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Brucella abortus strain 2308 produces brucebactin, a highly efficient catecholic siderophore

Departamento de Biología Molecular (Unidad Asociada al Centro de Investigaciones Biológicas, CSIC), Facultad de Medicina, Universidad de Cantabria, Cardenal Herrera Oria s/n, 39011-Santander, Spain¹

Author for correspondence: Manuel I. González Carreró. Tel: +34 42 201943. Fax: +34 42 201945. e-mail: carrerom{at}unican.es

Brucella abortus is known to produce 2,3-dihydroxybenzoate (2,3-DHBA) and to use this catechol as a siderophore to grow under iron-limited conditions. In this study a mutant (BAM41) is described that is deficient in siderophore production by insertion of Tn5 in the virulent B. abortus strain 2308. This mutant was unable to grow on iron-deprived medium and its growth could not be restored by addition of 2,3-DHBA. Production of catecholic compounds by both the Brucella mutant and parental strains under iron-deprivation conditions was assayed by TLC. Two catecholic substances were identified in the supernatant of the parental strain 2308. The faster migrating spot showed the same retention factor (R_f) as that of purified 2,3-DHBA. The mutant BAM41 overproduced 2,3-DHBA, but failed to form the slower migrating catechol. This defect could only be complemented by the addition of the slow-migrating catechol from strain 2308. The genomic region containing Tn5 in BAM41 was cloned and the position of the transposon was determined by nucleotide sequencing. The sequence revealed that the insertion had occurred at a gene with homology to Escherichia coli entF, a locus involved in the late steps of the biosynthesis of the complex catecholic siderophore enterobactin. Intracellular survival and growth rates of the B. abortus wild-type and entF mutant strains in mouse-derived J774 macrophages were similar, indicating that production of this siderophore was not essential in this model of infection. It is concluded that B. abortus synthesizes a previously unknown and highly efficient catecholic siderophore, different from 2,3-DHBA, for which the name brucebactin is proposed.

Keywords: iron acquisition, catechol, macrophage, 2,3-DHBA

Abbreviations: 2,3-DHBA, 2,3-dihydroxybenzoate; CAS, chrome-azurol-S; DIP, 2,2-dipyridyl; EDDA, diethylenediamine di(o-hydroxyphenylacetic acid); NTA Fe(III), ferric nitriletriacetate

The GenBank accession number for the sequence reported in this paper is AF361942.

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