The destination vector, pRH016 [31], carries a chloramphenicol resistance marker, and the toxic cassette is flanked by attR1 and attR2 recombinational sites. The recombinational cloning procedure was performed as recommended by the manufacturer, to produce pFJS243. nikO was amplified by PCR with oligonucleotides nikO_SalI.F and nikO_PstI.R, cloned into pGEM®-T Easy to obtain

pFJS244, and then subcloned into pBBR1 MCS/SalI &PstI to give pFJS245. Both pFJS243 CA4P mw and pFJS245 were transformed into E. coli S17-1 λ pir to be mobilized to Brucella. Complemented strains were selected in BAF Cm. In vitro susceptibility of Brucella to acid pH B. abortus strains were grown in BB until the end of the exponential phase, washed in sterile water

and resuspended at a concentration of 108 CFU/ml in citrate buffer pH 2.0 for 30 min in the presence or absence of different concentrations of urea. Bacteria this website were washed three times in phosphate-buffered saline (PBS), and survivors counted after dilution and plating. Measurement of urease activity Urease activity was determined by measuring the amount of ammonia released from urea. Exponential cultures of bacteria grown in BB, supplemented or not with 500 μM of NiCl2 as indicated, were recovered by Geneticin price centrifugation, washed, and resuspended in PBS to a concentration of 108 CFU/ml. The preparations were then lysed using three 10-s cycles with a FastPrep system (Bio 101, Vista, CA) at the maximum setting, cooled on ice, and centrifuged for 5 min at 25,000 × g at 4°C to remove the cell debris. Crude extracts were stored at -80°C until they were used. For standard urease

reactions, 5 to 10 μl of extract were added to a tube containing 200 μl of 50 mM urea in PBS and incubated for 5 min at 37°C. Urease activitiy was also measured in intact cells, in this case the pelleted bacteria were resuspended in 200 μl of either PBS (pH 7.7) or citrate buffer at different pH (3.8, 4.2, 4.6, 5.0, 5.4, 5.8, and 6.2), supplemented or not with urea at different concentrations ID-8 (0, 1, 5, 10, 20, 30, 40, 50, 75, and 100 mM), and incubated at 37°C for 1 hour. The amount of ammonia released from urea hydrolysis was determined colorimetrically by the modified Berthelot reaction [32], and the total protein concentration was measured by a Bradford assay [33]. Urease specific activity was expressed in μmol of NH3 min-1mg-1 protein (for crude extracts) and pmol of NH3 min-1 log10 cfu-1 (for intact cells). RNA isolation and reverse transcriptase PCR (RT-PCR) 3 ml of a bacterial culture in mid-log phase (OD600 = 0.6-0.7) were stabilized with RNAprotect Bacteria Reagent (Qiagen). After harvesting the cells, they were resuspended in 300 μl of TE containing lysozyme 1 mg/ml, and incubated for 15 min at room temperature.