Journal
MOLECULAR MICROBIOLOGY
Volume 54, Issue 3, Pages 808-822Publisher
WILEY
DOI: 10.1111/j.1365-2958.2004.04316.x
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Type IV secretion systems (T4SSs) are multicomponent machineries that play an essential role in pathogenicity of many facultative intracellular bacteria. The virB operon of Brucella abortus codes for a T4SS essential for virulence and intracellular multiplication. Here, virB expression analyses carried out using lacZ transcriptional fusions showed that virB promoter (P-virB) is temporally activated within J774 cells. Primer extension experiments revealed that virB transcription starts at 27 bp upstream of the first gene of the virB operon. Structural analyses showed that P-virB and regulatory sequences involved in intracellular regulation span 430 bp upstream of the transcription start site. A protein able to bind P-virB was isolated and identified. This protein, homologue to integration host factor (IHF), specifically interacts with P-virB and induces a DNA bending with an angle of 50.36degrees. DNAse I footprinting experiments showed that IHF protects a 51 bp region that contains two overlapped IHF binding consensus motifs. VirB expression experiments carried out with P-virB-lacZ fusions showed that in B. abortus IHF participates in the regulation of P-virB activity during the intracellular and vegetative growth in different media. A mutant strain with a 20 bp IHF binding site replacement failed to turn on the virB operon during the initial stages of macrophage infection and displayed severe intracellular multiplication defects. These data indicate that IHF plays a key role during intracellular virB operon expression being required for the biogenesis of the endoplasmic reticulum-derived replicative vacuole.
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