Arsenobetaine: an ecophysiologically important organoarsenical confers cytoprotection against osmotic stress and growth temperature extremes.

Arsenic, a highly cytotoxic and cancerogenic metalloid, is brought into the biosphere through geochemical sources and anthropogenic activities. A global biogeochemical arsenic biotransformation cycle exists in which inorganic arsenic species are transformed into organoarsenicals, which are subsequently mineralized again into inorganic arsenic compounds. Microorganisms contribute to this biotransformation process greatly and one of the organoarsenicals synthesized and degraded in this cycle is arsenobetaine. Its nitrogen-containing homologue glycine betaine is probably the most frequently used compatible solute on Earth. Arsenobetaine is found in marine and terrestrial habitats and even in deep-sea hydrothermal vent ecosystems. Despite its ubiquitous occurrence, the biological function of arsenobetaine has not been comprehensively addressed. Using Bacillus subtilis as a well-understood platform for the study of microbial osmostress adjustment systems, we ascribe here to arsenobetaine both a protective function against high osmolarity and a cytoprotective role against extremes in low and high growth temperatures. We define a biosynthetic route for arsenobetaine from the precursor arsenocholine that relies on enzymes and genetic regulatory circuits for glycine betaine formation from choline, identify the uptake systems for arsenobetaine and arsenocholine, and describe crystal structures of ligand-binding proteins from the OpuA and OpuB ABC transporters complexed with either arsenobetaine or arsenocholine.

Genetic control of osmoadaptive glycine betaine synthesis in Bacillus subtilis through the choline-sensing and glycine betaine-responsive GbsR repressor.

Synthesis of the compatible solute glycine betaine confers a considerable degree of osmotic stress tolerance to Bacillus subtilis. This osmoprotectant is produced through the uptake of the precursor choline via the osmotically inducible OpuB and OpuC ABC transporters and a subsequent two-step oxidation process by the GbsB and GbsA enzymes. We characterized a regulatory protein, GbsR, controlling the transcription of both the structural genes for the glycine betaine biosynthetic enzymes (gbsAB) and those for the choline-specific OpuB transporter (opuB) but not of that for the promiscuous OpuC transporter. GbsR acts genetically as a repressor and functions as an intracellular choline sensor. Spectroscopic analysis of the purified GbsR protein showed that it binds the inducer choline with an apparent K(D) (equilibrium dissociation constant) of approximately 165 µM. Based on the X-ray structure of a protein (Mj223) from Methanococcus jannaschii, a homology model for GbsR was derived. Inspection of this GbsR in silico model revealed a possible ligand-binding pocket for choline resembling those of known choline-binding sites present in solute receptors of microbial ABC transporters, e.g., that of the OpuBC ligand-binding protein of the OpuB ABC transporter. GbsR was not only needed to control gbsAB and opuB expression in response to choline availability but also required to genetically tune down glycine betaine production once cellular adjustment to high osmolarity has been achieved. The GbsR regulatory protein from B. subtilis thus records and integrates cellular and environmental signals for both the onset and the repression of the synthesis of the osmoprotectant glycine betaine.

Regulated secretion of a protease activates intercellular signaling during fruiting body formation in M. xanthus.

In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. There are two morphogenetic events in this program, aggregation and sporulation, which are temporally and spatially coordinated by the contact-dependent intercellular C-signal protein (p17). p17 is generated by proteolytic cleavage of the p25 precursor protein, which accumulates in the outer membrane of vegetative and starving cells. However, p17 generation is restricted to starving cells. Here we identify the subtilisin-like protease PopC that is directly responsible for cleavage of p25. PopC accumulates in the cytoplasm of vegetative cells but is selectively secreted during starvation coinciding with the generation of p17. Consequently, p25 and PopC only encounter each other in starving cells. Thus, restriction of p25 cleavage to starving cells occurs by a compartmentalization mechanism that depends on selective secretion of PopC during starvation. Our results provide evidence for regulated proteolysis via regulated secretion.

The Aspergillus nidulans putative kinase, KfsA (kinase for septation), plays a role in septation and is required for efficient asexual spore formation.

In Aspergillus nidulans nuclear division and cytokinesis are coupled processes during asexual sporulation. Metulae, phialides and conidia contain a single nucleus. Here we describe the role of a putative Saccharomyces cerevisiae Kin4-related kinase, KfsA (kinase for septation) in the control of septum formation in A. nidulans. The kfsA deletion caused an increase in the number of conidiophores with septa in their stalks from 20% in wild type to 60% in the mutant strain. Interestingly, 7% of metulae contained two nuclei and the corresponding phialides remained anucleate, suggesting septum formation before proper segregation of nuclei. This points to a checkpoint control of KfsA, which prevents septum formation before nuclear separation. KfsA localized to the cortex and septa in hyphae and in conidiophores but not to the spindle-pole bodies, as it was shown for Kin4 in yeast. KfsA appeared at septa after actin disappeared, suggesting an additional role of KfsA late during septum formation.