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.

Use of yeast two-hybrid system for detection of Bacillus subtilis FtsZ protein partners.

Yeast two-hybrid system was modified to allow easy detection of prokaryotic protein-protein interactions. Three plasmids (pGBR1, pGBR2, pGBR3) with the ClaI restriction site shifted in the three possible reading frames in fusion with GAL4 activating domain were constructed. The modified plasmids were used for identification of protein partners of FtsZ from Bacillus subtilis. Among partners of FtsZ the FtsA protein and a globular part of the SpoIIE protein were identified. The protein interactions were quantified by measurements of beta-galactosidase activity in yeast cells using 4-methylumbelliferyl beta-D-galactopyranoside as fluorogenic substrate.

Mechanisms of hippocampal reoxygenation injury.

Mechanisms of 12 min of hypoxia and subsequent reoxygenation were studied in rat hippocampal slices. General cell injury in reoxygenation was indicated by increased lactate dehydrogenase (LDH). Increase in conjugated dienes (CD) showed that oxygen radical burst induced lipid peroxidation (LPO). ATP increase was also involved in reoxygenation injury, since cyanide, an inhibitor of ATP synthesis, decreased this damage. The results obtained on using inhibitors of oxygen radicals generation, i.e., allopurinol, indomethacin, rotenone, and antimycin A, strongly suggest that the sources of oxygen radicals were the xanthine/xanthine oxidase system, prostaglandin synthesis, and mitochondrial respiratory chain. The involvement of oxygen radicals in oxidative stress was confirmed also by using chain-breaking antioxidants, trolox alpha-tocopherol and stobadine, [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido (4,3b)indole]. Stobadine added at the onset of reoxygenation was most effective, acting in a dose-dependent manner and found to be without effect when applied in hypoxia. Cytochrome-c oxidase was decreased in reoxygenated hippocampal slices treated with stobadine.

Mechanisms of hippocampal reoxygenation injury. Treatment with antioxidants.

The effects of hypoxia of different durations (8, 12 or 15 min) and of subsequent reoxygenation were studied in rat hippocampal slices by measuring enzyme activities related to oxidative stress: superoxide dismutase (SOD), cytochrome c oxidase and lactate dehydrogenase (LDH). Simultaneously the degree of lipid peroxidation was estimated by measuring conjugated dienes (CD). Reoxygenation after 8-min of hypoxia induced general cell injury indicated by increased LDH activity. Reoxygenation after 12-min of hypoxia started lipid peroxidation assessed by an increase in CD, and after 15-min of hypoxia followed by reoxygenation CD were found to be significantly decreased, suggesting lipid degradation. The injury induced by a hypoxia of 12 min and reoxygenation was reduced by SOD and catalase, indicating that oxygen radicals were involved in this process. The oxygen radicals originating from the xanthine/xanthine oxidase system, from the synthesis of prostaglandins, as well as from the mitochondrial respiratory chain, since allopurinol, indomethacin and rotenone decreased while antimycin increased reoxygenation injury. An increase in ATP may also have been involved as cyanide, an inhibitor of ATP synthesis, decreased the reoxygenation injury. The chain-breaking antioxidants trolox, alpha tocopherol and the pyridoindole stobadine were effective in preventing reoxygenation injury, indicating the involvement of lipid peroxidation in this process.