report the global transcriptional response of the marine ammonia-oxidizing thaumarchaeon ‘Candidatus Nitrosopelagicus brevis’ str. CN25 to ammonia limitation using RNA-Seq. We further describe the genome and transcriptome of Ca. N. brevis str. U25, a new strain capable of urea utilization. Ammonia limitation in CN25 resulted in reduced expression of transcripts coding for ammonia oxidation proteins, and increased expression of a gene coding an Hsp20-like chaperone. Despite significantly different transcript abundances across treatments, two ammonia monooxygenase subunits (amoAB), a nitrite reductase (nirK) and both ammonium transporter genes were always among the most abundant transcripts, regardless of growth state. Ca. N. brevis str. U25 cells expressed a urea transporter 139-fold more than the urease catalytic subunit ureC. Gene coexpression networks derived from culture transcriptomes and 10 thaumarchaea-enriched metatranscriptomes revealed a high degree of correlated gene expression across disparate environmental conditions and identified a module of coexpressed genes, including amoABC and nirK, that we hypothesize to represent the core ammonia oxidation machinery.
• Brewer, T. E., Handley, K. M., Carini, P., Gilbert, J. A., & Fierer, N. (2017). Genome reduction in an abundant and ubiquitous soil bacterium 'Candidatus Udaeobacter copiosus'. NATURE MICROBIOLOGY, 2(2).
• Carini, P. (2017). Patterns of thaumarchaeal gene expression in culture and diverse marine environments. Environmental Microbiology. doi:10.1101/175141
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  Abstract Thaumarchaea are ubiquitous in marine habitats where they participate in carbon and nitrogen cycling. Although metatranscriptomes suggest thaumarchaea are active microbes in marine waters, we understand little about how thaumarchaeal gene expression patterns relate to substrate utilization and activity. Here, we report the global transcriptional response of the marine ammonia-oxidizing thaumarchaeon ‘ Candidatus Nitrosopelagicus brevis’ str. CN25 to ammonia limitation using RNA-Seq. We further describe the genome and transcriptome of Ca . N. brevis str. U25, a new strain capable of urea utilization. Ammonia limitation in CN25 resulted in reduced expression of transcripts coding for ammonia oxidation proteins, and increased expression of a gene coding an Hsp20-like chaperone. Despite significantly different transcript abundances across treatments, two ammonia monooxygenase subunits ( amoAB ), a nitrite reductase ( nirK ), and both ammonium transporter genes were always among the most abundant transcripts, regardless of growth state. Ca . N. brevis str. U25 cells expressed a urea transporter 139-fold more than the urease catalytic subunit ureC . Gene co-expression networks derived from culture transcriptomes and ten thaumarchaea-enriched metatranscriptomes revealed a high degree of correlated gene expression across disparate environmental conditions and identified a module of genes, including amoABC and nirK , that we hypothesize to represent the core ammonia oxidation machinery. Originality-Significance Statement Discovering gene function in fastidious or uncultivated lineages remains one of the biggest challenges in environmental microbiology. Here, we use an approach that combines controlled laboratory experiments with in situ transcript abundance data from the environment to identify genes that share similar transcription patterns in marine ammonia-oxidizing thaumarchaea. These findings demonstrate how transcriptomes from microbial cultures can be used together with complex environmental samples to identify suites of co-expressed genes that are otherwise enigmatic and provide new insights into the mechanism of ammonia oxidation. Our results add to the growing body of literature showing that relatively small changes in transcript abundance are linked to large changes in growth in organisms with reduced genomes, suggesting they have limited capacity for metabolic regulation or that they rely on mechanisms other than transcriptional regulation to deal with a fluctuating environment.
• Carini, P., Marsden, P. J., Leff, J., Morgan, E. E., Strickland, M. S., & Fierer, N. (2017). Relic DNA is abundant in soil and obscures estimates of soil microbial diversity. NATURE MICROBIOLOGY, 2(3).
