Volume 19, Issue 9, Pages 1164-1174 (September 2012) Biosynthesis of the Respiratory Toxin Bongkrekic Acid in the Pathogenic Bacterium Burkholderia gladioli  Nadine Moebius, Claudia Ross, Kirstin Scherlach, Barbara Rohm, Martin Roth, Christian Hertweck  Chemistry & Biology  Volume 19, Issue 9, Pages 1164-1174 (September 2012) DOI: 10.1016/j.chembiol.2012.07.022 Copyright © 2012 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2012 19, 1164-1174DOI: (10. 1016/j. chembiol. 2012 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Respiratory Toxin Producers and Structures (A) Microscopic picture of a coculture of Burkholderia gladioli pv. cocovenenans and Rhizopus oligosporus used for tempe bongkrek production. (B) Structures of bongkrekic acid (BA) and iso-bongkrekic acid (IBA), and stable isotope labeling pattern. MS and NMR data used for structure elucidation can be found in Figures S3A–S3J. Chemistry & Biology 2012 19, 1164-1174DOI: (10.1016/j.chembiol.2012.07.022) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Genotype and Phenotype—Metabolic—Analysis of B. gladioli Wild-type and Mutant Strains (A) Architecture of the bon gene clusters in B. gladioli pv. cocovenenans and B. gladioli BSR3. For deduced gene functions, see Table 1. (B) Synteny dot plot showing a nucleotide-based comparison of the contigs of the draft genome of B. gladioli pv. cocovenenans DSMZ 11318 and the genome of B. gladioli BSR3. A high degree of similarity between both organisms is visible. A total of 5,576 matches were found, 393 queries were not matched. Location of the bon gene locus is indicated by a red box. (C) Bon gene expression assays. Expression of bonA in PDB (bongkrekic acid producing conditions, light gray bar) or NB medium (nonproducing conditions, dark gray bar) monitored by quantitative real-time PCR after 1 and 3 days. The rpoB gene was used as an internal standard for normalization. Relative quantification was carried out using the 2-ΔΔCt method (left y axis). HPLC-MS monitoring of BA production in cultures in production (red graph) and nonproduction media (green graph) (right y axis). (D) HPLC monitoring of production of bongkrekic acid (BA), iso-bongkrekic acid (IBA) and deoxybongkrekic acid (DOBA), and inhibition zone assay using A. nidulans as reporter strain; (a) B. gladioli WT, (b) B. gladioli ΔbonA, (c) B. gladioli ΔbonL. Gene replacement strategy using the λ RED system and confirmation of mutants can be found in Figure S1. Chemistry & Biology 2012 19, 1164-1174DOI: (10.1016/j.chembiol.2012.07.022) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 Analysis of the bon PKS (A) Deduced architecture of the bon PKS and model for BA biosynthesis. Abbreviations and details explained in the main text. (B) Neighbor-Joining phylogenetic tree of full-length KS domains from trans-AT PKSs with 10,000 bootstrap replicates for reliability estimation. Scale bar represents 0.1 substitutions per site. Bootstrap values above 90 are shown on branches. KS from bon gene cluster are highlighted in bold. Relevant substrates for KS clades including KS from bonPKS are displayed next to the individual clades. Clade types are shown in roman numbers referring to Nguyen et al. (2008) and explained in more detail in Results. Detailed analysis of the P450 monooxygenase BonL including a multiple sequence alignment of P450 protein sequences and a Neighbor-Joining tree of P450 orthologs can be found in Figure S2. Chemistry & Biology 2012 19, 1164-1174DOI: (10.1016/j.chembiol.2012.07.022) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Structure of Deoxybongkrekic Acid and Key 2D NMR Connectivities MS and NMR data used for structure elucidation can be found in Figures S3B–S3J. Chemistry & Biology 2012 19, 1164-1174DOI: (10.1016/j.chembiol.2012.07.022) Copyright © 2012 Elsevier Ltd Terms and Conditions