Tandem heterocyclization domains in a nonribosomal peptide synthetase essential for siderophore biosynthesis in Vibrio anguillarum.
Anguibactin, the siderophore produced by Vibrio anguillarum 775, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes required for anguibactin biosynthesis are harbored by the pJM1 plasmid. Complete sequencing of this plasmid identified an orf encoding a 108 kDa predicted protein, AngN. In this work we show that AngN is essential for anguibactin biosynthesis and possesses two domains with homology to cyclization (Cy) domains of NRPSs. Substitution by alanine of the aspartic acid residues within a conserved motif of either Cy1 or Cy2 domain demonstrated the importance of these two domains in AngN function during siderophore biosynthesis. Site-directed mutations in both domains (D133A/D575A and D138A/D580A) resulted in anguibactin-deficient phenotypes while mutations in each domain did not abolish siderophore production but caused a reduction in the amounts produced. The mutations D133A/D575A and D138A/D580A also resulted as expected in a dramatic attenuation of the virulence of V. anguillarum 775 highlighting the importance of this gene for the biosynthesis of anguibactin within the vertebrate host. Regulation of the angN gene follows the patterns observed at the iron transport-biosynthesis promoter with angN transcription repressed in the presence of iron and enhanced by AngR and trans-acting factor (TAF) under iron limitation.
Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR 97239-3098, USA. m.dilorenzo@nioo.knaw.nl
Manuela
M
Di Lorenzo
Michiel
M
Stork
Hiroaki
H
Naka
Marcelo E
ME
Tolmasky
Jorge H
JH
Crosa
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003500:Cyclization; D005393:Fish Diseases / Q000382:microbiology; D005798:Genes, Bacterial / Q000235:genetics; D007501:Iron / Q000008:administration & dosage / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017686:Oncorhynchus mykiss; D046908:Peptide Biosynthesis, Nucleic Acid-Independent / Q000502:physiology*; D010453:Peptide Synthases / Q000737:chemistry*; D010455:Peptides / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017262:Siderophores / Q000096:biosynthesis*; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D014735:Vibrio Infections / Q000662:veterinary; D014774:Virulence
D010455:Peptides; D017262:Siderophores; C049469:anguibactin; D007501:Iron; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase