The ure1 and ure2 operons are located on the chromosome I of B. suis strain 1330 (GenBank accession number NC_004310). The ure1 operon is 5284-bp long and composed of seven coding sequences (CDS). The ure2 operon is 6571-bp long and comprised eight CDS (Figure 1). The ureA gene was the same in size in both operons (302-bp). All the other genes of ure2 operon were slightly longer than their counterparts in ure1 operon. The ureC gene was the longest in each operon (1712-bp in operon-1 and 1721-bp in operon-2). The G+C content of each ure gene was compared with that of its counterpart of the other operon and found not differ substantially between ure genes of operon-1 and operon-2 (Table 1). The identity of each ure gene was compared with that of its counterpart of the other operon. The ureA, ureB, ureC, and ureG genes of the two operons exhibited 52 to 60% identity, whereas the ureD, ureE, and ureF genes did not share significant identity (Table 1).

The deduced amino acid sequences encoded by the ureA, ureB, and ureC genes in both operons displayed great identity with the structural urease subunits of a vast range of organisms including Gram-positive bacteria, Gram-negative bacteria, photosynthetic bacteria, fungi, and higher plants (see Table 5). For instance, urease subunits of other organisms exhibited up to 81% identity with the UreC of ure1 and up to 69% identity with the UreC of ure2. The ureases of alpha and beta-proteobacteria exhibited the greatest identity with UreC of operon-1, whereas, the ureases of all species of Yersinia exhibited the greatest identity with the UreC of operon-2.

Real-time PCR assays produced amplicons in sizes exactly similar to the expected sizes for each ure gene (data not shown). The UreB subunit in the ure2 operon contains a predicted hydrophobic signal sequence and suggests that the subunit may localize in the periplasmic space. All other Ure subunits lack any signal sequences and were predicted to localize in the cytoplasm (data not shown).

Four mutant B. suis strains were generated by allelic exchange, i.e., 1330Δure1K, 1330Δure2K, 1330Δure2C, and 1330Δure1KΔure2C. The PCR assays produced a predicted 2.2-kb amplicon from the wild type B. suis strain 1330 and an approximately 3.2-kb amplicon from the mutant strain 1330Δure1Kwith the ureONE-Forward and ureONE-Reverse primers (see Table 6); a predicted 2.2-kb-size amplicon from the strain 1330 and an approximately 2.8-kb product from strain1330Δure2K with ureTWO-Forward and ureTWo-Reverse primers; and a predicted 2.9-kb-sizeamplicon from the strain 1330 and an approximately 3.4-kb product from strain1330Δure2C with primers Ure-2-AB-Forward and Ure-2-AB-Reverse. The PCR assays with the primer pairs ureONE-Forward/ureONE-Reverse and Ure-2-AB-Forward/Ure-2-AB-Reverse confirmed that the double-mutant strain 1330Δure1KΔure2C carried a 575-bp deletion from the ure1DA region and a 1.2-kb deletion from the ure2ABC region (Figures 1A and 1B).

Native polyacrylamide gel electrophoresis revealed urease activity at approximately 95-kDa from strains 1330, 1330Δure2K and 1330Δure2C, but not from strains 1330Δure1K or 1330Δure1KΔure2C (Figure 2).

In a quantitative urease assay, mutants 1330Δure1K and 1330Δure1KΔure2C exhibited 0 activity, the wild type and the mutant 1330Δure2K displayed maximal activity, and mutant 1330Δure2C showed slightly reduced activity (Table 2). In qualitative urease assay, urease test broth started turning positive within 4 h after either strain 1330, 1330Δure2K, or 1330Δure2C were introduced, and acquired a bright pink color after approximately 24 h (Figure 3 and Table 2). In contrast, strains 1330Δure1K and 1330Δure1KΔure2C failed to cause a pink color in the urease test broth even after 96 h of incubation (Figure 3 and Table 2).

Strains 1330Δure1K and 1330Δure1KΔure2C, both urease negative, grew approximately 25% slower than wild type strain 1330. In contrast, strains 1330Δure2K and 1330Δure2C, both urease positive, did not display any measurable differences in growth rate compared to strain 1330 (Table 2).

When used to infect J774A.1 or H36.12a [Pixie 12a] mouse macrophage cell lines, the recovery of all the B. suis strains declined 2–3 log₁₀ cfu between 0 and 4 h post-inoculation. During the next 20 h, all the B. suis strains increased 1–2 log₁₀ cfu. There were no significant differences between the wild type and the urease mutant strains in terms of their ability to replicate in macrophages (data not shown).

Following an intraperitoneal inoculation, the recovery of ure mutants from spleens did not differ from the wild type strain at 6 wks post-infection (Table 3). When the mice were inoculated by gavage, one week after inoculation, strain 1330 was recovered from spleens (Figure 4) as well as from livers (Figure 5). When the mice were inoculated with strain 1330 supplemented with 10 mM urea, nearly 2.2 log₁₀ greater cfu was recovered from spleens and nearly 3.5 log₁₀ greater cfu was recovered from livers. However, when the mice were inoculated with strain 1330Δure1KΔure2C, with or without urea supplementation, no cfu were recovered from spleens (Figure 4) but nearly 2.5 log₁₀ cfu was recovered from livers only when the inoculum was supplemented with 10 mM urea (Figure 5).

The wild type and the ure mutants did not differ with respect to the survival after 90 min incubation at pH 4.0 or 7.0 (data not shown). All the strains including the wild type were killed when incubated at pH 2.0 for 90 min (Figure 6). When the strains were supplemented with 5 mM urea during incubation at pH 2.0, more than 6.0 log₁₀ cfu of strains 1330 and 1330Δure2K were recovered. In comparison to strain 1330, the recovery of the strain 1330Δure2C was nearly 1.5 log₁₀ lower at 5 mM urea concentration and nearly 1.0 log₁₀ lower at 10 mM urea. In contrast to strains 1330, 1330Δure2K and 1330Δure2C, strain 1330Δure1K was not recovered after incubation at pH 2.0 supplemented at any urea concentration (Figure 6). Addition of urea did not change the pH of the incubation media.

The nucleotide sequence of the urease genes was analyzed with DNASTAR software (DNASTAR, Inc., Madison, Wis.). The destination of the deduced proteins upon translation and processing was predicted using the Subloc v1.0 server of the Institute of Bioinformatics of the Tsinghua University http://www.bioinfo.tsinghua.edu.cn/. Identity of the ureA, ureB, and ureC genes of B. suis ure1 and ure2 operons with sequences of the EMBL/GenBank/DDBJ databases was analyzed using the BLAST software 22 at the National Center for Biotechnology Information (Bethesda, MD).

B. suis strain 1330 was obtained from our culture collection. Escherichia coli strain Top10 (Invitrogen Life Technologies, Carlsbad, Calif.) was used for producing plasmid constructs. E. coli were grown in Luria-Bertani (LB) broth or on LB agar (Difco Laboratories, Sparks, MD). Brucella were grown either in Trypticase soy broth(TSB) or on Trypticase soy agar (TSA) plates (Difco) at 37°C in the presence of 5% CO₂ as previously described 23. Theplasmids used in this study are listed in Table 4. Bacteria containing plasmids were grown in the presence of ampicillinor kanamycin at a 100-μg/ml concentration (Table 4).

All experiments with live Brucella were performed in a Biosafety Level 3 facility in the Infectious Disease Unit of the Virginia-Maryland Regional College of Veterinary Medicine per standard operating procedures approved by the Centers for Disease Control and Prevention.

Genomic DNA was isolated from B. suis strain 1330 by use ofa QIAGEN blood and tissue DNA kit (QIAGEN Inc., Valencia, CA). Plasmid DNA was isolated using either plasmid Mini- or Midiprep purification kits (QIAGEN). Restriction digests, Klenow reactions, and ligations of DNA were performed as described elsewhere 24. Restriction enzymes, Klenow fragment, and T4 DNA ligase enzyme were purchased from Promega Corporation (Madison, WI). Ligated plasmid DNAwas transformed into E. coli Top10 cells by heat shock per the guidelines of the supplier (Invitrogen). Plasmid DNA was electroporated into B. suis with a BTX ECM-600electroporator (BTX, San Diego, CA), as described previously25.

A 2,241-bp region including the whole length of the ure1D, ure1A, and ure1B genes and a portion of ure1C gene (Figure 1) was amplified via PCR using the genomic DNA of B. suis strain 1330 and the primers UreaseONE-Forward and UreaseONE-Reverse (RansomHill Bioscience, Inc., Ramona, CA) (see Table 6). The amplified gene fragment was cloned into the pCR2.1 vector of the TA cloning system (Invitrogen) to produce plasmid pCRure1. From this plasmid the ure1 region was isolated by EcoRI digestion and cloned into pGEM-3Z (Promega) and the resulting 5.0-kb plasmid was designated pGEMure1. The suicide vector pGEMure1K was constructed as follows: the plasmid pGEMure1 was digested with NcoI to delete a 575-bp region from the ure1 region. The NcoI sites on the 4.4-kb plasmid were filled in by reaction with Klenow enzyme and ligated to the1.6-kb PvuII fragment of pUC4K (also blunt ended) containing the Tn903 npt gene 26, which confers kanamycin resistance(Kan^r) to B. suis. The resulting suicide vector was designated pGEMure1K. The E. coli Top10 cells carrying the recombinant plasmid were picked from TSA plates containing kanamycin (100 μg/ml).

One ug of pGEMure1K was used to electroporate B. suis strain 1330; several colonies of strain 1330 were obtained from a TSA plate containing kanamycin (100 μg/ml). These colonies were streaked on TSA plates containing ampicillin (100 μg/ml) to determine whether a single- or double-crossover event had occurred. Five of the colonies did not grow on ampicillin-containingplates, suggesting that a double-crossover event had occurred. PCR with the primers UreaseONE-Forward and UreaseONE-Reverse (see Table 6) confirmed that a double-crossover event had taken place in all five transformants. One of these strains was chosen for further analyses and designated 1330Δure1K.

A 2,214-bp region including the whole length of the ure2A and ure2B genes, and a portion of the ure2C gene was amplified via PCR using the primers UreaseTWO-Forward and ureaseTWO-Reverse (see Table 6). The amplified gene fragment was cloned into the pCR2.1 vector to produce plasmid pCRure2ABC. From this plasmid, the ure2ABC region was isolated by BamHI and XbaI digestion and cloned into the same sites of plasmid pGEM-3Z (Promega). The resulting 5.0-kb plasmid was designated pGEMure2ABC. The suicide vector pGEMure2ABCK was constructed as follows: the plasmid pGEMure2ABC was digested with SacII to delete a 940-bp region from the ure2ABC region, sticky sites filled in with Klenow enzyme, and ligated to the 1.6-kb PvuII fragment of pUC4K. The resulting suicide vector was designated pGEMure2ABCK. One microgram of pGEMure2ABCK was used to electroporate B. suis strain 1330, and a transformed strain containing a double-crossover event was verified by PCR and designated 1330Δure2K.

A 2,923-bp region including the whole length of ure2A and ure2B genes, and a portion of ure2C gene (Figure 1) was amplified via PCR using the primers Ure-2-AB-Forward and Ure-2-AB-reverse (see Table 6). The amplified gene fragment was purified using a Qiagen PCR purification kit (Qiagen), digested with BamHI and XbaI, cloned into the same sites of plasmid pGEM-3Z (Promega) to produce the 5.7-kb plasmid pGEMure2ABC-2. The suicide vector pGEMure2ABCC was constructed as follows: the plasmid pGEMure2ABC-2 was digested with ClaI and MfeI to delete a 1215-bp region from the ure2ABC region, and the ends were filled in with Klenow enzyme. The 1.7-kb gene encoding resistance to chloramphenicol (Cm^r) was isolated by digesting the plasmid pBBR1MCS 27 with Eco52I plus KpnI, and the ends were filled in with Klenow enzyme. The larger fragment of the plasmid pGEMure2ABC-2 was ligated with the Cm^r gene, to make the suicide vector pGEMure2ABCC. One ug of pGEMure2ABCC was used to electroporate B. suis strain 1330 and the transformants were picked from TSA plates containing chloramphenicol (30 μg/ml). A transformed B. suis strain with a double-crossover event was verified by PCR and designated 1330Δure2C.

One ug of suicide vector pGEMure2ABCC was used to electroporate mutant B. suis strain 1330Δure1K. The transformants were picked from TSA plates containing kanamycin (100 μg/ml) plus chloramphenicol (30 μg/ml). A transformant B. suis strain with a double-crossover event was verified by PCR and designated 1330Δure1KΔure2C.

RNA was isolated from broth cultures of B. suis strain 1330 by the procedure described previously 31. After a 75% ethanol wash, the dried RNA pellet was resuspended in RNase- and DNase-free water (Sigma). The concentration of the RNA was be determined with the RiboGreen RNA Quantitation kit (Molecular Probes). Genomic DNA was digested with RNase-free DNase (Ambion), and precipitated with GlycoBlue (Ambion). RNA samples not treated with reverse transcriptase was also subjected to PCR to measure the level of contamination from genomic DNA. For each RNA sample, the control transcript (sigA) and the target mRNA were reverse-transcribed using the ReverTra Dash kit (Toyobo). The cDNA was amplified using the Light Cycler (Roche) in conjunction with the DNA Master SYBR Green I kit (Roche). Primers specific for the internal standard of ure genes were purchased from Sigma-Genosys. The target cDNA was normalized internally to the sigA cDNA level in the same sample 3233.

Single colonies of strains 1330, 1330Δure1K, 1330Δure2K, 1330Δure2C and 1330Δure1KΔure2C were grown at 37°C for 72 h to stationary phase in 5 ml of TSB. These cultures were used to inoculate 25 ml of TSB in a Klett side-arm flask to 12 to 16 Klett units. Cultures were grown at 37°C at 200 rpm; Klett readings were recorded every 2 h in a Klett-Summerson colorimeter (New York, NY).

Extracts were prepared from B. suis strains 1330,1330Δure1K, 1330Δure2K, 1330Δure2C and 1330Δure1KΔure2C using glass beads and vortex, in Tris-HCl 30 mM, pH 8.0). Buffer without SDS or mercaptoethanol were added to the extracts and the extracts were loaded into the gel that did not contain SDS. After running was complete, the gel was placed in 0.02% cresol red-0.1 EDTA, washed several times until it became yellow, and incubated with 1.5% urea at room temperature until pink bands appeared, i.e., the positive urease reaction.

Fifty μl of culture (grown for 72 h in TSB) volumes of strains 1330, 1330Δure1K, 1330Δure2K, 1330Δure2C and 1330Δure1KΔure2C were used to inoculate 5 ml volumes of urease test broth (Difco). The contents were incubated at 37°C with 200 rpm shaking. At 8, 24 and 48 hours after incubation, the cultures were centrifuged to remove the cells. The color change in the clarified urease test broth was measured using a Klett-Summerson colorimeter. The native color of the urease test broth was used as a blank.

The Coomassie Brilliant Blue G, TRIS, NADPH, 2-oxoglutarate and glutamate dehydrogenase (from bovine liver) were from Sigma-Aldrich. Urea was obtained from Qiagen. All other reagents were of analytical grade.

The concentration of protein in the extracts was determined using a Bradford-modified assay 34. All assays (final volume of 2 mL) were performed in 31 mM Tris-HCl pH 8.0 buffer at 28°C, using a Beckman DU 800 UV/Vis spectrophotometer, with a stirred, temperature-controlled multi-cell holder. Urease activity was determined using a coupled assay with glutamate dehydrogenase 35 Glutamate dehydrogenase 12 U/mL was incubated with 250 μM NADPH for 5 min. 2-Oxoglutarate (1 mM) and B. suis extracts were added and the reaction was followed at 340 nm. The observed decrease in absorbance monitored during this period is due to nonspecific oxidation of NADPH by several enzymes in the extract. When the absorbance was stable, urea (10 mM) was added and the decrease in absorbance at 340 due to urease activity was measured for 5 min. Initial rates were calculated from the linear portion of the curves, by linear regression using the least squares method. The absorption coefficient used for NADPH was 6.22 M^-1cm^-1 36. The volume of extracts was varied and the specific activity of urease was calculated. One unit of urease activity was defined as the amount of enzyme that hydrolyzes 1 μmol of urea per min. Specific activities were calculated as units of urease per mg of protein in the extract.

TSA plates were inoculated with single colonies of B. suis strains. After 4 days of incubation at 37°C with 5% CO₂, the cells were harvested from plates, washed with phosphate-buffered saline(PBS), resuspended in 20% glycerol, and frozen at -80°C. The number of viable cells or cfu was determined by counting after spreading of dilutions of the cell suspensions on TSA that were incubated at 37C with 5% CO₂. The cultures from these were used to inoculate mice and macrophages as described below.

The mouse macrophage-like cell lines J774 and H36.12a [Pixie 12a] were obtained from the American Type Culture Collection (Manassas, VA). The macrophage cells were seeded at a density of 5 × 10⁵/ml in Dulbecco's modified essential medium (DMEM) (Sigma-Aldrich) into 24-well tissue culture dishes and cultured at 37°C with 5% CO₂ until confluent. The tissue culture medium was removed, 200 μl (10⁸ cells) of the bacterial suspension in PBS was added, and the cells were incubated at 37°C for 4 h. The suspension above the cell monolayer was removed, and the macrophages washed three times with PBS. One milliliter of DMEM containing 25 μg of gentamicin was added, and the cells were incubated for 48 h at 37°C. At various time points (0, 1, 4, 24, and 48 h of incubation), the growth medium was removed, the cells were washed with PBS, and 500 μl of 0.25% sodium deoxycholate was added to lyse the infected macrophages. After 5 min the lysate was diluted in PBS, and the number of B. suis cfu was determined after growth at 37°C with 5% CO₂ for 72 h on TSA. Triplicate samples were taken at all time points, and the assay was repeated two times.

The Animal Care Committee of the Virginia Polytechnic Institute and State University approved the procedures used in handling research animals. Six-week-old female BALB/c mice (Charles River Laboratories, Wilmington, MA) were allowed 1 week of acclimatization. Groups of seven or eight mice each were intraperitoneally injected with 4.1 to 5.2 log₁₀ cfu of B. suis strains 1330,1330Δure1K, 1330Δure2K, 1330Δure2C and 1330Δure1KΔure2C. Six weeks after inoculation, mice were killed using CO₂ asphixiation, and the Brucella cfu per spleen determined as described previously 23. Briefly, spleens were collected and homogenized in TSB. Serial dilutions of each splenic homogenate were plated on TSA and the number ofcfu was determined after 4 days of incubation at 37°C with 5% CO₂.

Groups of seven to eight BALB/c mice each were dosed by gavage with 8.6 log₁₀ cfu of B. suis strain 1330 or strain 1330Δure1KΔure2C, with or without supplementation of 10 mM urea in approximately 0.5 ml PBS. Mice were sacrificed 1 week after inoculation, and the Brucella cfu per spleen or per liver was determined as described previously 23.

The pH of the phosphate buffered saline (PBS) was adjusted to 2, 4 or 7 by adding 1N HCl. The urea concentration of the PBS was adjusted to 5, 10, or 20 mM by adding urea. The PBS with different pH and urea contents were inoculated with 8.0 log₁₀ cfu of strains 1330,1330Δure1K, 1330Δure2K or 1330Δure2C, and incubated at 37°C. At the end of 90 min incubation, serial dilutions of each culture were plated on TSA. The number of cfu on plates was determined after 4 days of incubation at 37°C with 5% CO₂.

The mean and the standard deviation values from the mouse tissue clearance studies were calculated using the Microsoft Excel 2001 program (Microsoft Corporation). The Student t test was performed in the analysis of cfu data in the macrophage study. The cfu data from the splenic and liver clearance studies, and in vitro pH sensitivity study were analyzed by performing analysis of variance. The mean cfu counts among treatments were compared using the least-significance pair-wise comparison procedure 29.