• Carini, P. (2016). Genome reduction in an abundant and ubiquitous soil bacterial lineage. Nature Microbiology. doi:10.1101/053942
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  Abstract Although bacteria within the Verrucomicrobia phylum are pervasive in soils around the world, they are underrepresented in both isolate collections and genomic databases. Here we describe a single verrucomicrobial phylotype within the class Spartobacteria that is not closely related to any previously described taxa. We examined >1000 soils and found this spartobacterial phylotype to be ubiquitous and consistently one of the most abundant soil bacterial phylotypes, particularly in grasslands, where it was typically the most abundant phylotype. We reconstructed a nearly complete genome of this phylotype from a soil metagenome for which we propose the provisional name ‘ Candidatus Udaeobacter copiosus’. The Ca . U. copiosus genome is unusually small for soil bacteria, estimated to be only 2.81 Mbp compared to the predicted effective mean genome size of 4.74 Mbp for soil bacteria. Metabolic reconstruction suggests that Ca . U. copiosus is an aerobic chemoorganoheterotroph with numerous amino acid and vitamin auxotrophies. The large population size, relatively small genome and multiple putative auxotrophies characteristic of Ca . U. copiosus suggests that it may be undergoing streamlining selection to minimize cellular architecture, a phenomenon previously thought to be restricted to aquatic bacteria. Although many soil bacteria need relatively large, complex genomes to be successful in soil, Ca . U. copiosus appears to have identified an alternate strategy, sacrificing metabolic versatility for efficiency to become dominant in the soil environment.
• Carini, P. (2016). Microbial oxidation of DMS to DMSO: a biochemical surprise with geochemical implications. ENVIRONMENTAL MICROBIOLOGY, 18(8), 2302-2304.
• Carini, P. (2016). Microbial oxidation of DMS to DMSO: a biochemical surprise with geochemical implications. Environmental microbiology, 18(Issue 8). doi:10.1111/1462-2920.13317
• Handley, K. M., Gilbert, J. A., Fierer, N., Carini, P., & Brewer, T. E. (2016). Genome reduction in an abundant and ubiquitous soil bacterium 'Candidatus Udaeobacter copiosus'.. Nature microbiology, 2(2), 16198. doi:10.1038/nmicrobiol.2016.198
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  Although bacteria within the Verrucomicrobia phylum are pervasive in soils around the world, they are under-represented in both isolate collections and genomic databases. Here, we describe a single verrucomicrobial group within the class Spartobacteria that is not closely related to any previously described taxa. We examined more than 1,000 soils and found this spartobacterial phylotype to be ubiquitous and consistently one of the most abundant soil bacterial phylotypes, particularly in grasslands, where it was typically the most abundant. We reconstructed a nearly complete genome of this phylotype from a soil metagenome for which we propose the provisional name 'Candidatus Udaeobacter copiosus'. The Ca. U. copiosus genome is unusually small for a cosmopolitan soil bacterium, estimated by one measure to be only 2.81 Mbp, compared to the predicted effective mean genome size of 4.74 Mbp for soil bacteria. Metabolic reconstruction suggests that Ca. U. copiosus is an aerobic heterotroph with numerous putative amino acid and vitamin auxotrophies. The large population size, relatively small genome and multiple putative auxotrophies characteristic of Ca. U. copiosus suggest that it may be undergoing streamlining selection to minimize cellular architecture, a phenomenon previously thought to be restricted to aquatic bacteria. Although many soil bacteria need relatively large, complex genomes to be successful in soil, Ca. U. copiosus appears to use an alternative strategy, sacrificing metabolic versatility for efficiency to become dominant in the soil environment.
• Smith, D. P., Nicora, C. D., Carini, P., Lipton, M. S., Norbeck, A. D., Smith, R. D., & Giovannoni, S. J. (2016). Proteome Remodeling in Response to Sulfur Limitation in "Candidatus Pelagibacter ubique". MSYSTEMS, 1(4).
• Smith, D. P., Nicora, C. D., Carini, P., Lipton, M. S., Norbeck, A. D., Smith, R. D., & Giovannoni, S. J. (2016). Proteome remodeling in response to sulfur limitation in "Candidatus pelagibacter ubique". mSystems, 1(Issue 4). doi:10.1128/msystems.00068-16
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  The alphaproteobacterium "Candidatus Pelagibacter ubique" strain HTCC1062 and most other members of the SAR11 clade lack genes for assimilatory sulfate reduction, making them dependent on organosulfur compounds that occur naturally in seawater. To investigate how these cells adapt to sulfur limitation, batch cultures were grown in defined medium containing either limiting or nonlimiting amounts of dimethylsulfoniopropionate (DMSP) as the sole sulfur source. Protein and mRNA expression were measured before, during, and after the transition from exponential growth to stationary phase. Two distinct responses were observed, one as DMSP became exhausted and another as the cells acclimated to a sulfur-limited environment. The first response was characterized by increased transcription and translation of all "Ca. Pelagibacter ubique" genes downstream from the previously confirmed S-adenosyl methionine (SAM) riboswitches bhmT, mmuM, and metY. The proteins encoded by these genes were up to 33 times more abundant as DMSP became limiting. Their predicted function is to shunt all available sulfur to methionine. The secondary response, observed during sulfur-limited stationary phase, was a 6- to 10-fold increase in the transcription of the heme c shuttleencoding gene ccmC and two small genes of unknown function (SAR11-1163 and SAR11-1164). This bacterium's strategy for coping with sulfur stress appears to be intracellular redistribution to support methionine biosynthesis rather than increasing organosulfur import. Many of the genes and SAM riboswitches involved in this response are located in a hypervariable genome region (HVR). One of these HVR genes, ordL, is located downstream from a conserved motif that evidence suggests is a novel riboswitch.
