Structural basis for the regulatory interaction of the methylglyoxal synthase MgsA with the carbon flux regulator Crh in Bacillus subtilis.

Utilization of energy-rich carbon sources such as glucose is fundamental to the evolutionary success of bacteria. Glucose can be catabolized via glycolysis for feeding the intermediary metabolism. The methylglyoxal synthase MgsA produces methylglyoxal from the glycolytic intermediate dihydroxyacetone phosphate. Methylglyoxal is toxic, requiring stringent regulation of MgsA activity. In the Gram-positive bacterium Bacillus subtilis, an interaction with the phosphoprotein Crh controls MgsA activity. In the absence of preferred carbon sources, Crh is present in the nonphosphorylated state and binds to and thereby inhibits MgsA. To better understand the mechanism of regulation of MgsA, here we performed biochemical and structural analyses of B. subtilis MgsA and of its interaction with Crh. Our results indicated that MgsA forms a hexamer (i.e. a trimer of dimers) in the crystal structure, whereas it seems to exist in an equilibrium between a dimer and hexamer in solution. In the hexamer, two alternative dimers could be distinguished, but only one appeared to prevail in solution. Further analysis strongly suggested that the hexamer is the biologically active form. In vitro cross-linking studies revealed that Crh interacts with the N-terminal helices of MgsA and that the Crh-MgsA binding inactivates MgsA by distorting and thereby blocking its active site. In summary, our results indicate that dimeric and hexameric MgsA species exist in an equilibrium in solution, that the hexameric species is the active form, and that binding to Crh deforms and blocks the active site in MgsA.

The Rational Design, Synthesis, and Antimicrobial Properties of Thiophene Derivatives That Inhibit Bacterial Histidine Kinases.

The emergence of multidrug-resistant bacteria emphasizes the urgent need for novel antibacterial compounds targeting unique cellular processes. Two-component signal transduction systems (TCSs) are commonly used by bacteria to couple environmental stimuli to adaptive responses, are absent in mammals, and are embedded in various pathogenic pathways. To attenuate these signaling pathways, we aimed to target the TCS signal transducer histidine kinase (HK) by focusing on their highly conserved adenosine triphosphate-binding domain. We used a structure-based drug design strategy that begins from an inhibitor-bound crystal structure and includes a significant number of structurally simplifiying "intuitive" modifications to arrive at the simple achiral, biaryl target structures. Thus, ligands were designed, leading to a series of thiophene derivatives. These compounds were synthesized and evaluated in vitro against bacterial HKs. We identified eight compounds with significant inhibitory activities against these proteins, two of which exhibited broad-spectrum antimicrobial activity. The compounds were also evaluated as adjuvants for the treatment of resistant bacteria. One compound was found to restore the sensivity of these bacteria to the respective antibiotics.

Molecular Mechanisms of Two-Component Signal Transduction.

Two-component systems (TCS) comprising sensor histidine kinases and response regulator proteins are among the most important players in bacterial and archaeal signal transduction and also occur in reduced numbers in some eukaryotic organisms. Given their importance to cellular survival, virulence, and cellular development, these systems are among the most scrutinized bacterial proteins. In the recent years, a flurry of bioinformatics, genetic, biochemical, and structural studies have provided detailed insights into many molecular mechanisms that underlie the detection of signals and the generation of the appropriate response by TCS. Importantly, it has become clear that there is significant diversity in the mechanisms employed by individual systems. This review discusses the current knowledge on common themes and divergences from the paradigm of TCS signaling. An emphasis is on the information gained by a flurry of recent structural and bioinformatics studies.

Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16.

EIIA(Ntr) is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIA(Ntr) regulates K(+) homeostasis through interaction with the K(+) transporter TrkA and sensor kinase KdpD. In the ß-Proteobacterium Ralstonia eutropha H16, EIIA(Ntr) influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA(Ntr) increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA(Ntr), has the opposite effect. To clarify the role of EIIA(Ntr) in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA(Ntr) from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA(Ntr) interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIA(Ntr) with KdpD appears to be absent in R. eutropha, although R. eutropha EIIA(Ntr) can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary 'ancient' task of EIIA(Ntr) that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIA(Ntr) might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha.

SubtiWiki-a database for the model organism Bacillus subtilis that links pathway, interaction and expression information.

Genome annotation and access to information from large-scale experimental approaches at the genome level are essential to improve our understanding of living cells and organisms. This is even more the case for model organisms that are the basis to study pathogens and technologically important species. We have generated SubtiWiki, a database for the Gram-positive model bacterium Bacillus subtilis (http://subtiwiki.uni-goettingen.de/). In addition to the established companion modules of SubtiWiki, SubtiPathways and SubtInteract, we have now created SubtiExpress, a third module, to visualize genome scale transcription data that are of unprecedented quality and density. Today, SubtiWiki is one of the most complete collections of knowledge on a living organism in one single resource.

Determinants of interaction specificity of the Bacillus subtilis GlcT antitermination protein: functionality and phosphorylation specificity depend on the arrangement of the regulatory domains.

The control of several catabolic operons in bacteria by transcription antitermination is mediated by RNA-binding proteins that consist of an RNA-binding domain and two reiterated phosphotransferase system regulation domains (PRDs). The Bacillus subtilis GlcT antitermination protein regulates the expression of the ptsG gene, encoding the glucose-specific enzyme II of the phosphotransferase system. In the absence of glucose, GlcT becomes inactivated by enzyme II-dependent phosphorylation at its PRD1, whereas the phosphotransferase HPr phosphorylates PRD2. However, here we demonstrate by NMR analysis and mass spectrometry that HPr also phosphorylates PRD1 in vitro but with low efficiency. Size exclusion chromatography revealed that non-phosphorylated PRD1 forms dimers that dissociate upon phosphorylation. The effect of HPr on PRD1 was also investigated in vivo. For this purpose, we used GlcT variants with altered domain arrangements or domain deletions. Our results demonstrate that HPr can target PRD1 when this domain is placed at the C terminus of the protein. In agreement with the in vitro data, HPr exerts a negative control on PRD1. This work provides the first insights into how specificity is achieved in a regulator that contains duplicated regulatory domains with distinct dimerization properties that are controlled by phosphorylation by different phosphate donors. Moreover, the results suggest that the domain arrangement of the PRD-containing antitermination proteins is under selective pressure to ensure the proper regulatory output, i.e. transcription antitermination of the target genes specifically in the presence of the corresponding sugar.