Ecophysiology and interactions of a taurine-respiring bacterium in the mouse gut.

Taurine-respiring gut bacteria produce H2S with ambivalent impact on host health. We report the isolation and ecophysiological characterization of a taurine-respiring mouse gut bacterium. Taurinivorans muris strain LT0009 represents a new widespread species that differs from the human gut sulfidogen Bilophila wadsworthia in its sulfur metabolism pathways and host distribution. T. muris specializes in taurine respiration in vivo, seemingly unaffected by mouse diet and genotype, but is dependent on other bacteria for release of taurine from bile acids. Colonization of T. muris in gnotobiotic mice increased deconjugation of taurine-conjugated bile acids and transcriptional activity of a sulfur metabolism gene-encoding prophage in other commensals, and slightly decreased the abundance of Salmonella enterica, which showed reduced expression of galactonate catabolism genes. Re-analysis of metagenome data from a previous study further suggested that T. muris can contribute to protection against pathogens by the commensal mouse gut microbiota. Together, we show the realized physiological niche of a key murine gut sulfidogen and its interactions with selected gut microbiota members.

Nutritional and host environments determine community ecology and keystone species in a synthetic gut bacterial community.

A challenging task to understand health and disease-related microbiome signatures is to move beyond descriptive community-level profiling towards disentangling microbial interaction networks. Using a synthetic gut bacterial community, we aimed to study the role of individual members in community assembly, identify putative keystone species and test their influence across different environments. Single-species dropout experiments reveal that bacterial strain relationships strongly vary not only in different regions of the murine gut, but also across several standard culture media. Mechanisms involved in environment-dependent keystone functions in vitro include exclusive access to polysaccharides as well as bacteriocin production. Further, Bacteroides caecimuris and Blautia coccoides are found to play keystone roles in gnotobiotic mice by impacting community composition, the metabolic landscape and inflammatory responses. In summary, the presented study highlights the strong interdependency between bacterial community ecology and the biotic and abiotic environment. These results question the concept of universally valid keystone species in the gastrointestinal ecosystem and underline the context-dependency of both, keystone functions and bacterial interaction networks.

Bacteriophages targeting protective commensals impair resistance against Salmonella Typhimurium infection in gnotobiotic mice.

Gut microbial communities protect the host against a variety of major human gastrointestinal pathogens. Bacteriophages (phages) are ubiquitous in nature and frequently ingested via food and drinking water. Moreover, they are an attractive tool for microbiome engineering due to the lack of known serious adverse effects on the host. However, the functional role of phages within the gastrointestinal microbiome remain poorly understood. Here, we investigated the effects of microbiota-directed phages on infection with the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm), using a gnotobiotic mouse model (OMM14) for colonization resistance (CR). We show, that phage cocktails targeting Escherichia coli and Enterococcus faecalis acted in a strain-specific manner. They transiently reduced the population density of their respective target before establishing coexistence for up to 9 days. Infection susceptibility to S. Tm was markedly increased at an early time point after challenge with both phage cocktails. Surprisingly, OMM14 mice were also susceptible 7 days after a single phage inoculation, when the targeted bacterial populations were back to pre-phage administration density. Concluding, our work shows that phages that dynamically modulate the density of protective members of the gut microbiota can provide opportunities for invasion of bacterial pathogens, in particular at early time points after phage application. This suggests, that phages targeting protective members of the microbiota may increase the risk for Salmonella infection.

The Biological Significance of Pyruvate Sensing and Uptake in Salmonella enterica Serovar Typhimurium.

Pyruvate (CH3COCOOH) is the simplest of the alpha-keto acids and is at the interface of several metabolic pathways both in prokaryotes and eukaryotes. In an amino acid-rich environment, fast-growing bacteria excrete pyruvate instead of completely metabolizing it. The role of pyruvate uptake in pathological conditions is still unclear. In this study, we identified two pyruvate-specific transporters, BtsT and CstA, in Salmonella enterica serovar Typhimurium (S. Typhimurium). Expression of btsT is induced by the histidine kinase/response regulator system BtsS/BtsR upon sensing extracellular pyruvate, whereas expression of cstA is maximal in the stationary phase. Both pyruvate transporters were found to be important for the uptake of this compound, but also for chemotaxis to pyruvate, survival under oxidative and nitrosative stress, and persistence of S. Typhimurium in response to gentamicin. Compared with the wild-type cells, the ΔbtsTΔcstA mutant has disadvantages in antibiotic persistence in macrophages, as well as in colonization and systemic infection in gnotobiotic mice. These data demonstrate the surprising complexity of the two pyruvate uptake systems in S. Typhimurium.

In vitro interaction network of a synthetic gut bacterial community.

A key challenge in microbiome research is to predict the functionality of microbial communities based on community membership and (meta)-genomic data. As central microbiota functions are determined by bacterial community networks, it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on the growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as a model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments as well as in community batch culture. Metabolic network reconstruction in combination with metabolomics analysis of bacterial culture supernatants provided insights into the metabolic potential and activity of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both exploitative and interference competition in vitro in nutrient-rich culture media and demonstrate how community structure can be shifted by changing the nutritional environment. In particular, Enterococcus faecalis KB1 was identified as an important driver of community composition by affecting the abundance of several other consortium members in vitro. As a result, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network in vitro, which serves as a knowledge base for future mechanistic in vivo studies.

E. coli enhance colonization resistance against Salmonella Typhimurium by competing for galactitol, a context-dependent limiting carbon source.