• Strickland, M. S., Morgan, E. E., Marsden, P. J., Leff, J. W., Fierer, N., & Carini, P. (2016). Relic DNA is abundant in soil and obscures estimates of soil microbial diversity.. Nature microbiology, 2(3), 16242. doi:10.1038/nmicrobiol.2016.242
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  Extracellular DNA from dead microorganisms can persist in soil for weeks to years1-3. Although it is implicitly assumed that the microbial DNA recovered from soil predominantly represents intact cells, it is unclear how extracellular DNA affects molecular analyses of microbial diversity. We examined a wide range of soils using viability PCR based on the photoreactive DNA-intercalating dye propidium monoazide4. We found that, on average, 40% of both prokaryotic and fungal DNA was extracellular or from cells that were no longer intact. Extracellular DNA inflated the observed prokaryotic and fungal richness by up to 55% and caused significant misestimation of taxon relative abundances, including the relative abundances of taxa integral to key ecosystem processes. Extracellular DNA was not found in measurable amounts in all soils; it was more likely to be present in soils with low exchangeable base cation concentrations, and the effect of its removal on microbial community structure was more profound in high-pH soils. Together, these findings imply that this 'relic DNA' remaining in soil after cell death can obscure treatment effects, spatiotemporal patterns and relationships between microbial taxa and environmental conditions.
• Carini, P., Van, M., Thrash, J. C., White, A., Zhao, Y., Campbell, E. O., Fredricks, H. F., & Giovannoni, S. J. (2015). SAR11 lipid renovation in response to phosphate starvation. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 112(25), 7767-7772.
• Orsi, W. D., Smith, J. M., Wilcox, H. M., Swalwell, J. E., Carini, P., Worden, A. Z., & Santoro, A. E. (2015). Ecophysiology of uncultivated marine euryarchaea is linked to particulate organic matter. ISME JOURNAL, 9(8), 1747-1763.
• Santoro, A. E., Dupont, C. L., Richter, R. A., Craig, M. T., Carini, P., McIlvin, M. R., Yang, Y., Orsi, W. D., Moran, D. M., & Saito, M. A. (2015). Genomic and proteomic characterization of "Candidatus Nitrosopelagicus brevis": An ammonia-oxidizing archaeon from the open ocean. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 112(4), 1173-1178.
• Santoro, A. E., Dupont, C. L., Richter, R. A., Craig, M. T., Carini, P., McIlvin, M. R., Yang, Y., Orsi, W. D., Moran, D. M., & Saito, M. A. (2015). Genomic and proteomic characterization of "Candidatus Nitrosopelagicus brevis": An ammonia-oxidizing archaeon from the open ocean. Proceedings of the National Academy of Sciences of the United States of America, 112(Issue 4). doi:10.1073/pnas.1416223112
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  Thaumarchaeota are among the most abundant microbial cells in the ocean, but difficulty in cultivating marine Thaumarchaeota has hindered investigation into the physiological and evolutionary basis of their success. We report here a closed genome assembled from a highly enriched culture of the ammonia-oxidizing pelagic thaumarchaeon CN25, originating from the open ocean. The CN25 genome exhibits strong evidence of genome streamlining, including a 1.23-Mbp genome, a high coding density, and a low number of paralogous genes. Proteomic analysis recovered nearly 70% of the predicted proteins encoded by the genome, demonstrating that a high fraction of the genome is translated. In contrast to other minimal marine microbes that acquire, rather than synthesize, cofactors, CN25 encodes and expresses near-complete biosynthetic pathways for multiple vitamins. Metagenomic fragment recruitment indicated the presence of DNA sequences >90% identical to the CN25 genome throughout the oligotrophic ocean. We propose the provisional name "Candidatus Nitrosopelagicus brevis" str. CN25 for this minimalist marine thaumarchaeon and suggest it as a potentialmodel system for understanding archaeal adaptation to the open ocean.
