GenBank access DQ532441 (Table 4) pLac36: mgoB, mgoC, mgoA and mgoD cloned R428 solubility dmso in pBBR1MCS-5 (Table 4) pLac56: mgoA and mgoD cloned in pBBR1MCS-5 (Table 4) pLac6: mgoD cloned in pBBR1MCS-5 (Table 4) Mangotoxin production in mutants derived from Pseudomonas syringae pv. syringae UMAF0158 To further support our results, we determined the amount of mangotoxin production in the insertional and miniTn5 mutants relative to wild-type UMAF0158 (Table 2).

The production of the syringomycin complex by the insertional mutants confirmed that only mangotoxin production was affected (data not shown). The results obtained from the quantitative mangotoxin analysis indicated that the two miniTn5 mutants that were complemented with pCG2-6, UMAF2-6A and UMAF2-6-3H1, and the insertion mutant UMAF0158::ORF1 were able to produce mangotoxin at the same level as wild-type UMAF0158. Upon complementation with pLac56 (mgoA and mgoD), mangotoxin production was restored in find more the mutants UMAF0158::ORF2 and UMAF0158::mgoB and the miniTn5 mutant UMAF0158-6γF6; however, the production was slightly lower and could be detected only until a 1:4 dilution (Table 2). Promoter and terminator localisation in the mgo operon Promoter

expression and terminator localisation experiments were performed to characterise the structure of the operon. The promoter prediction software

BPROM (SoftBerry Inc.) was used to identify possible promoters in the putative mgo operon. The best candidates were found in the nucleotide sequence (814 bp) of the non-coding region located upstream of the mgoB gene. Two possible promoters were predicted and designated as P mgo . The first predicted promoter was located at position 134 from 5′-end with a linear discriminant function (LDF) of 0.59, a -10 box, CGTTTTTAT, at position 119 (score: 37) and a -35 box, TCGCCA, at position 95 (score: 24). P-type ATPase The second predicted promoter was located at position 549 from the 5′-end of the sequence, with an LDF of 4.38, a -10 box, TGATAAATT, at position 534 (score: 55) and a -35 box, TTAAAA, at position 513 (score: 37) (Figure 3C). The scores of the first predicted promoter were lower than those of the second promoter. According to the in silica prediction, the 814 bp sequence containing both putative promoters was cloned into pMP220, and its activity was measured with a β-galactosidase assay (β-Gal) [17, 18]. The P mgo studies were performed in Pseudomonas fluorescens Pf-5, which contains no genomic sequences that are homologous to the mgo operon, and P. syringae pv.