Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in Pseudomonas putida KT2440.

There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules ("functional modules"), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida. Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE: This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

Richness and density jointly determine context dependence in bacterial interactions.

Pairwise interactions are often used to predict features of complex microbial communities due to the challenge of measuring multi-species interactions in high dimensional contexts. This assumes that interactions are unaffected by community context. Here, we used synthetic bacterial communities to investigate that assumption by observing how interactions varied across contexts. Interactions were most often weakly negative and showed a phylogenetic signal among genera. Community richness and total density emerged as strong predictors of interaction strength and contributed to an attenuation of interactions as richness increased. Population level and per-capita measures of interactions both displayed such attenuation, suggesting factors beyond systematic changes in population size were involved; namely, changes to the interactions themselves. Nevertheless, pairwise interactions retained some explanatory power across contexts, provided those contexts were not substantially divergent in richness. These results suggest that understanding the emergent properties of microbial interactions can improve our ability to predict the features of microbial communities.

Metabolite diversity among representatives of divergent Prochlorococcus ecotypes.

IMPORTANCE: Approximately half of the annual carbon fixation on Earth occurs in the surface ocean through the photosynthetic activities of phytoplankton such as the ubiquitous picocyanobacterium Prochlorococcus. Ecologically distinct subpopulations (or ecotypes) of Prochlorococcus are central conduits of organic substrates into the ocean microbiome, thus playing important roles in surface ocean production. We measured the chemical profile of three cultured ecotype strains, observing striking differences among them that have implications for the likely chemical impact of Prochlorococcus subpopulations on their surroundings in the wild. Subpopulations differ in abundance along gradients of temperature, light, and nutrient concentrations, suggesting that these chemical differences could affect carbon cycling in different ocean strata and should be considered in models of Prochlorococcus physiology and marine carbon dynamics.

Environmental and Taxonomic Drivers of Bacterial Extracellular Vesicle Production in Marine Ecosystems.

Extracellular vesicles are small (approximately 50 to 250 nm in diameter), membrane-bound structures that are released by cells into their surrounding environment. Heterogeneous populations of vesicles are abundant in the global oceans, and they likely play a number of ecological roles in these microbially dominated ecosystems. Here, we examine how vesicle production and size vary among different strains of cultivated marine microbes as well as explore the degree to which this is influenced by key environmental variables. We show that both vesicle production rates and vesicle sizes significantly differ among cultures of marine Proteobacteria, Cyanobacteria, and Bacteroidetes. Further, these properties vary within individual strains as a function of differences in environmental conditions, such as nutrients, temperature, and light irradiance. Thus, both community composition and the local abiotic environment are expected to modulate the production and standing stock of vesicles in the oceans. Examining samples from the oligotrophic North Pacific Gyre, we show depth-dependent changes in the abundance of vesicle-like particles in the upper water column in a manner that is broadly consistent with culture observations: the highest vesicle abundances are found near the surface, where the light irradiances and the temperatures are the greatest, and they then decrease with depth. This work represents the beginnings of a quantitative framework for describing extracellular vesicle dynamics in the oceans, which is essential as we begin to incorporate vesicles into our ecological and biogeochemical understanding of marine ecosystems. IMPORTANCE Bacteria release extracellular vesicles that contain a wide variety of cellular compounds, including lipids, proteins, nucleic acids, and small molecules, into their surrounding environment. These structures are found in diverse microbial habitats, including the oceans, where their distributions vary throughout the water column and likely affect their functional impacts within microbial ecosystems. Using a quantitative analysis of marine microbial cultures, we show that bacterial vesicle production in the oceans is shaped by a combination of biotic and abiotic factors. Different marine taxa release vesicles at rates that vary across an order of magnitude, and vesicle production changes dynamically as a function of environmental conditions. These findings represent a step forward in our understanding of bacterial extracellular vesicle production dynamics and provide a basis for the quantitative exploration of the factors that shape vesicle dynamics in natural ecosystems.

Novel integrative elements and genomic plasticity in ocean ecosystems.

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.

Prochlorococcus extracellular vesicles: molecular composition and adsorption to diverse microbes.