• Carini, P., Campbell, E. O., Morre, J., Sanudo-Wilhelmy, S. A., Thrash, J. C., Bennett, S. E., Temperton, B., Begley, T., & Giovannoni, S. J. (2014). Discovery of a SAR11 growth requirement for thiamin's pyrimidine precursor and its distribution in the Sargasso Sea. ISME JOURNAL, 8(8), 1727-1738.
• Carini, P., Campbell, E. O., Morré, J., Sañudo-Wilhelmy, S. A., Cameron Thrash, J., Bennett, S. E., Temperton, B., Begley, T., & Giovannoni, S. J. (2014). Discovery of a SAR11 growth requirement for thiamin's pyrimidine precursor and its distribution in the Sargasso Sea. ISME Journal, 8(Issue 8). doi:10.1038/ismej.2014.61
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  Vitamin traffic, the production of organic growth factors by some microbial community members and their use by other taxa, is being scrutinized as a potential explanation for the variation and highly connected behavior observed in ocean plankton by community network analysis. Thiamin (vitamin B 1), a cofactor in many essential biochemical reactions that modify carbon-carbon bonds of organic compounds, is distributed in complex patterns at subpicomolar concentrations in the marine surface layer (0-300 m). Sequenced genomes from organisms belonging to the abundant and ubiquitous SAR11 clade of marine chemoheterotrophic bacteria contain genes coding for a complete thiamin biosynthetic pathway, except for thiC, encoding the 4-amino-5-hydroxymethyl-2- methylpyrimidine (HMP) synthase, which is required for de novo synthesis of thiamin's pyrimidine moiety. Here we demonstrate that the SAR11 isolate 'Candidatus Pelagibacter ubique', strain HTCC1062, is auxotrophic for the thiamin precursor HMP, and cannot use exogenous thiamin for growth. In culture, strain HTCC1062 required 0.7 zeptomoles per cell (ca. 400 HMP molecules per cell). Measurements of dissolved HMP in the Sargasso Sea surface layer showed that HMP ranged from undetectable (detection limit: 2.4 pM) to 35.7 pM, with maximum concentrations coincident with the deep chlorophyll maximum. In culture, some marine cyanobacteria, microalgae and bacteria exuded HMP, and in the Western Sargasso Sea, HMP profiles changed between the morning and evening, suggesting a dynamic biological flux from producers to consumers. © 2014 International Society for Microbial Ecology All rights reserved.
• Carini, P., White, A. E., Campbell, E. O., & Giovannoni, S. J. (2014). Methane production by phosphate-starved SAR11 chemoheterotrophic marine bacteria. NATURE COMMUNICATIONS, 5.
• Carini, P., White, A. E., Campbell, E. O., & Giovannoni, S. J. (2014). Methane production by phosphate-starved SAR11 chemoheterotrophic marine bacteria. Nature Communications, 5(Issue). doi:10.1038/ncomms5346
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  The oxygenated surface waters of the world's oceans are supersaturated with methane relative to the atmosphere, a phenomenon termed the 'marine methane paradox'. The production of methylphosphonic acid (MPn) by marine archaea related to Nitrosopumilus maritimus and subsequent decomposition of MPn by phosphate-starved bacterioplankton may partially explain the excess methane in surface waters. Here we show that Pelagibacterales sp. strain HTCC7211, an isolate of the SAR11 clade of marine α-proteobacteria, produces methane from MPn, stoichiometric to phosphorus consumption, when starved for phosphate. Gene transcripts encoding phosphonate transport and hydrolysis proteins are upregulated under phosphate limitation, suggesting a genetic basis for the methanogenic phenotype. Strain HTCC7211 can also use 2-aminoethylphosphonate and assorted phosphate esters for phosphorus nutrition. Despite strain-specific differences in phosphorus utilization, these findings identify Pelagibacterales bacteria as a source of biogenic methane and further implicate phosphate starvation of chemoheterotrophic bacteria in the long-observed methane supersaturation in oxygenated waters. © 2014 Macmillan Publishers Limited. All rights reserved.
