Sometimes, to move ahead, you must take a look at where you have been. Culturing microbes is a foundational underpinning of microbiology. Before genome sequencing, researchers spent countless hours ...tediously deducing the nutritional requirements of bacterial isolates and tinkering with medium formulations to entice new microbes into culture. This art of cultivation took a back seat to the powerful molecular tools of the last 25 years, and as a result, many researchers have forgotten the utility of having a culture in hand. This perception is changing, as there is clearly a renewed interest in isolating microbes from various environments. Here, I suggest three focus areas to ensure continued growth and success of this "cultural" renaissance, including (i) setting clear cultivation goals, (ii) funding exploratory cultivation, and (iii) culturing and studying unusual organisms. "Unculturable" is a frame of mind, not a state of microbiology; it is time to dust off the bottle of yeast extract.
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.
Summary
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.
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 potential model system for understanding archaeal adaptation to the open ocean.
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.
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.
Particles in aquatic environments host distinct communities of microbes, yet the evolution of particle-specialized taxa and the extent to which specialized microbial metabolism is associated with ...particles is largely unexplored. Here, we investigate the hypothesis that a widely distributed and uncultivated microbial group--the marine group II euryarchaea (MGII)--interacts with living and detrital particulate organic matter (POM) in the euphotic zone of the central California Current System. Using fluorescent in situ hybridization, we verified the association of euryarchaea with POM. We further quantified the abundance and distribution of MGII 16 S ribosomal RNA genes in size-fractionated seawater samples and compared MGII functional capacity in metagenomes from the same fractions. The abundance of MGII in free-living and >3 μm fractions decreased with increasing distance from the coast, whereas MGII abundance in the 0.8-3 μm fraction remained constant. At several offshore sites, MGII abundance was highest in particle fractions, indicating that particle-attached MGII can outnumber free-living MGII under oligotrophic conditions. Compared with free-living MGII, the genome content of MGII in particle-associated fractions exhibits an increased capacity for surface adhesion, transcriptional regulation and catabolism of high molecular weight substrates. Moreover, MGII populations in POM fractions are phylogenetically distinct from and more diverse than free-living MGII. Eukaryotic phytoplankton additions stimulated MGII growth in bottle incubations, providing the first MGII net growth rate measurements. These ranged from 0.47 to 0.54 d(-1). However, MGII were not recovered in whole-genome amplifications of flow-sorted picoeukaryotic phytoplankton and heterotrophic nanoflagellates, suggesting that MGII in particle fractions are not physically attached to living POM. Collectively, our results support a linkage between MGII ecophysiology and POM, implying that marine archaea have a role in elemental cycling through interactions with particles.
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 spatiotemporal 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. IMPORTANCE The SAR11 clade is the most abundant group of marine microorganisms worldwide, making them key players in the global carbon cycle. Growing knowledge about their biochemistry and metabolism is leading to a more mechanistic understanding of organic carbon oxidation and sequestration in the oceans. The discovery of small genomes in SAR11 provided crucial support for the theory that streamlining selection can drive genome reduction in low-nutrient environments. Study of isolates in culture revealed atypical organic nutrient requirements that can be attributed to genome reduction, such as conditional auxotrophy for glycine and its precursors, a requirement for reduced sulfur compounds, and evidence for widespread cycling of C1 compounds in marine environments. However, understanding the genetic variation and distribution of such pathways and characteristics like streamlining throughout the group has required the isolation and genome sequencing of diverse SAR11 representatives, an analysis of which we provide here.
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
. 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.
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