Each gene studied in this study was given a specific name. The ORFs upstream of the mgo operon are illustrated by white arrows, and the 5S and 23S

ribosomal RNAs are indicated by black arrows. Results The gene cluster containing mgoA may constitute an operon composed of four ORFs. Our current study provides insight into the organisation of the operon and the involvement of the genes in the production of mangotoxin. The construction and characterisation of insertion mutants derived from Pseudomonas syringae pv. syringae UMAF0158 Each ORF that was cloned into plasmid pCG2-6 (Figure 1) was subjected to insertional inactivation mutagenesis in the P. syringae pv. syringae UMAF0158 chromosome by integration of the appropriately cloned PCR products. The ORFs were 92%-98%

HM781-36B identical to the homologous genes in P. syringae pv. syringae strain B728a (accession no. CP000075, Table 1). The deduced ORF0 and ORF1 protein products are homologous to proteins of the HAD hydrolase family and aldo-keto oxidoreductases, respectively. The mutation of these ORFs by insertional inactivation did not affect mangotoxin production. ORF2 is located just Carfilzomib cost upstream of the putative mgo operon (Figure 1) and contains a putative ribosomal binding site (RBS) at nucleotide -6 (AAGAAGT). This gene is 97% identical to Psyr_5008 from P. syringae pv. syringae B728a (Table 1), PSPTO_5454 from P. syringae pv. tomato DC3000 and PSPPH_5087 from P. syringae pv. phaseolicola 1448A. The protein products of the genes from each of these bacteria were annotated in the database as members of the GntR family of transcriptional regulators [16]. When ORF2 was disrupted, the corresponding mutant UMAF0158::ORF2 still produced mangotoxin (Tables Demeclocycline 1 and 2). Table 1 Characterization

of disrupted genes surrounding the mgo operon in derivates miniTn5 and insertional mutants from the wild type Pseudomonas syringae pv.syringae UMAF0158 mangotoxin producer Bacterial strains ORF disrupted Mangotoxin productiona Putative homology of disrupted gene Comparison ncl-nclb with Pss B728a         % of identity gene name miniTn5 mutants c           UMAF0158-3νH1 mgoC – Conserved hypothetical protein 95 Psyr_5010 UMAF0158-6νF6 mgoA – Nonribosomal peptide synthetase 93 Psyr_5011 Insertional mutants         UMAF0158::ORF0 ORF0 + HAD hydrolase 92 Psyr_5006 UMAF0158::ORF1 ORF1 + Aldo-keto oxidoreductase 98 Psyr_5007 UMAF0158::ORF2 ORF2 + Transcriptional regulator GntR family 97 Psyr_5008 UMAF0158::mgoB mgoB (+) Haem-oxigenase-likee 96 Psyr_5009 UMAF0158::mgoC mgoC – p-aminobenzoate N-oxygenase AurFe 95 Psyr_5010 UMAF0158::mgoA mgoA – Nonribosomal peptide synthetase 93 Psyr_5011 UMAF0158::mgoD mgoD – Poliketide_cyc2d 94 Psyr_5012 a) Presence of inhibition halo around the bacterial growth point in E. coli growth inhibition test.