• Carini, P., Steindler, L., Beszteri, S., & Giovannoni, S. J. (2013). Nutrient requirements for growth of the extreme oligotroph 'Candidatus Pelagibacter ubique' HTCC1062 on a defined medium. ISME JOURNAL, 7(3), 592-602.
• Smith, D. P., Thrash, J. C., Nicora, C. D., Lipton, M. S., Burnum-Johnson, K. E., Carini, P., Smith, R. D., & Giovannoni, S. J. (2013). Proteomic and Transcriptomic Analyses of "Candidatus Pelagibacter ubique" Describe the First P-II-Independent Response to Nitrogen Limitation in a Free-Living Alphaproteobacterium. MBIO, 4(6).
• Smith, D. P., Thrash, J. C., Nicora, C. D., Lipton, M. S., Burnum-Johnson, K. E., Carini, P., Smith, R. D., & Giovannoni, S. J. (2013). Proteomic and transcriptomic analyses of "Candidatus Pelagibacter ubique" describe the first PII-independent response to nitrogen limitation in a free-living alphaproteobacterium. mBio, 4(Issue 6). doi:10.1128/mbio.00133-12
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  Nitrogen is one of the major nutrients limiting microbial productivity in the ocean, and as a result, most marine microorganisms have evolved systems for responding to nitrogen stress. The highly abundant alphaproteobacterium "Candidatus Pelagibacter ubique," a cultured member of the order Pelagibacterales (SAR11), lacks the canonical GlnB, GlnD, GlnK, and NtrB/NtrC genes for regulating nitrogen assimilation, raising questions about how these organisms respond to nitrogen limitation. A survey of 266 Alphaproteobacteria genomes found these five regulatory genes nearly universally conserved, absent only in intracellular parasites and members of the order Pelagibacterales, including "Ca. Pelagibacter ubique." Global differences in mRNA and protein expression between nitrogen-limited and nitrogen-replete cultures were measured to identify nitrogen stress responses in "Ca. Pelagibacter ubique" strain HTCC1062. Transporters for ammonium (AmtB), taurine (TauA), amino acids (YhdW), and opines (OccT) were all elevated in nitrogen-limited cells, indicating that they devote increased resources to the assimilation of nitrogenous organic compounds. Enzymes for assimilating amine into glutamine (GlnA), glutamate (GltBD), and glycine (AspC) were similarly upregulated. Differential regulation of the transcriptional regulator NtrX in the two-component signaling system NtrY/NtrX was also observed, implicating it in control of the nitrogen starvation response. Comparisons of the transcriptome and proteome supported previous observations of uncoupling between transcription and translation in nutrientdeprived "Ca. Pelagibacter ubique" cells. Overall, these data reveal a streamlined, PII-independent response to nitrogen stress in "Ca. Pelagibacter ubique," and likely other Pelagibacterales, and show that they respond to nitrogen stress by allocating more resources to the assimilation of nitrogen-rich organic compounds. © 2013 Smith et al.
• Carini, P. (2012). Nutrient requirements for growth of the extreme oligotroph ‘Candidatus Pelagibacter ubique’ HTCC1062 on a defined medium. ISME. doi:10.1038/ismej.2012.122
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  Chemoheterotrophic marine bacteria of the SAR11 clade are Earth's most abundant organisms. Following the first cultivation of a SAR11 bacterium, 'Candidatus Pelagibacter ubique' strain HTCC1062 (Ca. P. ubique) in 2002, unusual nutritional requirements were identified for reduced sulfur compounds and glycine or serine. These requirements were linked to genome streamlining resulting from selection for efficient resource utilization in nutrient-limited ocean habitats. Here we report the first successful cultivation of Ca. P. ubique on a defined artificial seawater medium (AMS1), and an additional requirement for pyruvate or pyruvate precursors. Optimal growth was observed with the collective addition of inorganic macro- and micronutrients, vitamins, methionine, glycine and pyruvate. Methionine served as the sole sulfur source but methionine and glycine were not sufficient to support growth. Optimal cell yields were obtained when the stoichiometry between glycine and pyruvate was 1:4, and incomplete cell division was observed in cultures starved for pyruvate. Glucose and oxaloacetate could fully replace pyruvate, but not acetate, taurine or a variety of tricarboxylic acid cycle intermediates. Moreover, both glycine betaine and serine could substitute for glycine. Interestingly, glycolate partially restored growth in the absence of glycine. We propose that this is the result of the use of glycolate, a product of phytoplankton metabolism, as both a carbon source for respiration and as a precursor to glycine. These findings are important because they provide support for the hypothesis that some micro-organisms are challenging to cultivate because of unusual nutrient requirements caused by streamlining selection and gene loss. Our findings also illustrate unusual metabolic rearrangements that adapt these cells to extreme oligotrophy, and underscore the challenge of reconstructing metabolism from genome sequences in organisms that have non-canonical metabolic pathways.
