Between these two segments is the nucleotide sequence in which the two putative terminators

were identified by the bioinformatic analysis (SoftBerry Inc.), which are indicated as terminator 1 (T1) and terminator 2 (T2). C) The secondary structure of the two putative Rho-independent terminators within the mgo operon (terminator 1 (T1) and terminator 2 (T2)), as predicted by FindTerm software (SoftBerry Inc.). D) A diagram of the experimental design for locating the functional mgo operon terminator. The amplicon sizes and primer directions are indicated. Agarose electrophoresis of the RT-PCR experiments. HyperLadder IV (Bioline) was used as the loading buffer. The hypothetical function of the mgo operon Our study of the mgo locus demonstrates that the mgo operon is involved in the biosynthesis or regulation of mangotoxin. Entospletinib Recent studies APR-246 datasheet of the pvf genes,

which share high homology with the mgo operon, have indicated a possible regulatory function for those genes [21]. Given these findings, it should be possible to isolate a signalling molecule that is required for virulence gene expression and use it to restore the virulence of an mgoA mutant (defective in the nonribosomal peptide synthetase [15]) by adding this molecule to the growth medium. Growing the UMAF0158 mutant, which mTOR inhibitor possesses a deletion of mgoA (UMAF0158ΔmgoA) and is defective in mangotoxin production, in media supplemented with an extract from wild-type UMAF0158 restored mangotoxin production. why An extract from the mgoA mutant did not restore toxin production. Strains that were defective in other regulatory genes were also used. Extracts from wild-type Pss UMAF0158 and the reference strain Pss B728a were used to complement UMAF0158-2βB7, which contains a miniTn5 disruption of the gacA gene, and UMAF0158-3αE10, which contains a miniTn5 disruption of the gacS gene (Table 4). Mangotoxin production was restored in the defective mutants when an extract from UMAF0158 was added. By contrast, an extract from Pss B728a only restored mangotoxin production in the gacS mutant (Table 4).

These results suggest a possible regulatory role for the mgo operon. Table 4 Extract complementation of defective mutants in mangotoxin production using extract obtained from Pseudomonas syringae pv.syringae wild-type UMAF0158 and references train B728a   Controls Extracts Complemented strains Standard methanol UMAF0158 B728a P. syringae pv. syringae         UMAF0158 + + nd nd B728a – - nd nd Defective mutants         UMAF0158ΔmgoA – - + – UMAF0158-2βB7 (gacA) – - + – UMAF0158-3αE10 (gacS) – - + + Discussion The focus of the present study was to characterise the transcriptional organisation that is directly involved in mangotoxin production. We had previously identified the mgo operon (Mangotoxin-Generating Operon) [15].