Beyond Toxin Transport: Novel Role of ABC Transporter for Enzymatic Machinery of Cereulide NRPS Assembly Line.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) play a pivotal role in the production of bioactive natural products, such as antibiotics and cytotoxins. Despite biomedical and pharmaceutical importance, the molecular mechanisms and architectures of these multimodular enzyme complexes are not fully understood. Here, we report on an ABC transporter that forms a vital part of the nonribosomal peptide biosynthetic machinery. Emetic Bacillus cereus produces the highly potent, mitochondrial active nonribosomal depsipeptide cereulide, synthesized by the NRPS Ces. The ces gene locus includes, next to the structural cesAB genes, a putative ABC transporter, designated cesCD Our study demonstrates that tethering of CesAB synthetase to the cell membrane by CesCD is critical for peptide assembly. In vivo studies revealed that CesAB colocalizes with CesCD on the cell membrane, suggesting direct involvement of this ABC transporter in the biosynthesis of a nonribosomal peptide. Mutation of cesCD, disrupting the assembly of the CesCD complex, resulted in decreased interaction with CesAB and, as a consequence, negatively affected cereulide biosynthesis. Specific domains within CesAB synthetase interacting with CesC were identified. Furthermore, we demonstrated that the structurally similar BerAB transporter from Bacillus thuringiensis complements CesCD function in cereulide biosynthesis, suggesting that the direct involvement of ABC transporter in secondary metabolite biosynthesis could be a widespread mechanism. In summary, our study revealed a novel, noncanonical function for ABC transporter, which is essential for megaenzyme functionality of NRPS. The new insights into natural product biosynthesis gained may facilitate the discovery of new metabolites with bioactive potential.IMPORTANCE This study revealed a novel, potentially conserved mechanism involved in the biosynthesis of microbial natural products, exemplified by the mitochondrial active depsipeptide cereulide. Similar to other bioactive substances, such as the last-resort antibiotics vancomycin and daptomycin, the antitumor drug cryptophycin or the cholesterol-lowering agent lovastatin, cereulide is synthesized nonribosomally by multienzyme machinery, requiring the concerted actions of multiple proteins to ensure correct product assembly. Given the importance of microbial secondary metabolites in human and veterinary medicine, it is critical to understand how these processes are orchestrated within the host cells. By revealing that tethering of a biosynthetic enzyme to the cell membrane by an ABC transporter is essential for nonribosomal peptide production, our study provides novel insights into synthesis of microbial secondary metabolites, which could contribute to isolation of novel compounds from cryptic secondary metabolite clusters or improve the yield of produced pharmaceuticals.

[EU rules for surveillance and control of communicable diseases]. / EU-Strukturen zur Uberwachung und Bekämpfung von Infektionskrankheiten. Wer sie macht, wie sie entstehen und wie sie funktionieren.

Since 1999, EU Member States have had a number of obligations in the area of surveillance and control of communicable diseases (Decision 2119/98/EC). Specific technical standards to be applied by Member States are determined by the Commission, following an opinion of the network committee. Those concern among others the list of diseases under surveillance, case definitions, epidemiological and microbiological surveillance methods, and guidelines for emergency situations. Since the founding of the European Centre for Disease Prevention and Control (ECDC) in 2005, capacity at the EU level to support Member States has been extended considerably. At the same time at the international level, the World Health Organization (WHO) issued new International Health Regulations (2005) (IHR), which include broader obligations for notification than the old International Health Regulations. In order to address this new situation, the Commission is working on a revision of the network decision and the ECDC regulation. These proposals should be submitted to the Council and Parliament in 2010. Important aims are simplified decision- making procedures, more clarity about the distribution of tasks between Member States, Commission, and ECDC, fewer working groups and committees, as well as an adaptation to the IHR of WHO.