Microbial communities often undergo intricate compositional changes yet also maintain stable coexistence of diverse species. The mechanisms underlying long-term coexistence remain unclear as system-wide studies have been largely limited to engineered communities, ex situ adapted cultures, or synthetic assemblies. Here we show how kefir, a natural milk-fermenting community of prokaryotes (predominantly lactic and acetic acid bacteria) and yeasts (family Saccharomycetaceae), realizes stable coexistence through spatiotemporal orchestration of species and metabolite dynamics. During milk fermentation, kefir grains (a polysaccharide matrix synthesized by kefir microbes) grow in mass but remain unchanged in composition. In contrast, the milk is colonized in a sequential manner in which early members open the niche for the followers by making available metabolites like amino acids and lactate. Through metabolomics, transcriptomics and large-scale mapping of inter-species interactions, we show how microbes poorly suited for milk survive in — and even dominate — the community, through metabolic cooperation and uneven partitioning between grain and milk. Overall, our findings reveal how inter-species interactions partitioned in space and time lead to stable coexistence.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. Yet, the systematic mapping of the respective interactions has only started recently 1 and the main underlying mechanism proposed is chemical transformation of drugs by microbes (biotransformation). Here, we investigated the depletion of 15 structurally diverse drugs by 25 representative gut bacterial strains. This revealed 70 bacteria-drug interactions, 29 of which had not been reported before. Over half of the new interactions can be ascribed to bioaccumulation, that is bacteria storing the drug intracellularly without chemically modifying it, and in most cases without their growth being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using clickchemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the community composition through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioral response of Caenorhabditis elegans to duloxetine. Taken together, bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, likely in an individual manner.Therapeutic drugs can have a strong impact on the gut microbiome and vice versa 2-5 . The underlying drug-bacteria interactions can reduce microbial fitness 6 or alter the drug availability through biotransformation 7-14 . The latter can have either a positive or a negative impact on drug activity and efficacy. While drugs like lovastatin and sulfasalazine are chemically transformed by gut bacteria into their active forms, bacterial metabolism can inactivate drugs such as digoxin 15,16 , or cause toxic effects as in the case of irinotecan 17 .Furthering the diversity of susceptible drugs, over one hundred molecules were recently reported to be chemically modified by gut bacteria 1 . Yet, the mechanistic view on these interactions is largely confined to drug biotransformation 12,13 . Drug accumulation without metabolizationTo expand the knowledge of bacterial effect on drug availability, we systematically profiled interactions between 15 human-targeted drugs and 25 representative human gut bacterial strains (21 species; with additional subspecies or conspecific strains of Bifidobacterium longum, Escherichia coli and Bacteroides uniformis) (Supplementary Table 1). The bacterial species were selected to cover a broad phylogenetic and metabolic diversity representative of the healthy microbiota 18 (Extended Data Fig. 1a, Supplementary Table 1). On the drug side, 12 orally administered small molecule drugs (MW<500 Da), amenable to UPLC-UV-based quantificat...

Enterohemorrhagic and enteropathogenic E. coli (EHEC and EPEC) can cause severe and potentially life-threatening infections. Their pathogenicity is mediated by at least 40 effector proteins which they inject into their host cells by a type-III secretion system leading to the subversion of several cellular pathways. However, the molecular function of several effectors remains unknown, even though they contribute to virulence. Here we show that one of them, NleF, binds to caspase-4, -8, and -9 in yeast two-hybrid, LUMIER, and direct interaction assays. NleF inhibits the catalytic activity of the caspases in vitro and in cell lysate and prevents apoptosis in HeLa and Caco-2 cells. We have solved the crystal structure of the caspase-9/NleF complex which shows that NleF uses a novel mode of caspase inhibition, involving the insertion of the carboxy-terminus of NleF into the active site of the protease. In conformance with our structural model, mutagenized NleF with truncated or elongated carboxy-termini revealed a complete loss in caspase binding and apoptosis inhibition. Evasion of apoptosis helps pathogenic E. coli and other pathogens to take over the host cell by counteracting the cell’s ability to self-destruct upon infection. Recently, two other effector proteins, namely NleD and NleH, were shown to interfere with apoptosis. Even though NleF is not the only effector protein capable of apoptosis inhibition, direct inhibition of caspases by bacterial effectors has not been reported to date. Also unique so far is its mode of inhibition that resembles the one obtained for synthetic peptide-type inhibitors and as such deviates substantially from previously reported caspase-9 inhibitors such as the BIR3 domain of XIAP.

ObjectiveThe composition of the healthy human adult gut microbiome is relatively stable over prolonged periods, and representatives of the most highly abundant and prevalent species have been cultured and described. However, microbial abundances can change on perturbations, such as antibiotics intake, enabling the identification and characterisation of otherwise low abundant species.DesignAnalysing gut microbial time-series data, we used shotgun metagenomics to create strain level taxonomic and functional profiles. Community dynamics were modelled postintervention with a focus on conditionally rare taxa and previously unknown bacteria.ResultsIn response to a commonly prescribed cephalosporin (ceftriaxone), we observe a strong compositional shift in one subject, in which a previously unknown species, U Borkfalki ceftriaxensis, was identified, blooming to 92% relative abundance. The genome assembly reveals that this species (1) belongs to a so far undescribed order of Firmicutes, (2) is ubiquitously present at low abundances in at least one third of adults, (3) is opportunistically growing, being ecologically similar to typical probiotic species and (4) is stably associated to healthy hosts as determined by single nucleotide variation analysis. It was the first coloniser after the antibiotic intervention that led to a long-lasting microbial community shift and likely permanent loss of nine commensals.ConclusionThe bloom of U B. ceftriaxensis and a subsequent one of Parabacteroides distasonis demonstrate the existence of monodominance community states in the gut. Our study points to an undiscovered wealth of low abundant but common taxa in the human gut and calls for more highly resolved longitudinal studies, in particular on ecosystem perturbations.

Microbial communities are composed of cells of varying metabolic capacity, and regularly include auxotrophs that lack essential metabolic pathways. Through analysis of auxotrophs for amino acid biosynthesis pathways in microbiome data derived from >12,000 natural microbial communities obtained as part of the Earth Microbiome Project (EMP), and study of auxotrophic–prototrophic interactions in self-establishing metabolically cooperating yeast communities (SeMeCos), we reveal a metabolically imprinted mechanism that links the presence of auxotrophs to an increase in metabolic interactions and gains in antimicrobial drug tolerance. As a consequence of the metabolic adaptations necessary to uptake specific metabolites, auxotrophs obtain altered metabolic flux distributions, export more metabolites and, in this way, enrich community environments in metabolites. Moreover, increased efflux activities reduce intracellular drug concentrations, allowing cells to grow in the presence of drug levels above minimal inhibitory concentrations. For example, we show that the antifungal action of azoles is greatly diminished in yeast cells that uptake metabolites from a metabolically enriched environment. Our results hence provide a mechanism that explains why cells are more robust to drug exposure when they interact metabolically.

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dAlthough most of the 73 open reading frames (ORFs) in bacteriophage have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast twohybrid screens. More than 60 years ago, Esther Lederberg discovered phage lambda (1). Since this seminal discovery, lambda has become a model organism, contributing to our current understanding of the ways genes work and viruses take control of their hosts. However, phage lambda is still far from being completely understood given that the function of several genes in its 48.5-kb genome remain only vaguely defined. For instance, 14 of 73 predicted lambda proteins are still largely uncharacterized (2).Some of the best-characterized aspects of lambda biology are the genetic switch that determines whether a phage lyses the cell or integrates into its host genome as a prophage and the mechanism of transcriptional antitermination (3). Also, lambda continues to provide new insights into its gene regulatory circuits (4, 5), with recent studies of its DNA packaging motor as the vanguard of nanomotor research (6).Key to lambda biology is a detailed understanding of the phage's proteins, including their interactions. We recently analyzed 95 protein-protein interactions (PPIs) between ϳ70 lambda proteins, capturing 16 of 33 previously published interactions (2). Although protein interactions are reasonably well characterized within the virus particle, host-phage interactions remain incompletely known during the replication and recombination stages of the lambda life cycle in the host cell. In addition, the molecular details of virion assembly are still largely mysterious. Altogether, about 30 host-lambda interactions have been discovered in the past 50 years (Table 1).To improve our understanding of lambda PPIs, we have cloned 68 lambda open reading frames (ORFs) (2) and performed screens against a pooled Escherichia coli library using a multivector yeast two-hybrid (Y2H) strategy. Among a total of 631 raw interactions, we found 244 reproducible interactions. Furthermore, we confirmed 162 interactions in an additional round of retesting, allowing us to obtain an o...

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