Brucella abortus ure2 region contains an acid-activated urea transporter and a nickel transport system

Abstract

Background: Urease is a virulence factor that plays a role in the resistance of Brucella to low pH conditions, both in vivo and in vitro. Brucella contains two separate urease gene clusters, ure1 and ure2. Although only ure1 codes for an active urease, ure2 is also transcribed, but its contribution to Brucella biology is unknown.

Results: Re-examination of the ure2 locus showed that the operon includes five genes downstream of ureABCEFGDT that are orthologs to a nikKMLQO cluster encoding an ECF-type transport system for nickel. ureT and nikO mutants were constructed and analyzed for urease activity and acid resistance. A non-polar ureT mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The nikO mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium.

Conclusions: Based on these results, we concluded that the operon ure2 codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the ure1 operon.

Figure 1

Evidence of transcription and redefinition of the ure2 operon of Brucella abortus 2308. The map on top of the figure shows the ure2 region of the large chromosome of Brucella abortus 2308. Below the map the arrows indicate primers designed to check transcription of the region. For each pair of primers marked with a number, three separate PCR reactions were performed: a positive control using genomic DNA as template; a test reaction using cDNA as template, and a control using RNA as template. M, 1 Kb Plus DNA ladder.

Figure 2

Urease activity of B. abortus 2308-derived strains. Urease activity was determined in bacterial extracts obtained from the indicated strains and growth conditions, and expressed in μmol of NH3 min^-1 mg^-1 protein The experiments were performed by triplicate with three technical measures per replica. The data shown correspond to one representative experiment and the error bar indicates the standard deviation. An unpaired t-test was performed to determine if the urease activity of each mutant was significantly different than the corresponding wild type control. A. Protein extracts from cultures of the indicated strains grown in BB. B: Protein extracts from cultures grown in BB supplemented with 0.5 mM NiCl₂. * indicates p < 0.05.

Figure 3

pH dependency of urease activity in intact Brucella cells. Intact cells were exposed to the indicated pH for 15 minutes, in buffer containing 5 mM urea and then urease activity determined, and expressed in pmol of NH₃ min^-1 log10 cfu^-1 (diamond) 2308, (white square) 2308 ΔureT, (black square) 2308 ΔureT (pFJS243).

Figure 4

Urease activity in a urea gradient. Intact cells exposed to buffer pH 4.2 with increasing amounts of urea. (diamond) 2308, (white square) 2308 ΔureT, (black square) 2308 ΔureT (pFJS243).

Figure 5

Survival of B. abortus urease mutants to acid exposure. Log n° of bacteria surviving an acid shock of 30 minutes at pH 2.0 in the presence of different amounts of urea. The arithmetic media from three separate experiments was plotted with standard deviations. An unpaired t-test was performed to determine if survival of each strain was significantly different than the corresponding wild type control. * indicates p < 0.05, ** p < 0.01.