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Infection and Immunity, April 2003, p. 1794-1803, Vol. 71, No. 4
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.4.1794-1803.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genetic Organization and Iron-Responsive Regulation of the Brucella abortus 2,3-Dihydroxybenzoic Acid Biosynthesis Operon, a Cluster of Genes Required for Wild-Type Virulence in Pregnant Cattle

Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932,¹ Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803,² Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003,³ Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354⁴

Received 16 September 2002/ Returned for modification 12 November 2002/ Accepted 10 January 2003

Brucella abortus reportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulent B. abortus 2308 and genetic complementation of defined Escherichia coli mutants were used to identify the B. abortus genes (designated dhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA with dhb-specific primers demonstrated that dhbC, -B, and -A are transcribed as components of an operon, together with dhbE, a functional homolog of the Escherichia coli entE gene. Homologs of the E. coli entD and Vibrio cholerae vibH genes were also detected in the flanking regions immediately adjacent to the B. abortus dhbCEBA operon, suggesting that B. abortus has the genetic capacity to produce a more complex 2,3-DHBA-based siderophore. Slot blot hybridization experiments and primer extension analysis showed that transcription of the B. abortus dhbCEBA operon originates from two iron-regulated promoters located upstream of dhbC. Consistent with their iron-dependent regulation, both of the dhbCEBA promoter sequences contain typical consensus Fur-binding motifs. Although previously published studies have shown that 2,3-DHBA production is not required for the establishment and maintenance of chronic spleen infection by B. abortus in mice, experimental infection of pregnant cattle with the B. abortus dhbC mutant BHB1 clearly showed that production of this siderophore is essential for wild-type virulence in the natural ruminant host.

Editor: J. T. Barbieri

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