The composition of intrinsic microbial communities determines if invading pathogens will find a suitable niche for colonization and cause infection or be eliminated. Here, we investigate how commensal E. coli mediate colonization resistance (CR) against Salmonella Typhimurium (S. Tm). Using synthetic bacterial communities, we show that the capacity of E. coli Mt1B1 to block S. Tm colonization depends on the microbial context. In an infection-permissive context, E. coli utilized a high diversity of carbon sources and was unable to block S. Tm invasion. In mice that were stably colonized by twelve phylogenetically diverse murine gut bacteria (OMM12), establishing a protective context, E. coli depleted galactitol, a substrate otherwise fueling S. Tm colonization. Here, Lachnospiraceae, capable of consuming C5 and C6 sugars, critically contributed to CR. We propose that E. coli provides CR by depleting a limited carbon source when in a microbial community adept at removing simple sugars from the intestine.

Evolutionary Stabilization of Cooperative Toxin Production through a Bacterium-Plasmid-Phage Interplay.

Colicins are toxins produced and released by Enterobacteriaceae to kill competitors in the gut. While group A colicins employ a division of labor strategy to liberate the toxin into the environment via colicin-specific lysis, group B colicin systems lack cognate lysis genes. In Salmonella enterica serovar Typhimurium (S. Tm), the group B colicin Ib (ColIb) is released by temperate phage-mediated bacteriolysis. Phage-mediated ColIb release promotes S. Tm fitness against competing Escherichia coli It remained unclear how prophage-mediated lysis is realized in a clonal population of ColIb producers and if prophages contribute to evolutionary stability of toxin release in S. Tm. Here, we show that prophage-mediated lysis occurs in an S. Tm subpopulation only, thereby introducing phenotypic heterogeneity to the system. We established a mathematical model to study the dynamic interplay of S. Tm, ColIb, and a temperate phage in the presence of a competing species. Using this model, we studied long-term evolution of phage lysis rates in a fluctuating infection scenario. This revealed that phage lysis evolves as bet-hedging strategy that maximizes phage spread, regardless of whether colicin is present or not. We conclude that the ColIb system, lacking its own lysis gene, is making use of the evolutionary stable phage strategy to be released. Prophage lysis genes are highly prevalent in nontyphoidal Salmonella genomes. This suggests that the release of ColIb by temperate phages is widespread. In conclusion, our findings shed new light on the evolution and ecology of group B colicin systems.IMPORTANCE Bacteria are excellent model organisms to study mechanisms of social evolution. The production of public goods, e.g., toxin release by cell lysis in clonal bacterial populations, is a frequently studied example of cooperative behavior. Here, we analyze evolutionary stabilization of toxin release by the enteric pathogen Salmonella The release of colicin Ib (ColIb), which is used by Salmonella to gain an edge against competing microbiota following infection, is coupled to bacterial lysis mediated by temperate phages. Here, we show that phage-dependent lysis and subsequent release of colicin and phage particles occurs only in part of the ColIb-expressing Salmonella population. This phenotypic heterogeneity in lysis, which represents an essential step in the temperate phage life cycle, has evolved as a bet-hedging strategy under fluctuating environments such as the gastrointestinal tract. Our findings suggest that prophages can thereby evolutionarily stabilize costly toxin release in bacterial populations.

Dynamics of ColicinE2 production and release determine the competitive success of a toxin-producing bacterial population.

The release of toxins is one mechanism used by bacterial species to establish dominance over competitors, but how the dynamics of toxin expression determine the competitive success of a toxin-producing population is largely unknown. Here, we investigate how the expression dynamics of ColicinE2 - a toxic bacteriocin - affect competition between toxin-producing and toxin-sensitive strains of Escherichia coli. We demonstrate that, in addition to genetic modifications in the toxin expression system, alterations of the growth medium can be used to modulate the timing of toxin production and the amount of toxin released. Thus cells that release the toxin at later times can accumulate more colicin. In experiments, we found that delaying toxin release does not significantly alter competition outcome. However, our theoretical analysis allowed us to assess the relative contributions of release time and toxin level to the competitive success of the producer strain, that might counteract each other in experiments. The results reveal that the importance of delaying toxin release lies in increasing the toxin amount. This is a more effective strategy for the toxin-producing strain than prompt discharge of the colicin. In summary, our study shows how the toxin release dynamics influence the competitive success of the toxin-producing bacterial population.

Gene expression noise in a complex artificial toxin expression system.

Gene expression is an intrinsically stochastic process. Fluctuations in transcription and translation lead to cell-to-cell variations in mRNA and protein levels affecting cellular function and cell fate. Here, using fluorescence time-lapse microscopy, we quantify noise dynamics in an artificial operon in Escherichia coli, which is based on the native operon of ColicinE2, a toxin. In the natural system, toxin expression is controlled by a complex regulatory network; upon induction of the bacterial SOS response, ColicinE2 is produced (cea gene) and released (cel gene) by cell lysis. Using this ColicinE2-based operon, we demonstrate that upon induction of the SOS response noise of cells expressing the operon is significantly lower for the (mainly) transcriptionally regulated gene cea compared to the additionally post-transcriptionally regulated gene cel. Likewise, we find that mutations affecting the transcriptional regulation by the repressor LexA do not significantly alter the population noise, whereas specific mutations to post-transcriptionally regulating units, strongly influence noise levels of both genes. Furthermore, our data indicate that global factors, such as the plasmid copy number of the operon encoding plasmid, affect gene expression noise of the entire operon. Taken together, our results provide insights on how noise in a native toxin-producing operon is controlled and underline the importance of post-transcriptional regulation for noise control in this system.