The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress.

SUMMARY Cellular turgor is of fundamental importance to bacterial growth and survival. Changes in external osmolarity as a consequence of fluctuating environmental conditions and colonization of diverse environments can significantly impact cytoplasmic water content, resulting in cellular lysis or plasmolysis. To ensure maintenance of appropriate cellular turgor, bacteria import ions and small organic osmolytes, deemed compatible solutes, to equilibrate cytoplasmic osmolarity with the extracellular environment. Here, we show that elevated levels of c-di-AMP, a ubiquitous second messenger among bacteria, result in significant susceptibility to elevated osmotic stress in the bacterial pathogen Listeria monocytogenes. We found that levels of import of the compatible solute carnitine show an inverse correlation with intracellular c-di-AMP content and that c-di-AMP directly binds to the CBS domain of the ATPase subunit of the carnitine importer OpuC. Biochemical and structural studies identify conserved residues required for this interaction and transport activity in bacterial cells. Overall, these studies reveal a role for c-di-AMP mediated regulation of compatible solute import and provide new insight into the molecular mechanisms by which this essential second messenger impacts bacterial physiology and adaptation to changing environmental conditions.

BackgroundCross-species comparisons of gene neighborhoods (also called genomic contexts) in microbes may provide insight into determining functionally related or co-regulated sets of genes, suggest annotations of previously un-annotated genes, and help to identify horizontal gene transfer events across microbial species. Existing tools to investigate genomic contexts, however, lack features for dynamically comparing and exploring genomic regions from multiple species. As DNA sequencing technologies improve and the number of whole sequenced microbial genomes increases, a user-friendly genome context comparison platform designed for use by a broad range of users promises to satisfy a growing need in the biological community.ResultsHere we present JContextExplorer: a tool that organizes genomic contexts into branching diagrams. We implement several alternative context-comparison and tree rendering algorithms, and allow for easy transitioning between different clustering algorithms. To facilitate genomic context analysis, our tool implements GUI features, such as text search filtering, point-and-click interrogation of individual contexts, and genomic visualization via a multi-genome browser. We demonstrate a use case of our tool by attempting to resolve annotation ambiguities between two highly homologous yet functionally distinct genes in a set of 22 alpha and gamma proteobacteria.ConclusionsJContextExplorer should enable a broad range of users to analyze and explore genomic contexts. The program has been tested on Windows, Mac, and Linux operating systems, and is implemented both as an executable JAR file and java WebStart. Program executables, source code, and documentation is available at http://www.bme.ucdavis.edu/facciotti/resources_data/software/.

Two-component signal transduction involves phosphoryl transfer between a histidine kinase sensor and a response regulator effector. The nitrate-responsive two-component signal transduction systems in Escherichia coli represent a paradigm for a cross-regulation network, in which the paralogous sensor-response regulator pairs, NarX-NarL and NarQ-NarP, exhibit both cognate (e.g. NarX-NarL) and non-cognate (e.g. NarQ-NarL) interactions to control output. Here, we describe results from bacterial adenylate cyclase two-hybrid (BACTH) analysis to examine sensor dimerization as well as interaction between sensor-response regulator cognate and non-cognate pairs. Although results from BACTH analysis indicated that the NarX and NarQ sensors interact with each other, results from intragenic complementation tests demonstrate that they do not form functional heterodimers. Additionally, intragenic complementation shows that both NarX and NarQ undergo intermolecular autophosphorylation, deviating from the previously reported correlation between DHp (dimerization and histidyl phosphotransfer) domain loop handedness and autophosphorylation mode. Results from BACTH analysis revealed robust interactions for the NarX-NarL, NarQ-NarL and NarQ-NarP pairs but a much weaker interaction for the NarX-NarP pair. This demonstrates that asymmetrical cross-regulation results from differential binding affinities between different sensor-regulator pairs. Finally, results indicate that the NarL effector (DNA-binding) domain inhibits NarX-NarL interaction. Missense substitutions at receiver domain residue Ser-80 enhanced NarX-NarL interaction, apparently by destabilizing the NarL receiver-effector domain interface.