The AraC-like transcriptional regulator DhbR is required for maximum expression of the 2,3-dihydroxybenzoic acid biosynthesis genes in Brucella abortus 2308 in response to iron deprivation

Abstract

Phenotypic evaluation of isogenic mutants derived from Brucella abortus 2308 indicates that the AlcR homolog DhbR (2,3-dihydroxybenzoic acid [2,3-DHBA] biosynthesis regulator) modulates the expression of the genes involved in 2,3-DHBA production, employing 2,3-DHBA or brucebactin as a coinducer.

FIG. 1.

Genetic organization of the BAB2_1150-BAB2_1153 locus in B. abortus 2308. BAB2_1150 encodes a TonB-dependent outer membrane protein (BhuA) that is involved in heme transport (26), BAB2_1151 is predicted to encode a hypothetical protein, and BAB2_1153 is predicted to encode a homolog of LivK, a periplasmic protein involved in branched-chain amino acid transport in Escherichia coli and other bacteria. The black bars above the locus represent the upstream and downstream regions flanking the cat gene in the mutated BAB2_1152 locus in B. abortus BEA6. The transcriptional start for the BAB2_1152 gene (dhbR) is noted by +1, the corresponding G is shown in boldface and is underlined, and the −10 and −35 regions relative to the transcriptional site are also shown. A putative ribosome binding site and the assigned ATG start codon for the dhbR coding region (BAB2_1152) are shown in boldface and italics and in boldface and underlined, respectively.

FIG. 2.

DhbR is required for maximal siderophore production (A) and dhbCEBA transcription (B) by B. abortus 2308 in response to iron limitation. B. abortus strains 2308, BEA6 (2308 dhbR), BEA6.C [BEA6(pEA17)], and BHB2 (2308 dhbC) were grown in 100 ml low-iron minimal medium (hatched bars) or low-iron minimal medium supplemented with 50 μM FeCl₃ (black bars) in 500-ml flasks at 37°C with shaking. (A) Following 48 h of cultivation in these media, samples of the supernatants of these cultures were evaluated for the presence of siderophores using the Arnow assay (3). (B) β-Galactosidase production in Miller units (24) by derivatives of B. abortus 2308, BEA6, BEA6.D4 [BEA6(pEAD4)], and BHB2 carrying plasmid pP_dhbC::lacZ (6) following 48 h of growth under the same cultural conditions is also shown. The results presented are from single experiments that are representative of multiple (three or more) experiments performed for each analysis from which equivalent results were obtained. *, significance at a P of <0.05 for comparisons of BEA6 or BHB2 versus 2308, BEA6.C, or BEA6.D4 in a two-tailed Student t test.

FIG. 3.

DhbR binds to the dhbCEBA promoter region in B. abortus 2308 in an EMSA. Lane 1, 0.125 μg ³³P-labeled dhbC promoter-specific DNA fragment; lane 2, 0.125 μg ³³P-labeled dhbC promoter-specific DNA fragment plus 2 μg MBP-DhbR fusion protein; lane 3, 0.125 μg ³³P-labeled dhbC promoter-specific DNA fragment plus 10 μg MBP-DhbR fusion protein; lane 4, 0.125 μg ³³P-labeled dhbC-promoter-specific DNA fragment plus 2 μg MBP-DhbR fusion protein plus 6.25 μg unlabeled dhbC promoter-specific DNA fragment; lane 5, 0.125 μg ³³P-labeled dhbC promoter-specific DNA fragment plus 2 μg MBP-DhbR fusion protein plus 6.25 μg unlabeled nonspecific competitor DNA; lane 6, 0.125 μg ³³P-labeled dhbC promoter-specific DNA fragment plus 2 μg unfused MBP. The results presented are from a single experiment that is representative of multiple (three or more) experiments performed from which equivalent results were obtained.