Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.
Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.
Barghouthi
S
S
Department of Microbiology, University of Mississippi Medical Center, Jackson 39216.
Payne
S M
SM
Arceneaux
J E
JE
Byers
B R
BR
D000333:Aeromonas / Q000235:genetics* / Q000472:pathogenicity; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004758:Enterobactin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D062385:Hydroxybenzoates / Q000378:metabolism*; D019751:Intramolecular Transferases; D007502:Iron Chelating Agents / Q000378:metabolism*; D007535:Isomerases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics* / Q000378:metabolism; D009876:Operon / Q000235:genetics*; D015183:Restriction Mapping; D017262:Siderophores
D062385:Hydroxybenzoates; D007502:Iron Chelating Agents; D009842:Oligopeptides; D017262:Siderophores; C065805:amonabactin P; C065804:amonabactin T; D004758:Enterobactin; C009135:2,3-dihydroxybenzoic acid; D007535:Isomerases; D019751:Intramolecular Transferases; C058486:isochorismate synthase