Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila

Abstract

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.

Discovery of a Nonclassical Siderophore, Legiobactin, Produced by Strains ofLegionella pneumophila

“…To explore this further, we extracted the 130b and Philadelphia-1 supernatants with several organic solvents and then assayed for loss of CAS reactivity. The siderophore activity was not extracted by either ethyl acetate or dichloromethane, further indicating that it is not a classic catecholate or phenolate (5,12,15,37,68,74,85). The reactivity was also not extractable with butanol, confirming that it is not a typical alcohol-soluble hydroxamate (68,74).…”

“…Indeed, a number of other aquatic bacteria elaborate siderophores; e.g., species of Aeromonas, Alcaligenes, Alteromonas, Pseudomonas, and Vibrio all utilize this type of iron scavenger (94). Similarly, a variety of other (5,18,19,28,35,38,56,62,97). For some of these pathogens, including intraphagosomal, intracellular parasites, the siderophore is known to be critical for disease (19,28,38,62,67,97).…”

“…For a number of reasons, we strongly believe that this CAS-reactive substance represents a bona fide siderophore. First, the CAS assay has reliably identified and characterized a range of siderophores encompassing the iron chelators of over 20 bacterial genera (2,3,5,12,18,20,21,27,33,36,37,38,47,48,51,58,66,68,74,80,82,85,98). Second, the presence of CAS-reactive material correlated with enhanced aerobic growth in an iron-depleted defined medium (68).…”

Analysis of the pmsCEAB Gene Cluster Involved in Biosynthesis of Salicylic Acid and the Siderophore Pseudomonine in the Biocontrol Strain Pseudomonas fluorescens WCS374

“…This compound is utilized for the biosynthesis of some quinones and phenolic acid derivatives such as 2,3-dihydroxybenzoic acid (2,3-DHB). ICSs cited above have been reported as key enzymes in the biosynthetic pathway of siderophores such as enterobactin (19), pyochelin and SA (56), amonabactin (6), and vibriobactin (O07898).…”

An isochorismate hydroxymutase isogene in Escherichia coli

“…5), are known in bacteria other than E. coli. Thus, amonabactin is a phenolate siderophor produced by Aeromonas hydrophila [29]. This microorganism contains the amoA gene which is homologous to entC and menF (Fig.…”