Impact of mutations in hemA and hemH genes on pyoverdine production by Pseudomonas fluorescens ATCC17400

Abstract

A Pseudomonas fluorescens Tn5 mutant, with decreased production of the siderophore pyoverdine, was obtained, with the transposon inserted in the hemA gene coding for glutamyl tRNA reductase, the enzyme that catalyzes the first step of heme biosynthesis. Since this mutant was leaky, a second round of transposition was needed to obtain a second mutant completely auxotrophic for the heme precursor Naminolevulinate (ALA). Pyoverdine production by this mutant is ALA-dependent at concentrations above those needed to sustain growth. A transposon mutant in the hemH gene that encodes the enzyme ferrochelatase showing a characteristic red fluorescence upon UV exposure as a result of porphyrins accumulation, was obtained by selecting transconjugants on LB medium containing hemin. The vhemH mutant was characterized and the corresponding hemH gene sequenced. Antibodies against P. fluorescens HemH detected the protein both in soluble and membrane fractions of the wild-type and confirmed the absence of the enzyme in the mutant. The vhemH mutant failed to produce pyoverdine, but the production of the siderophore was restored by introduction of the Pseudomonas aeruginosa hemH gene in trans. These results indicate that de novo heme biosynthesis is needed for a normal level of siderophore pyoverdine production. ß

Co-ordination of iron acquisition, iron porphyrin chelation and iron–protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens

“…The auxotrophy of this mutant for haemin was confirmed by its inability to growth on CAA medium without externally added haemin. The regions flanking the transposon insertion were isolated as described previously (Baysse et al, 2001) and sequenced. The position of the Tn5 insertion in the hemH gene is described by Baysse et al (2001).…”

“…The regions flanking the transposon insertion were isolated as described previously (Baysse et al, 2001) and sequenced. The position of the Tn5 insertion in the hemH gene is described by Baysse et al (2001). In the double pvsA hemH mutant the insertion occurred at 127 bases from the 59 end of the hemH ORF.…”

“…The vector pPAhemH, containing the hemH gene from PAO1 cloned in the expression vector pMMB208 (Baysse et al, 2001), was introduced into the double mutant by conjugation with the helper strain carrying pRK2013. The transconjugants were selected on CAA medium without haemin to confirm the functional complementation.…”

“…Polyclonal antibodies against P. fluorescens HemH were generated as described previously (Baysse et al, 2001). Western blot analysis was performed on cytoplasmic and inner membrane fractions of both wild-type and ccmC mutant using the BM Chromogenic Western Blotting Kit from Roche.…”

“…Ferribactin, the precursor of PVD, is probably exported to the periplasm where it is matured into PVD with a chromophore . Recently, we demonstrated that de novo haem biosynthesis is required for the production of PVD in P. fluorescens (Baysse et al, 2001). …”