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

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.

A functional water channel protein in the pathogenic bacterium Brucella abortus.

The gene for a new bacterial aquaporin, AqpX, was cloned from the pathogenic Gram-negative bacterium Brucella abortus. The gene was mapped on the large chromosome of B. abortus. It is flanked by one upstream and two downstream copies of the Brucella repeated sequence Bru-RS. Prediction from the nucleotide sequence indicated that the protein is a member of the MIP family, which comprises channels for water and/or solute transport. Expression in Xenopus oocytes and cryoelectron microscopy of Escherichia coli cells transformed with the aqpX gene confirmed that the protein is an efficient water channel. Glycerol uptake experiments in E. coli also showed that the protein is not able to transport glycerol.

The genes for erythritol catabolism are organized as an inducible operon in Brucella abortus.

Erythritol utilization is a characteristic of pathogenic Brucella abortus strains. The attenuated vaccine strain B19 is the only Brucella strain that is inhibited by erythritol, so a role for erythritol metabolism in virulence is suspected. A chromosomal fragment from the pathogenic strain B. abortus 2308 containing genes for the utilization of erythritol was cloned taking advantage of an erythritol-sensitive Tn5 insertion mutant. The nucleotide sequence of the complete 7714 bp fragment was determined. Four ORFs were identified in the sequence. The four genes were closely spaced, suggesting that they were organized as a single operon (the ery operon). The first gene (eryA) encoded a 519 aa putative erythritol kinase. The second gene (eryB) encoded an erythritol phosphate dehydrogenase. The function of the third gene (eryC) product was tentatively assigned as D-erythrulose-1-phosphate dehydrogenase and the fourth gene (eryD) encoded a regulator of ery operon expression. The operon promoter was located 5' to eryA, and contained an IHF (integration host factor) binding site. Transcription from this promoter was repressed by EryD, and stimulated by erythritol. Functional IHF was required for expression of the operon in Escherichia coli, suggesting a role for IHF in its regulation in B. abortus. The results obtained will be helpful in clarifying the role of erythritol metabolism in the virulence of Brucella spp.