Gram-positive bacteria synthesize the second messenger cyclic di-AMP (c-di-AMP) to control cell wall and potassium homeostasis and to secure the integrity of their DNA. In the firmicutes, c-di-AMP is essential for growth. The model organism Bacillus subtilis encodes three diadenylate cyclases and two potential phosphodiesterases to produce and degrade c-di-AMP, respectively. Among the three cyclases, CdaA is conserved in nearly all firmicutes, and this enzyme seems to be responsible for the c-di-AMP that is required for cell wall homeostasis. Here, we demonstrate that CdaA localizes to the membrane and forms a complex with the regulatory protein CdaR and the glucosamine-6-phosphate mutase GlmM. Interestingly, cdaA, cdaR, and glmM form a gene cluster that is conserved throughout the firmicutes. This conserved arrangement and the observed interaction between the three proteins suggest a functional relationship. Our data suggest that GlmM and GlmS are involved in the control of c-di-AMP synthesis. These enzymes convert glutamine and fructose-6-phosphate to glutamate and glucosamine-1-phosphate. c-di-AMP synthesis is enhanced if the cells are grown in the presence of glutamate compared to that in glutamine-grown cells. Thus, the quality of the nitrogen source is an important signal for c-di-AMP production. In the analysis of c-di-AMP-degrading phosphodiesterases, we observed that both phosphodiesterases, GdpP and PgpH (previously known as YqfF), contribute to the degradation of the second messenger. Accumulation of c-di-AMP in a gdpP pgpH double mutant is toxic for the cells, and the cells respond to this accumulation by inactivation of the diadenylate cyclase CdaA. IMPORTANCEBacteria use second messengers for signal transduction. Cyclic di-AMP (c-di-AMP) is the only second messenger known so far that is essential for a large group of bacteria. We have studied the regulation of c-di-AMP synthesis and the role of the phosphodiesterases that degrade this second messenger. c-di-AMP synthesis strongly depends on the nitrogen source: glutamategrown cells produce more c-di-AMP than glutamine-grown cells. The accumulation of c-di-AMP in a strain lacking both phosphodiesterases is toxic and results in inactivation of the diadenylate cyclase CdaA. Our results suggest that CdaA is the critical diadenylate cyclase that produces the c-di-AMP that is both essential and toxic upon accumulation. In order to process environmental information in the cell, many organisms are capable of synthesizing so-called second messengers. These small molecules are formed in response to primary signals and perceived by cellular targets. Bacteria often use specific nucleotides as second messengers. These nucleotides include cyclic mononucleotides, such as cyclic AMP (cAMP) and cyclic GMP (cGMP), as well as cyclic dinucleotides, such as cyclic di-AMP (c-di-AMP), cyclic di-GMP (c-di-GMP), and (p) ppGpp (1-3).The investigation of c-di-AMP-mediated signaling has recently attracted much attention (2). This molecule is formed by many bacteria a...

Background: Bacillus subtilis encodes three diadenylate cyclases. Results: Cyclic di-AMP is essential for the viability of B. subtilis; however, excess c-di-AMP also harms the cells. The activity of the cyclases is subject to regulation. Conclusion:The control of c-di-AMP homeostasis is crucial for B. subtilis. Significance: c-di-AMP is the first essential signaling nucleotide in bacteria.

Background:Bacillus subtilis CdaS is a sporulation-specific diadenylate cyclase. Results: Activity of CdaS is regulated by its N-terminal autoinhibitory domain. Conclusion:The synthesis of c-di-AMP is under tight control in B. subtilis. Significance: The activity of CdaS is governed by a hexamer/dimer transition.

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate.

For the medico-psychological assessment (MPA) during driving licence re-granting in Germany, abstinence control including urine samples is required. In these programmes, even small amounts of markers for drug or alcohol abuse have to be detected. Thus, the concentrations of the target compounds are very low, and, in consequence, the sensitivity of the applied screening method has to be much higher than for clinical use. Modified drugs of abuse and ethyl glucuronide immunoassays on a Roche cobas c 501 analyzer were evaluated for precision, accuracy, onboard calibration stability, cross reactivity, sensitivity, and specificity using authentic urine samples. Precision (intra-day and inter-day relative standard deviation (RSD) and accuracy (bias) at three concentrations were 12% or lower for all parameters. The calibrations remained stable (deviations <25%) for at least 28 days for all assays except amphetamines (21 days). Satisfactory cross reactivity was determined for the relevant analytes and also for several new psychoactive substances (NPS). The sensitivity was 100% for all parameters except methadone metabolite EDDP (92%) and fully met the sensitivity criteria for MPA urine testing. The presented kinetic interaction of microparticles in a solution (KIMS) immunoassays on a cobas c 501 thus provide a new method to reliably detect drug or alcohol consumption in abstinence control programmes requiring high sensitivity. Copyright © 2016 John Wiley & Sons, Ltd.