Transcriptional regulation of the Brucella abortus virB promoter

RODRIGO SIEIRA, DIEGO J. COMERCI, AND RODOLFO A. UGALDE.

Giens, Toulon, France.

Congreso; EuroConference on the Mechanisms and Applications in Biotechnology-Biology of Type IV Secretion Processes; 2003

EURESCO Conferences (European Science Fundation)

In the last years several studies demonstrated that the Brucella virulence is directly related to a type-IV secretion system encoded by the virB operon. In B. abortus 2308 this system is essential for virulence and intracellular multiplication, and it is necessary for the bacterium to redirect the intracellular traffic and to reach the intracellular replication niche. We have analyzed the virB promoter (PvirB) activity at different levels. The in vitro PvirB activity in rich medium was studied using different techniques. Both chromosomal and plasmid lacZ transcriptional fusions, and western blot experiments, showed that the PvirB is induced in the stationary phase of growth, which is different to that observed in other Brucella species. Low copy number plasmids containing lacZ transcriptional fusions to PvirB were sued to analyze the in vitro promoter activity in minimal medium under diffrerent conditions. We show that in minimal medium PvirB is induced during the exponential phase of growth and remains active during the stationary phase. This induction is higher when a carbon source (glucose, glycerol) or amino acids are added to the minimal medium. We also show that PvirB is not induced at low pH. This behaviour is different from that described for other species of Brucella. PvirB intracellular activity was analyzed using J774 cells infected with bacteria carrying lacZ fusions. We show that PvirB is induced early during cell infection and that it is turned off 15 post infection. Primer extension experiments revealed that B. abortus virB transcription starts 27 bp upstream of the virB1 start codon. A 237 bp probe from position -199 to +38 was designed in order to perform gel shift experiments using B. abortus 2308 cell extracts. Specific retarded bands were observed, and a purification protocol was used to isolate pvirB transcription factors. A histone-like protein was isolated and identified by tandem mass spectrometry. This protein showed 49% identifty with the Escherichia coli Integration Host Factor (IHF) alpha subunit. Gel shift and Dna foot0printing experiments carried out with a rexombinant Bl. Abortus IHF prootein (recIHF) showed that recIHF specifricalli binds on three sites within the PvirB. All the binding sites contain two overlapped sequences thjat partially maths the E. coli IHF-binding consensus sequence and are located in the center of the protected regions. lacZ transcriptional fusions to different regions of PvirB were used to identify the minimal region necessary to wil type activity both in culture and inside the J774 cells. The results showed that the B. abortus IHF protein is probably involved in B. abortus PvirB regulation.