The class 4 P-type ATPases ("flippases") maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MAT A: cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection ("shmoo") tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MAT A: dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2⋅Cdc28-initiated degradation. Similarly, a MAT A: dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus.

The widespread CBASS (cyclic oligonucleotide-based anti-phage signaling system) immune systems in bacteria protect their hosts from bacteriophage infection by triggering programmed cell death. CBASS systems all encode a cyclic oligonucleotide synthase related to eukaryotic cGAS but use diverse regulators and effector proteins including nucleases, phospholipases, and membrane-disrupting proteins to effect cell death. Cap18 is a predicted 3'-5' exonuclease associated with hundreds of CBASS systems, whose structure, biochemical activities, and biological roles remain unknown. Here we show that Cap18 is a DEDDh-family exonuclease related to the bacterial exonucleases RNase T and Orn and has nonspecific 3'-5' DNA exonuclease activity. Cap18 is commonly found in CBASS systems with associated CapW or CapH+CapP transcription factors, suggesting that it may coordinate with these proteins to regulate CBASS transcription in response to DNA damage. These data expand the repertoire of enzymatic activities associated with bacterial CBASS systems and provide new insights into the regulation of these important bacterial immune systems.

Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how they are regulated to avoid cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW suggest that the protein switches from an unliganded, DNA binding-competent state to a ligand-bound state unable to bind DNA. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that phage infection causes increased CBASS expression in a CapW-dependent manner. Unexpectedly, this CapW-dependent increase in CBASS expression is not required for robust anti-phage activity, suggesting that CapW may mediate CBASS activation and cell death in response to a signal other than phage infection. Our results parallel concurrent reports on the structure and activity of BrxR, a transcription factor associated with the BREX anti-phage system, suggesting that CapW and BrxR are members of a family of universal defense signaling proteins.

Background

Mixed cultures of different microbial species are increasingly being used to carry out a specific biochemical function in lieu of engineering a single microbe to do the same task. However, knowing how different species' metabolisms will integrate to reach a desired outcome is a difficult problem that has been studied in great detail using steady-state models. However, many biotechnological processes, as well as natural habitats, represent a more dynamic system. Examining how individual species use resources in their growth medium or environment (exometabolomics) over time in batch culture conditions can provide rich phenotypic data that encompasses regulation and transporters, creating an opportunity to integrate the data into a predictive model of resource use by a mixed community.

Results

Here we use exometabolomic profiling to examine the time-varying substrate depletion from a mixture of 19 amino acids and glucose by two Pseudomonas and one Bacillus species isolated from ground water. Contrary to studies in model organisms, we found surprisingly few correlations between resource preferences and maximal growth rate or biomass composition. We then modeled patterns of substrate depletion, and used these models to examine if substrate usage preferences and substrate depletion kinetics of individual isolates can be used to predict the metabolism of a co-culture of the isolates. We found that most of the substrates fit the model predictions, except for glucose and histidine, which were depleted more slowly than predicted, and proline, glycine, glutamate, lysine and arginine, which were all consumed significantly faster.

Conclusions

Our results indicate that a significant portion of a model community's overall metabolism can be predicted based on the metabolism of the individuals. Based on the nature of our model, the resources that significantly deviate from the prediction highlight potential metabolic pathways affected by species-species interactions, which when further studied can potentially be used to modulate microbial community structure and/or function.

Background

As microbiome research becomes increasingly prevalent in the fields of human health, agriculture and biotechnology, there exists a need for a resource to better link organisms and environmental chemistries. Exometabolomics experiments now provide assertions of the metabolites present within specific environments and how the production and depletion of metabolites is linked to specific microbes. This information could be broadly useful, from comparing metabolites across environments, to predicting competition and exchange of metabolites between microbes, and to designing stable microbial consortia. Here, we introduce Web of Microbes (WoM; freely available at: http://webofmicrobes.org ), the first exometabolomics data repository and visualization tool.

Description

WoM provides manually curated, direct biochemical observations on the changes to metabolites in an environment after exposure to microorganisms. The web interface displays a number of key features: (1) the metabolites present in a control environment prior to inoculation or microbial activation, (2) heatmap-like displays showing metabolite increases or decreases resulting from microbial activities, (3) a metabolic web displaying the actions of multiple organisms on a specified metabolite pool, (4) metabolite interaction scores indicating an organism's interaction level with its environment, potential for metabolite exchange with other organisms and potential for competition with other organisms, and (5) downloadable datasets for integration with other types of -omics datasets.

Conclusion

We anticipate that Web of Microbes will be a useful tool for the greater research community by making available manually curated exometabolomics results that can be used to improve genome annotations and aid in the interpretation and construction of microbial communities.

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.

Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.