• Grote, J., Cameron Thrash, J., Huggett, M. J., Landry, Z. C., Carini, P., Giovannoni, S. J., & Rappé, M. S. (2012). Streamlining and core genome conservation among highly divergent members of the SAR11 clade. mBio, 3(Issue 5). doi:10.1128/mbio.00252-12
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  SAR11 is an ancient and diverse clade of heterotrophic bacteria that are abundant throughout the world's oceans, where they play a major role in the ocean carbon cycle. Correlations between the phylogenetic branching order and spatiotem-poral patterns in cell distributions from planktonic ocean environments indicate that SAR11 has evolved into perhaps a dozen or more specialized ecotypes that span evolutionary distances equivalent to a bacterial order. We isolated and sequenced genomes from diverse SAR11 cultures that represent three major lineages and encompass the full breadth of the clade. The new data expand observations about genome evolution and gene content that previously had been restricted to the SAR11 Ia subclade, providing a much broader perspective on the clade's origins, evolution, and ecology. We found small genomes throughout the clade and a very high proportion of core genome genes (48 to 56%), indicating that small genome size is probably an ancestral characteristic. In their level of core genome conservation, the members of SAR11 are outliers, the most conserved free-living bacteria known. Shared features of the clade include low GC content, high gene synteny, a large hypervariable region bounded by rRNA genes, and low numbers of paralogs. Variation among the genomes included genes for phosphorus metabolism, glycolysis, and C1 metabolism, suggesting that adaptive specialization in nutrient resource utilization is important to niche partitioning and ecotype divergence within the clade. These data provide support for the conclusion that streamlining selection for efficient cell replication in the planktonic habitat has occurred throughout the evolution and diversification of this clade. © 2012 Grote et al.
• Grote, J., Thrash, J. C., Huggett, M. J., Landry, Z. C., Carini, P., Giovannoni, S. J., & Rappe, M. S. (2012). Streamlining and Core Genome Conservation among Highly Divergent Members of the SAR11 Clade. MBIO, 3(5).
• Thrash, J. C., Boyd, A., Huggett, M. J., Grote, J., Carini, P., Yoder, R. J., Robbertse, B., Spatafora, J. W., Rappe, M. S., & Giovannoni, S. J. (2011). Phylogenomic evidence for a common ancestor of mitochondria and the SAR11 clade. SCIENTIFIC REPORTS, 1.
• Thrash, J. C., Boyd, A., Huggett, M. J., Grote, J., Carini, P., Yoder, R. J., Robbertse, B., Spatafora, J. W., Rappé, M. S., & Giovannoni, S. J. (2011). Phylogenomic evidence for a common ancestor of mitochondria and the SAR11 clade. Scientific Reports, 1(Issue). doi:10.1038/srep00013
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  Mitochondria share a common ancestor with the Alphaproteobacteria, but determining their precise origins is challenging due to inherent difficulties in phylogenetically reconstructing ancient evolutionary events. Nonetheless, phylogenetic accuracy improves with more refined tools and expanded taxon sampling. We investigated mitochondrial origins with the benefit of new, deeply branching genome sequences from the ancient and prolific SAR11 clade of Alphaproteobacteria and publicly available alphaproteobacterial and mitochondrial genome sequences. Using the automated phylogenomic pipeline Hal, we systematically studied the effect of taxon sampling and missing data to accommodate small mitochondrial genomes. The evidence supports a common origin of mitochondria and SAR11 as a sister group to the Rickettsiales. The simplest explanation of these data is that mitochondria evolved from a planktonic marine alphaproteobacterial lineage that participated in multiple inter-specific cell colonization events, in some cases yielding parasitic relationships, but in at least one case producing a symbiosis that characterizes modern eukaryotic life.
• Kraetzer, C., Carini, P., Hovey, R., & Deppenmeier, U. (2009). Transcriptional Profiling of Methyltransferase Genes during Growth of Methanosarcina mazei on Trimethylamine. JOURNAL OF BACTERIOLOGY, 191(16), 5108-5115.
