Bacteria-Killing Type IV Secretion Systems

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I–IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitor

A Minimal Threshold of c-di-GMP Is Essential for Fruiting Body Formation and Sporulation in Myxococcus xanthus

Generally, the second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-diGMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be—at least partially—functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus

Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (K d) and Stoichiometry of Interaction (n) of C-di-GMP Binding Proteins

Isothermal titration calorimetry (ITC) is a commonly used biophysical technique that enables the quantitative characterization of intermolecular interactions in solution. Based on enthalpy changes (ΔH) upon titration of the binding partner (e.g., a small-molecule ligand such as c-di-GMP) to the molecule of interest (e.g., a receptor protein), the resulting binding isotherms provide information on the equilibrium association/dissociation constants (K a, K d) and stoichiometry of binding (n), as well as on changes in the Gibbs free energy (ΔG) and entropy (ΔS) along the interaction. Here we present ITC experiments used for the characterization of c-di-GMP binding proteins and discuss advantages and potential caveats in the interpretation of results

Regulation by c-di-GMP in Myxococcus xanthus

Myxococcus xanthus has a complex lifecycle that is regulated by nutrient availability. In the presence of nutrients, M. xanthus cells grow, divide, and move to assemble into colonies that feed cooperatively either saprophytically or on prey. In response to starvation, a developmental program is initiated that culminates in formation of multicellular spore-filled fruiting bodies. The nucleotide-based second messenger cyclic di-GMP accumulates in M. xanthus and has critical functions in both stages of the lifecycle. Here, we describe the roles of cyclic di-GMP, its metabolizing proteins, and receptor proteins. During growth, the correct level of cyclic di-GMP is important for type IV pili-dependent motility. During development, the cyclic di-GMP level increases and a threshold concentration of cyclic di-GMP is essential for completion of the developmental program. By individually inactivating the genes involved in cyclic di-GMP synthesis or degradation, two diguanylate cyclases, DmxA and DmxB, were identified to function at specific stages of the lifecycle with DmxA involved in type IV pili-dependent motility and DmxB in development. Similarly, the phosphodiesterase PmxA is specifically important for development but functions independently of DmxB. Bioinformatics analyses suggest the existence of various cyclic di-GMP receptor proteins, a few of which have been confirmed experimentally while the remainder are still uncharacterized. We are only just beginning to understand regulation by cyclic di-GMP in M. xanthus and it will be exciting to identify all the processes regulated by cyclic di-GMP and the underlying mechanisms