Enzymatic, immunological and phylogenetic characterization of Brucella suis urease

Abstract

Background: The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined.

Results: Urease encoded by the ure-1 operon of Brucella suis strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28-35 degrees C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a Km of 5.60 +/- 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with Ki of 1.04 +/- 0.31 mM and 26.12 +/- 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130-135 kDa by gel filtration chromatography and 157 +/- 7 kDa using 5-10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by ureC1. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, ureC1 was related to the ureC typically present in the Rhizobiales; in contrast, the ureC2 encoded in the ure-2 operon is more related to distant species.

Conclusion: We have for the first time purified and characterized an active urease from B. suis. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of B. suis is a product of ure-1 operon.

Figure 1

High Performance Q Sepharose and Source PHE chromatography of Brucella suis urease. Column elautes were monitored for absorbance 280 nm (solid lines), and fractions were assayed for urease activity (solid circles), as described in Methods. A) High Performance Q Sepharose chromatography was performed using a NaCl gradient (dotted line); fractions with urease activity where precipitated with (NH₄)₂SO₄ (1.5 M) and applied to the Source PHE column. B) Source PHE chromatography was performed using a (NH₄)₂SO₄ gradient (dotted line). Urease was eluted in 180 mM of (NH₄)₂SO₄.

Figure 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15%) of Brucella suis urease after two steps of purification. Lanes: 1, High Performance Q-sepharose (Ion-Exchange Chromatography), 5 μg of protein; 2, Source PHE (Hydrophobic Interaction Chromatography), 7 μg of protein; 3, standard molecular mass markers (size are indicated). Gel was stained with Coomasie blue.

Figure 3

5–10% polyacrylamide gradient nondenaturing gel electroforesis. Lane 1, albumin from chicken egg white, 45,000 Da; lane 2, albumin from bovine serum, 66,000 Da (monomer) and 132,000 Da (dimer); lane 3, jack bean urease, 272,000 Da (trimer), and 545,000 Da (hexamer). Lanes 1–3 were stained with Coomassie blue. Lane 4, jack bean urease; lane 5, Brucella suis urease (157,000 Da ± 7010). Determination of urease activitiy was performed as described in Methods section. Four different gels were done to calculate the molecular weight of Brucella suis urease.

Figure 4

Kinetics of the B. suis urease. The graph shows the rectangular hyperbola obtained by nonlinear regression to the initial rate values, which yielded a K_m (urea) of 5.60 ± 0.69 mM. The insert graph shows the Lineweaver-Burk plot. Initial rate values are the average of three determinations. Results shown are average ± standard deviation of triplicates of initial rate values for each substrate concentration, using the same enzyme preparation.

Figure 5

Inhibition of B. suis urease by thiourea. The concentrations of urea were 4 mM (□), 8 mM, 10 mM (Δ) and 20 mM (×). The combination of the two graphs, Dixon plot (A) and the parallel lines in graph (B) indicate a competitive inhibition, with a K_i of 26.12 ± 2.30 mM determined from the intersection point of the lines in (A) [47]. Results are from a representative experiment using a single enzyme preparation.

Figure 6

Phylogenetic and cluster structure analysis. Left. Phylogenetic tree inferred for UreC proteins. The tree topology and branch lengths were obtained with Maximum Likelihood method (as implemented in PhyML) with the JTT matrix. 100 bootstrap replicates were performed and the values, other than 100, are shown. S. coelicolor (SCO1234) was taken as outgroup. Right. Genetic structure of the clusters in each of the versions and species analyzed in the phylogenetic tree. Structures were obtained mainly from the MBGD database (see Methods). Boxes with arrows represent genes with relative direction of transcription. Fill-in code: dotted, ureA; dark gray, ureB; horizontal stripped, fusion of ureA and ureB genes; black, ureC; diagonal stripped, ureD; light gray, accessory genes ureE, ureF and ureG. When only two appear, lacking one is ureE. In Bradyrhizobium BTAi1, in the cluster of gene 1962, ureH appears instead of ureD. A fourth accessory gene, an urea transporter, appears in B. suis cluster 2 and Y. pseudotuberculosis near to ureD. White; hypothetical or unrelated genes. Slashes for P. syringae tomato denote that these genes are not contiguous.