We also used the insertional mutant UMAF0158::ORF2, which contains a disruption in the putative transcriptional regulator gene, and wild-type UMAF0158. P mgo activity was measured in three different culture media (LB, KB and PMS) and at two growth temperatures (28°C and 22°C). In the minimal medium PMS, the P mgo promoter was active in the wild-type strain at both temperatures and in the insertional mutant at 22°C (Figure 4). The β-Gal assays

of the strains grown in rich LB and KB media did not indicate activity in any of the strains at either temperature (data not shown). Flavopiridol Figure 3 Localisation and analysis of the promoter in the mgo operon. A) The design of the 5′ RACE experiment, including the upstream and downstream sequences of the mgoB gene. B) The results obtained from the 5′ RACE experiment. Lane 1, amplification from the primer GSP1; lane 2, amplification from the primer GSP2;

lane 3, amplification from the primer GSP3; lane L, loading buffer and HyperLadder I (Bioline), with the different sizes indicated. C) The 3′-end of ORF2, with the stop codon in bold type, and the LXH254 price 5′-end of mgoB, with the start codon also in bold type, are indicated. The nucleotide sequence (814 bp) located between these two ORFs was analysed. The two putative promoters found in this sequence by the in silico analysis are indicated by the locations of the respective -10 and -35 boxes (in red); moreover, the sequence of the alternative -35 and -10 boxes, which are more closely related to Pseudomonas promoters, are marked in blue. The start of the transcript is marked as nucleotide +1 (with black point under the nucleotide). The putative ribosomal binding site (RBS) of HM781-36B purchase mgoB is also indicated. Figure 4 The β-galactosidase (β-Gal) expression of Pseudomonas syringae pv. syringae wild-type UMAF0158, the UMAF0158::ORF2 insertional mutant, Pseudomonas syringae pv. syringae B728a and Pseudomonas fluorescens Pf5 was detected on PMS minimal medium Nintedanib (BIBF 1120) (without manipulation ( □ ), transformed with empty promoter-probe vector pMP220 (Grey Column) and transformed with pMPmgo, which contains the putative promoter P mgo (■)).

The cultures were tested at 28°C and 22°C. The results are indicative of three experiments performed in triplicate. The data were analysed by an analysis of variance (ANOVA) using SPSS 8.0 software for Windows (SPSS Inc., Chicago, IL, USA). The columns labelled with an asterisk are significantly different (P < 0.01) according to the least significant difference (LSD) test. Once the presence of promoter activity in the analysed sequence was confirmed, the 5′RACE method was used to determine the transcript start point of the mgo operon (Figure 3A, B). With this method, we could determine which of the two putative promoters of the mgo operon was the functional promoter and also analyse the presence of an additional promoter between mgoB and mgoC, which was suggested by the results of the polarity and mangotoxin production experiments.