3 Kb Pst 1 fragment in fur:kanP mutant but not in the wild type. These results confirm that a single copy

of Kmr was correctly inserted in the Fur box Trichostatin A order located in the promoter region of NE0616 gene of the N. europaea genome (Figure 4A). A fur transcript was not detected in the fur:kanP mutant by either RT-PCR or qRT-PCR analysis (up to 28 cycles) indicating the inactivation of fur gene due to Kmr insertion in its promoter region. Transcripts of ammonia monooxygenase C (amoC) component used as positive control both for the efficiency of the RT-PCR procedure and for RNA and cDNA recovery showed no significant difference in expression in wild type and the fur:kanP mutant (data not shown). Figure 4 In vitro transposon mutagenesis scheme and mutant confirmation.

(A) The physical structure of a 5,810-bp fragment Selleck Ku-0059436 of the N. europaea chromosome is shown in the center (heavy black line), with positions of NE0616 (fur) gene shown as grey arrow, the fur box (fb) located in NE0616 promoter region shown as white rectangle. The regions covered by the plasmids pFur616, pFur616-kanP, pFur616-kanC whose DNA sequences were determined are shown as thin black lines with the names of the respective plasmids shown below each line. The position and relative orientation of each in vitro-constructed Tn5-Kan2 cassette insertion mutation are indicated by a flag on the lines. The restriction endonuclease sites P (Pst 1) and E (Eco R1) used for Southern blot confirmation are indicated. (B) Verification of mutagenesis of fur:kanP in N. europaea by Southern hybridization. Genomic DNA from the

wild type (WT), fur:kanP mutant (MT) were digested with E (Eco RI) and Phospholipase D1 P (Pst 1), and probed with (left) fur ORF sequence and (right) kan sequence. Effect of fur:kanP mutation on growth of N. europaea Growth of the N. europaea fur:kanP strain was compared to that of the wild-type strain in both Fe-replete (10 μM Fe) and Fe-limited (0.2 μM Fe) media. Surprisingly, there was no significant difference in growth of fur:kanP in both Fe-replete and Fe-limited media compared to the wild-type strain (Figure 5A). The fur:kanP mutant did not exhibit a growth advantage over the wild type when iron was limiting or show increased sensitivity to iron-induced redox stress when grown in the presence of Fe (up to 250 μM Fe; data not shown). However, growth of fur:kanP mutant was affected when grown in medium containing 500 μM Fe (Figure 5B). The mutant was unable to grow in media containing more than 500 μM Fe (data not shown). Growth of wild type was inhibited only when concentrations of Fe exceeded 1 mM [14]. Figure 5 Growth curves of the N. europaea wild type (solid lines, filled symbols) and fur:kanP mutant (dotted lines, open symbols) as measured by OD. (A) Fe-replete (squares) and Fe-limited (triangles) medium. (B) 500 μM Fe medium (circles) and in Fe-limited medium with 10 μM ferrioxamine (diamonds).