Extracellular vesicles are small (~50-200 nm diameter) membrane-bound structures released by cells from all domains of life. While vesicles are abundant in the oceans, their functions, both for cells themselves and the emergent ecosystem, remain a mystery. To better characterize these particles - a prerequisite for determining function - we analysed the lipid, protein, and metabolite content of vesicles produced by the marine cyanobacterium Prochlorococcus. We show that Prochlorococcus exports a diverse array of cellular compounds into the surrounding seawater enclosed within discrete vesicles. Vesicles produced by two different strains contain some materials in common, but also display numerous strain-specific differences, reflecting functional complexity within vesicle populations. The vesicles contain active enzymes, indicating that they can mediate extracellular biogeochemical reactions in the ocean. We further demonstrate that vesicles from Prochlorococcus and other bacteria associate with diverse microbes including the most abundant marine bacterium, Pelagibacter. Together, our data point toward hypotheses concerning the functional roles of vesicles in marine ecosystems including, but not limited to, possibly mediating energy and nutrient transfers, catalysing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species.

Coping with darkness: The adaptive response of marine picocyanobacteria to repeated light energy deprivation.

The picocyanobacteria Prochlorococcus and Synechococcus are found throughout the ocean's euphotic zone, where the daily light:dark cycle drives their physiology. Periodic deep mixing events can, however, move cells below this region, depriving them of light for extended periods of time. Here, we demonstrate that members of these genera can adapt to tolerate repeated periods of light energy deprivation. Strains kept in the dark for 3 d and then returned to the light initially required 18-26 d to resume growth, but after multiple rounds of dark exposure they began to regrow after only 1-2 d. This dark-tolerant phenotype was stable and heritable; some cultures retained the trait for over 132 generations even when grown in a standard 13:11 light:dark cycle. We found no genetic differences between the dark-tolerant and parental strains of Prochlorococcus NATL2A, indicating that an epigenetic change is likely responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation-one potential mechanism mediating epigenetic inheritance in bacteria-occurs in Prochlorococcus. LC-MS/MS analysis showed that while DNA methylations, including 6 mA and 5 mC, are found in some other Prochlorococcus strains, there were no methylations detected in either the parental or dark-tolerant NATL2A strains. These findings suggest that Prochlorococcus utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation, and highlights phenotypic heterogeneity as an additional dimension of Prochlorococcus diversity.

Publisher Correction: Marine microbial metagenomes sampled across space and time.

Due to a typesetting error, 25 rows were omitted from Table 3 in the original version of this Data Descriptor. These missing rows correspond to the following sample names.

Investigating the Heterogeneous Ice Nucleation of Sea Spray Aerosols Using Prochlorococcus as a Model Source of Marine Organic Matter.

Sea spray is the largest aerosol source on Earth. Bubble bursting mechanisms at the ocean surface create smaller film burst and larger jet drop particles. This study quantified the effects of particle chemistry on the depositional ice nucleation efficiency of laboratory-generated sea spray aerosols under the cirrus-relevant conditions. Cultures of Prochlorococcus, the most abundant phytoplankton species in the global ocean, were used as a model source of organic sea spray aerosols. We showed that smaller particles generated from lysed Prochlorococcus cultures are organically enriched and nucleate more effectively than larger particles generated from the same cultures. We then quantified the ice nucleation efficiency of single component organic molecules that mimic Prochlorococcus proteins, lipids, and saccharides. Amylopectin, agarose, and aspartic acid exhibited similar critical ice saturations, fractional activations, and ice nucleation active site number densities to particles generated from Prochlorococcus cultures. These findings indicate that saccharides and proteins with numerous and well-ordered hydrophilic functional groups may determine the ice nucleation abilities of organic sea spray aerosols.

Marine microbial metagenomes sampled across space and time.

Recent advances in understanding the ecology of marine systems have been greatly facilitated by the growing availability of metagenomic data, which provide information on the identity, diversity and functional potential of the microbial community in a particular place and time. Here we present a dataset comprising over 5 terabases of metagenomic data from 610 samples spanning diverse regions of the Atlantic and Pacific Oceans. One set of metagenomes, collected on GEOTRACES cruises, captures large geographic transects at multiple depths per station. The second set represents two years of time-series data, collected at roughly monthly intervals from 3 depths at two long-term ocean sampling sites, Station ALOHA and BATS. These metagenomes contain genomic information from a diverse range of bacteria, archaea, eukaryotes and viruses. The data's utility is strengthened by the availability of extensive physical, chemical, and biological measurements associated with each sample. We expect that these metagenomes will facilitate a wide range of comparative studies that seek to illuminate new aspects of marine microbial ecosystems.