syringae UMAF0158 and its derived miniTn5 and insertion mutants grown in liquid minimal medium (PMS). Bacterial strains Mangotoxin production Dilutions of cultures filtratesa     1:1 1:2 1:4 1:8 + ornithine Wild type             UMAF0158 + 21.7 ± 0.4 18.2 ± 0.4 13.7 ± 0.4 9.5 ± 0.5 < 7 miniTn5 mutants             UMAF0158-3νH1 - < 7

< 7 < 7 < 7 < 7 UMAF0158-6νF6 - < 7 < 7 < 7 < 7 < 7 pCG2-6 complementation           UMAF2-6-3H1 + 19.0 ± 1.0 15.5 ± 0.5 13.5 ± 0.5 9.5 ± 0.5 < 7 UMAF2-6A + 19.0 ± 0.7 16.2 ± 0.4 12.7 ± 1.3 10.5 ± 0.5 Selleck Torin 2 < 7 Insertion mutants           UMAF0158::ORF1 + 20.2 ± 1.3 17.0 ± 0.7 14.7 ± 0.8 11.0 ± 0.8 < 7 UMAF0158::ORF2 + 19.7 ± 1.5 16.2 ± 0.8 12.2 ± 1.1 < 7 < 7 UMAF0158::mgoB + 17.7 ± 0.8 14.2 Pifithrin-�� molecular weight ± 0.8 12.0 ± 0.8 < 7 < 7 UMAF0158::mgoC - < 7 < 7 < 7 < 7 < 7 UMAF0158::mgoA - < 7 < 7 < 7 < 7 < 7 UMAF0158::mgoD - < 7 < 7 < 7 < 7 < 7 pLac complementation         UMAF0158-6νF6 containing pLac56 + 19.2 ± 0.4 15.7 ± 0.8 12.7 ± 1.2 < 7 < 7 UMAF0158-6νF6 containing pLac6 - < 7 < 7 < 7 < 7 < 7 The inhibition analysis was performed by Escherichia coli growth inhibition test a) Toxic activity is expressed as diameter of inhibition zone

(in mm). Average and standard deviation values were obtained from three replicate of three independent experiments The four genes downstream of ORF2 exhibit a high degree of identity to four consecutive P. syringae pv. syringae B728a genes (Psyr_5009 to Psyr_5012) (Table 1). The mgoB gene, which contains a putative RBS at nucleotide -8 (AGGA), is 96% similar to Psyr_5009, which encodes a conserved hypothetical protein. The mgoB mutant UMAF0158::mgoB produced mangotoxin (Table 1), although the level of mangotoxin was decreased slightly (Table 2). A search of the Pfam database revealed a similarity to DUF3050, a protein of unknown function, between amino acids 15 and 244 with an e-value of 3.1e-97. Searches in the InterProScan

(EMBL-EBI) database revealed that the theoretical MgoB protein product is similar to the haem oxygenase-like, multi-helical superfamily 3-mercaptopyruvate sulfurtransferase between amino acids 128 and 245 (e-value of 1.3e-8). The inactivation of the mgoC, mgoA and mgoD genes yielded mutants (UMAF0158::mgoC, UMAF0158::mgoA and UMAF0158::mgoD) that were completely unable to produce mangotoxin (Tables 1 and 2). The mgoC gene, which contains a putative RBS at -7 (AAGGA), exhibits 95% similarity to the Psyr_5010 gene of P. syringae pv. syringae B728a, a conserved hypothetical protein (Table 1). Homology searches for the MgoC protein product in the Pfam database revealed a significant match with the p-aminobenzoate N-oxygenase AurF from Streptomyces thioluteus. The alignment was between amino acids 2 and 295 with an e-value of 7.2e-88.