The 17-kDa protein (Skp) of Escherichia coli is a homotrimeric periplasmic chaperone for newly synthesized outer-membrane proteins. Here we present its X-ray structure at a resolution of 2.35 A. Three hairpin-shaped alpha-helical extensions reach out by approximately 60 A from a trimerization domain, which is composed of three intersubunit beta-sheets that wind around a central axis. The alpha-helical extensions approach each other at their distal turns, resulting in a fold that resembles a 'three-pronged grasping forceps'. The overall shape of Skp is reminiscent of the cytosolic chaperone prefoldin, although it is based on a radically different topology. The peculiar architecture, with apparent plasticity of the prongs and distinct electrostatic and hydrophobic surface properties, supports the recently proposed biochemical mechanism of this chaperone: formation of a Skp(3)-Omp complex protects the outer membrane protein from aggregation during passage through the bacterial periplasm.

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The s A -promoted transcription of spo0A was found to be crucial for cereulide production, while the s H -driven transcription of spo0A did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription. INTRODUCTIONThe endospore-forming bacterium Bacillus cereus is increasingly recognized as a food-borne pathogen causing diarrhoea or emesis. These two types of gastrointestinal disease are associated with very different virulence factors. Several enterotoxins such as haemolysin (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin CytK, which are known to be produced during vegetative growth in the small intestine of the host, are responsible for the diarrhoeal symptoms. In contrast, emesis is triggered by the single, heat-stable peptide toxin cereulide, which is preformed in contaminated food (for reviews see EhlingSchulz et al., 2004;Stenfors Arnesen et al., 2008). Cereulide is a cyclic dodecadepsipeptide that causes cellular damage by acting as a potassium ionophore (Agata et al., 1995;Mikkola et al., 1999). It is synthesized enzymically by a non-ribosomal peptide synthetase encoded by the ces genes (Ehling-Schulz et al., 2005;Magarvey et al., 2006). The 24 kb ces gene cluster is located on a megaplasmid (pBCE) and is flanked by genetic regions homologous to the pXO1 toxin plasmid of Bacillus anthracis . Detailed sequence analysis of the entire pBCE revealed its close relation to a 272 kb plasmid from B. cereus isolates linked to periodontal disease. This new group of pXO1-like plasmids share a highly conserved core region containing genes involved in plasmid replication and maintenance, sporulation and germination, and a formaldehyde-detoxification locus (Rasko et al., 2007).The ces structural genes show little molecular diversity; however, the toxicity of the emetic B. cereus varies strongly among different strains (Carlin et al., 2006;Ehling-Schulz et al., 2005). This suggests the existence of precise mechanisms governing c...

Cereulide, the emetic Bacillus cereus toxin, is synthesized by cereulide synthetase via a nonribosomal peptide synthetase (NRPS) mechanism. Previous studies focused on the identification, structural organization, and biochemical characterization of the ces gene locus encoding cereulide synthetase; however, detailed information about the transcriptional organization of the ces genes was lacking. The present study shows that the ces-PTABCD genes are transcribed as a 23-kb polycistronic transcript, while cesH, encoding a putative hydrolase, is transcribed from its own promoter. Transcription initiation was mapped by primer extension and rapid amplification of cDNA ends (RACE). Deletion analysis of promoter elements revealed a main promoter located upstream of the cesP coding sequence, encoding a 4-phosphopantetheinyl transferase. This promoter drives transcription of cesPTABCD. In addition, intracistronic promoter regions in proximity to the translational start sites of cesB and cesT were identified but were only weakly active under the chosen assay conditions. The identified main promoter was amplified from the emetic reference strain B. cereus F4810/72 and fused to luciferase genes in order to study promoter activity in complex environments and to establish a biomonitoring system to assess cereulide production in different types of foods. ces promoter activity was strongly influenced by the food matrix and varied by 5 orders of magnitude. The amount of cereulide toxin extracted from spiked foods correlated well with the bioluminescence data, thus illustrating the potential of the established reporter system for monitoring of ces gene expression in complex matrices.Bacillus cereus is the causative agent of two types of food poisoning: diarrhea and emesis. The toxicoinfection referred to as diarrheal disease occurs after consumption of B. cereus spores or vegetative cells, most likely due to the action of heat-labile enterotoxins, which are produced by cells multiplying in the small intestine (4,22,30,39). Contrarily, the emetic type of food-borne illness is caused by intoxication with the heat-stable peptide cereulide, which is preformed in foods and elicits vomiting a few hours after ingestion (18,51,58).Regulation of enterotoxin expression of B. cereus has been studied in some detail (e.g., references 16, 29, and 46; for a review, see reference 58), revealing a major role of the pleiotropic transcription regulator PlcR in activation of enterotoxin genes and other virulence factors (1,29,45). Cereulide synthesis in emetic B. cereus, in contrast to synthesis of B. cereus enterotoxins, is not controlled by PlcR but by the Spo0A phosphorelay. The global transition state factor AbrB was identified as one factor repressing cereulide production in early exponential phase (38). Although B. cereus enterotoxins and the emetic toxin cereulide seem to belong to completely different regulatory networks, production of both types of toxins depends substantially on nutritional and environmental factors (3,46,55), and it is expe...

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The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria. Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins. Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution. Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions. Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found. A first data set was collected to 2.60 A resolution.