• Krätzer, C., Carini, P., Hovey, R., & Deppenmeier, U. (2009). Transcriptional profiling of methyltransferase genes during growth of Methanosarcina mazei on trimethylamine. Journal of Bacteriology, 191(Issue 16). doi:10.1128/jb.00420-09
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  The genomic expression patterns of Methanosarcina mazei growing with trimethylamine were measured in comparison to those of cells grown with methanol. We identified a total of 72 genes with either an increased level (49 genes) or a decreased level (23 genes) of mRNA during growth on trimethylamine with methanolgrown cells as the control. Major differences in transcript levels were observed for the mta, mtb, mtt, and mtm genes, which encode enzymes involved in methane formation from methanol and trimethylamine, respectively. Other differences in mRNA abundance were found for genes encoding enzymes involved in isopentenyl pyrophosphate synthesis and in the formation of aromatic amino acids, as well as a number of proteins with unknown functions. The results were verified by in-depth analysis of methyltransferase genes using specific primers for real-time quantitative reverse transcription-PCR (RT-PCR). The monitored transcript levels of genes encoding corrinoid proteins involved in methyl group transfer from methylated C1 compounds (mtaC, mtbC, mttC, and mtmC) indicated increased amounts of mRNA from the mtaBC1, mtaBC2, and mtaBC3 operons in methanol-grown cells, whereas mRNA of the mtb1-mtt1 operon was found in high concentrations during trimethylamine consumption. The genes of the mtb1-mtt1 operon encode methyltransferases that are responsible for sequential demethylation of trimethylamine. The analysis of product formation of trimethylamine-grown cells at different optical densities revealed that large amounts of dimethylamine and monomethylamine were excreted into the medium. The intermediate compounds were consumed only in the very late exponential growth phase. RT-PCR analysis of key genes involved in methanogenesis led to the conclusion that M. mazei is able to adapt to changing trimethylamine concentrations and the consumption of intermediate compounds. Hence, we assume that the organism possesses a regulatory network for optimal substrate utilization. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
• Carini, P. (2007). Fine scale control of microbial communities in deep marine sediments that contain hydrates and high concentrations of methane. conference proceeding.

Presentations

• Carini, P. (2021). Surviving a dry spell: using microbial culture collections to understand the complex phenotype of actinobacterial dehydration-rehydration tolerance.. University of Innsbruck Online seminar series. Innsbruck, Austria.
• Carini, P. (2019, June). High Throughput Cultivation of Bacteria from Shallow Subsurface Soils. ASM Microbe. San Francisco.
• Carini, P. (2020, April). Leveraging microbial cultivation to identify core principles in soil microbial ecology and evolution. MicroSeminar. Live & archived on YouTube: MicroSeminar Series.
• Carini, P. (2020, Fall). Developing microbial culture collections to understand the complex phenotype of actinobacterial desiccation (and rehydration!) tolerance. Invited Seminar; University of California Riverside Plant Pathology Seminar Series. Virtual from my garage in Tucson: University of California Riverside.
• Carini, P. (2020, February). Leveraging microbial cultivation to identify core principles in soil microbial ecology and evolution. Invited Seminar; Department of Plant, Soil, and Microbial Sciences. Michigan State University.
• Carini, P. (2020, November). High-throughput dilution-to-extinction cultivation of bacterial from soil microbiomes. Invited Seminar; International Union of Microbiological Societies Congress (IUMS). South Korea/Virtual: International Union of Microbiological Societies Congress (IUMS).
• Carini, P. (2020, September). High-throughput dilution-to-extinction cultivation of bacterial from soil microbiomes. Invited Seminar; Microbiome Center Arizona State University. Virtual Seminar: Arizona State university Microbiome Center.

Others

• Carini, P. (2017, February). Hindsight 20/20: What I really learned in graduate school. Nature Microbiology Community. https://naturemicrobiologycommunity.nature.com/users/15756-paul-carini/posts/14799-hindsight-20-20-what-i-really-learned-in-graduate-school
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  Blog Post on Nature Microbiology Community Website
• Carini, P. (2016, December). A census of the dead: the story behind microbial relic DNA in soil.. Nature microbiology Community. https://naturemicrobiologycommunity.nature.com/users/15756-paul-carini/posts/14107-a-census-of-the-dead-the-story-behind-relic-dna-in-soil