Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-tocell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slowgrowing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.ultrahigh-throughput screening | microfluidic encapsulation | butyrylcholinesterase | Staphylococcus aureus | cell-cell interactions T he ultrahigh-throughput (1, 2) technique of screening (uHTS) in a double emulsion was applied in directed enzyme evolution (3, 4) to investigate the idea that a universal genotypephenotype linkage was provided by compartmentalization. Artificial compartments of double emulsions were produced with high polydispersity by shear stress (5, 6), which significantly decreased the portion of uniform droplets and thereby reduced the sensitivity and the maximal sorting rate. By contrast, sophisticated custom sorters demonstrated the screening of precise monodisperse droplets of water-in-oil emulsions generated by microfluidic technology (1, 7). However, it is not always convenient to use custom devices, and the use of oil as a continuous phase limits the sorting rate. Alternatively, compartmentalization in microfluidic double emulsion (MDE) enables uHTS of >10,000 events/s using commercially available cell sorters (8, 9). Furthermore, biocompatible oil and water phases provide viability and proliferation of Escherichia coli cells (10) inside the microenvironment of a double emulsion.Here, we propose an MDE-FACS platform that combines the benefits of previously reported systems based on compartmentalization in MDE and FACS selection together with modern omics (Fig. 1). We succeeded in assembling this platform using commercially available parts, which included straightforward microfluidics (Fig. S1) for MDE generation, multiparametric FACS for uHTS, next-generation sequencing (NGS) for bioinformatic predictions, and mass spectrometry for proteome and secretome analysis. We demonstrated this idea with several single-cell methods (Fig. S2), including the selection of di...

SignificanceAnalyzing complex microbial communities is the milestone of modern microbiology, calling for “deep functional profiling” techniques. While next generation sequencing revolutionized our understanding of microbiota communities, we still lack high-throughput technologies to precisely determine their functionality. Here we show how cultivation of individual bacteria inside droplets of microfluidic double water-in-oil-in-water emulsion enables us to isolate the clones with a desired activity. This approach allows us not only to select the potent antibiotic producer but also to discover a distinct mechanism of self-resistance as well as assess its efficiency on entire microbiomes. The outcome of this methodology shows that it could be effectively transferred to numerous applications in microbiology and biotechnology.

Genes coding for small peptides have been frequently misannotated as long noncoding RNA (lncRNA) genes. Here we have demonstrated that one such transcript is translated into a 56-amino-acid-long peptide conserved in chordates, corroborating the work published while this manuscript was under review. The Mtln peptide could be detected in mitochondria of mouse cell lines and tissues. In line with its mitochondrial localization, lack of the Mtln decreases the activity of mitochondrial respiratory chain complex I. Unlike the integral components and assembly factors of NADH:ubiquinone oxidoreductase, Mtln does not alter its enzymatic activity directly. Interaction of Mtln with NADH-dependent cytochrome b5 reductase stimulates complex I functioning most likely by providing a favorable lipid composition of the membrane. Study of Mtln illuminates the importance of small peptides, whose genes might frequently be misannotated as lncRNAs, for the control of vitally important cellular processes.

Numerous studies are devoted to the intestinal microbiota and intercellular communication maintaining homeostasis. In this regard, vesicles secreted by bacteria represent one of the most popular topics for research. For example, the outer membrane vesicles (OMVs) of Bacteroides fragilis play an important nutritional role with respect to other microorganisms and promote anti-inflammatory effects on immune cells. However, toxigenic B. fragilis (ETBF) contributes to bowel disease, even causing colon cancer. If nontoxigenic B. fragilis (NTBF) vesicles exert a beneficial effect on the intestine, it is likely that ETBF vesicles can be utilized for potential pathogenic implementation. To confirm this possibility, we performed comparative proteomic HPLC-MS/MS analysis of vesicles isolated from ETBF and NTBF. Furthermore, we performed, for the first time, HPLC-MS/MS and GS-MS comparative metabolomic analysis for the vesicles isolated from both strains with subsequent reconstruction of the vesicle metabolic pathways. We utilized fluxomic experiments to validate the reconstructed biochemical reaction activities and finally observed considerable difference in the vesicle proteome and metabolome profiles. Compared with NTBF OMVs, metabolic activity of ETBF OMVs provides their similarity to micro reactors that are likely to be used for long-term persistence and implementing pathogenic potential in the host.

To date, no genome of any of the species from the genus Spiroplasma has been completely sequenced. Long repetitive sequences similar to mobile units present a major obstacle for current genome sequencing technologies. Here, we report the assembly of the Spiroplasma melliferum KC3 genome into 4 contigs, followed by proteogenomic annotation and metabolic reconstruction based on the discovery of 521 expressed proteins and comprehensive metabolomic profiling. A systems approach allowed us to elucidate putative pathogenicity mechanisms and to discover major virulence factors, such as Chitinase utilization enzymes and toxins never before reported for insect pathogenic spiroplasmas.

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with yet incompletely uncovered molecular determinants. Alterations in the abundance of low molecular weight compounds (metabolites) in ASD could add to our understanding of the disease. Indeed, such alterations take place in the urine, plasma and cerebellum of ASD individuals. In this work, we investigated mass-spectrometric signal intensities of 1,366 metabolites in the prefrontal cortex grey matter of 32 ASD and 40 control individuals. 15% of these metabolites showed significantly different intensities in ASD and clustered in 16 metabolic pathways. Of them, ten pathways were altered in urine and blood of ASD individuals (Fisher test, p < 0.05), opening an opportunity for the design of new diagnostic instruments. Furthermore, metabolic measurements conducted in 40 chimpanzees and 40 macaques showed an excess of metabolite intensity differences unique to humans, supporting the hypothesized disruption of evolutionary novel cortical mechanisms in ASD.

We present a systematic study of three bacterial species that belong to the class Mollicutes, the smallest and simplest bacteria, Spiroplasma melliferum, Mycoplasma gallisepticum, and Acholeplasma laidlawii. To understand the difference in the basic principles of metabolism regulation and adaptation to environmental conditions in the three species, we analyzed the metabolome of these bacteria. Metabolic pathways were reconstructed using the proteogenomic annotation data provided by our lab. The results of metabolome, proteome and genome profiling suggest a fundamental difference in the adaptation of the three closely related Mollicute species to stress conditions. As the transaldolase is not annotated in Mollicutes, we propose variants of the pentose phosphate pathway catalyzed by annotated enzymes for three species. For metabolite detection we employed high performance liquid chromatography coupled with mass spectrometry. We used liquid chromatography method - hydrophilic interaction chromatography with silica column - as it effectively separates highly polar cellular metabolites prior to their detection by mass spectrometer.

What strategies do bacteria employ for adaptation to their hosts and are these strategies different for varied hosts? To date, many studies on the interaction of the bacterium and its host have been published. However, global changes in the bacterial cell in the process of invasion and persistence, remain poorly understood. In this study, we demonstrated phase transition of the avian pathogen Mycoplasma gallisepticum upon invasion of the various types of eukaryotic cells (human, chicken, and mouse) which was stable during several passages after isolation of intracellular clones and recultivation in a culture medium. It was shown that this phase transition is manifested in changes at the proteomic, genomic and metabolomic levels. Eukaryotic cells induced similar proteome reorganization of M. gallisepticum during infection, despite different origins of the host cell lines. Proteomic changes affected a broad range of processes including metabolism, translation and oxidative stress response. We determined that the activation of glycerol utilization, overproduction of hydrogen peroxide and the upregulation of the SpxA regulatory protein occurred during intracellular infection. We propose SpxA as an important regulator for the adaptation of M. gallisepticum to an intracellular environment.