Salt-sensitivity of σHand Spo0A prevents sporulation of Bacillus subtilisat high osmolarity avoiding death during cellular differentiation

The spore-forming bacterium Bacillus subtilis frequently experiences high osmolarity as a result of desiccation in the soil. The formation of a highly desiccation-resistant endospore might serve as a logical osmostress escape route when vegetative growth is no longer possible. However, sporulation efficiency drastically decreases concomitant with an increase in the external salinity. Fluorescence microscopy of sporulation-specific promoter fusions to gfp revealed that high salinity blocks entry into the sporulation pathway at a very early stage. Specifically, we show that both Spo0A- and SigH-dependent transcription are impaired. Furthermore, we demonstrate that the association of SigH with core RNA polymerase is reduced under these conditions. Suppressors that modestly increase sporulation efficiency at high salinity map to the coding region of sigH and in the regulatory region of kinA, encoding one the sensor kinases that activates Spo0A. These findings led us to discover that B. subtilis cells that overproduce KinA can bypass the salt-imposed block in sporulation. Importantly, these cells are impaired in the morphological process of engulfment and late forespore gene expression and frequently undergo lysis. Altogether our data indicate that B. subtilis blocks entry into sporulation in high-salinity environments preventing commitment to a developmental program that it cannot complete

Platelet FcγRIIa Expression in Ischemic Stroke: A Marker of Increased Platelet Reactivity

Background Platelet FcγRIIa amplifies platelet activation and, thus, increased expression identifies patients with increased platelet reactivity. Previous work has demonstrated that platelet FcγRIIa can identify patients at high and low risk of subsequent cardiovascular events after myocardial infarction (MI). This study was designed to compare platelet expression of FcγRIIa in patients with stroke and transient ischemic attack (TIA) with that in patients with a recent MI. Methods Patients were enrolled based on an admitting diagnosis of stroke/TIA, and the discharge diagnosis was used to categorize patients into stroke/TIA (n=99) and other causes of neurologic dysfunction (hemorrhagic, trauma, toxic, and seizure; n=14). Patients with stroke/TIA were divided into embolic (both cardioembolic and thromboembolic; n=32) and not embolic causes (n=67). Results were compared with platelet FcγRIIa expression in patients with recent MI from a previous study (n=197). Platelet expression of FcγRIIa (molecules of FcγRIIa/platelet) was quantified with the use of flow cytometry. Results are mean±SD. Results Platelet expression of FcγRIIa was similar in patients with ischemic (both embolic and nonembolic) stroke/TIA (11 332±4127), embolic (11 204±3889) and nonembolic (11 393±4263) causes, and MI (11 479±2405). Patients with other causes of neurologic dysfunction had modestly but not significantly lower platelet expression of FcγRIIa (9389±2883; P=0.13). Conclusions Platelet expression of FcγRIIa was similar in patients with stroke/TIA and recent MI. These results support future studies designed to determine whether platelet FcγRIIa expression can discriminate risk of subsequent stroke/TIA and its potential use as a precision tool capable of guiding individualized treatment decisions

Polynucleotide Phosphorylase Activity May Be Modulated by Metabolites in Escherichia coli*♦

RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency