c-di-AMP: An essential molecule in the signaling pathways that regulate the viability and virulence of gram-positive bacteria

Signal transduction pathways enable organisms to monitor their external environment and adjust gene regulation to appropriately modify their cellular processes. Second messenger nucleotides including cyclic adenosine monophosphate (c-AMP), cyclic guanosine monophosphate (c-GMP), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) play key roles in many signal transduction pathways used by prokaryotes and/or eukaryotes. Among the various second messenger nucleotides molecules, c-di-AMP was discovered recently and has since been shown to be involved in cell growth, survival, and regulation of virulence, primarily within Gram-positive bacteria. The cellular level of c-di-AMP is maintained by a family of c-di-AMP synthesizing enzymes, diadenylate cyclases (DACs), and degradation enzymes, phosphodiesterases (PDEs). Genetic manipulation of DACs and PDEs have demonstrated that alteration of c-di-AMP levels impacts both growth and virulence of microorganisms. Unlike other second messenger molecules, c-di-AMP is essential for growth in several bacterial species as many basic cellular functions are regulated by c-di-AMP including cell wall maintenance, potassium ion homeostasis, DNA damage repair, etc. c-di-AMP follows a typical second messenger signaling pathway, beginning with binding to receptor molecules to subsequent regulation of downstream cellular processes. While c-di-AMP binds to specific proteins that regulate pathways in bacterial cells, c-di-AMP also binds to regulatory RNA molecules that control potassium ion channel expression in Bacillus subtilis. c-di-AMP signaling also occurs in eukaryotes, as bacterially produced c-di-AMP stimulates host immune responses during infection through binding of innate immune surveillance proteins. Due to its existence in diverse microorganisms, its involvement in crucial cellular activities, and its stimulating activity in host immune responses, c-di-AMP signaling pathway has become an attractive antimicrobial drug target and therefore has been the focus of intensive study in several important pathogens

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

BacillOndex: An Integrated Data Resource for Systems and Synthetic Biology

BacillOndex is an extension of the Ondex data integration system, providing a semantically annotated, integrated knowledge base for the model Gram-positive bacterium Bacillus subtilis. This application allows a user to mine a variety of B. subtilis data sources, and analyse the resulting integrated dataset, which contains data about genes, gene products and their interactions. The data can be analysed either manually, by browsing using Ondex, or computationally via a Web services interface. We describe the process of creating a BacillOndex instance, and describe the use of the system for the analysis of single nucleotide polymorphisms in B. subtilis Marburg. The Marburg strain is the progenitor of the widely-used laboratory strain B. subtilis 168. We identified 27 SNPs with predictable phenotypic effects, including genetic traits for known phenotypes. We conclude that BacillOndex is a valuable tool for the systems-level investigation of, and hypothesis generation about, this important biotechnology workhorse. Such understanding contributes to our ability to construct synthetic genetic circuits in this organism

Biochemical Characterization of Diamide Inhibitors with N-acetylglucosaminidases LytG from Bacillus subtilis

In recent years the frequency of antibiotic resistance has been on the rise creating a need for antibiotic development with specific and lethal targets. It has been recently reported that glycosyl trizole are a novel class of antibacterial agents (1). Further investigation on the antibacterial ability of glycosyl triazole inhibitors has shown that targets include exo-acting N-acetylglucosaminidases (GlcNAcase) LytG (Bacillus subtilis) and FlgJ (Salmonella enterica) of the GH73 family (2). The Glycoside Hydrolase Family 73 (GH73) is characterized by bacterial and viral glycoside hydrolase. This enzyme cleaves the β-1,4-glycosidic linkage between N-acetylglucosaminyl (NAG) and N-acetylmuramyl (NAM) of the carbohydrate backbone in bacterial peptidoglycan. Glycoside hydrolase can occur as an endo- or exo- process, depending on the region of the chain that is cleaved. Endo-acting refers to activity in the middle of the chain, whereas exo-acting refers to the ends (typically the non-reducing end) (3). Currently, there is no kinetic parameters that have been determined for any member of the GH73 family, however binding and kinetic characterization will be performed for select glycosyl triazole inhibitors and GH73 targets interactions. Further studies will involve crystallization and GlcNAcase activity assays to identify GH73 family members as the target of glycosyl triazole inhibitors. Through these studies the interaction between the non-competing inhibitor and the GH73 target will be characterized. Additionally, it will be demonstrated that these Ugi- derived compounds are competitive inhibitors of GH73 enzymes

Structure and function of bacterial dynamin-like proteins

Membrane dynamics are essential for numerous cellular processes in eukaryotic and prokaryotic cells. In eukaryotic cells, membrane fusion and fission are often catalyzed by large GTPases of the dynamin protein family. These proteins couple GTP hydrolysis to membrane deformation, which eventually leads to fusion or fission of the lipid bilayer. Mutations in eukaryotic dynamin-like proteins (DLPs) are associated with various diseases underscoring the importance to fully understand the biochemistry of these proteins. In recent years, a wealth of structural and biochemical data have been published that allow a detailed analysis of how dynamins or DLPs modulate biological membranes. However, less is known about the function of bacterial DLPs, although structural data exist. This review summarizes current knowledge about bacterial dynamins and discusses structural and functional properties in comparison to their eukaryotic counterparts

Bistable forespore engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall

To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast engulfment in $\sim$ 60 $$ of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment. Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions. Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes