Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins

Abstract

Bacillus subtilis contains urease structural genes but lacks the accessory genes typically required for GTPdependent incorporation of nickel. Nevertheless, B. subtilis was shown to possess a functional urease, and the recombinant enzyme conferred low levels of nickel-dependent activity to Escherichia coli. Additional investigations of the system lead to the suggestion that B. subtilis may use unidentified accessory proteins for in vivo urease activation.Urease is a Ni-containing enzyme found in plants, fungi, and bacteria (15). This protein participates in the recycling of environmental nitrogen and serves as a virulence factor in pathogenic microorganisms associated with gastric ulceration and urinary stone formation (22). Most bacterial ureases possess three structural subunits (encoded by ureABC) associated into a trimer of trimers [(␣␤␥) 3 ], with each UreC subunit containing a dinuclear Ni active site bridged by a carbamylated lysine (4,16,28). Helicobacter species have only two subunits (UreA, a fusion of the small subunits [␤ and ␥] in other bacteria, and the large subunit, designated UreB) in a (␣ 3 ␤ 3 ) 4 macromolecular structure (14). Fungi and plants contain a homohexamer (␣ 6 ) of a fusion of the three bacterial sequences (30). Synthesis of active urease requires the action of several accessory proteins (23), with the best-studied system found in Klebsiella aerogenes, in which the structural genes are found in a gene cluster containing four accessory genes (ureDABCEFG). By use of this system, UreD-UreF-UreG was identified as a GTPdependent molecular chaperone that binds urease apoprotein (8, 32), while UreE was shown to function as a metallochaperone that delivers Ni 2ϩ (11,25,31). Genome sequence analysis has revealed that, in contrast to other ureolytic microorganisms, Bacillus subtilis contains only urease structural genes (ureABC) and lacks homologues to any accessory genes (18). Despite this dearth of urease genes, the organism exhibits urease activity and grows with urea as the sole nitrogen source unless ureC is inactivated (12).Urease activity in B. subtilis. B. subtilis SF10 cells (wild type, SMY derivative; from Susan Fisher) (3) were cultured at 37°C in S7 minimal medium (37) plus 0.2% glutamate. A low but detectable level of urease activity (0.113 Ϯ 0.006 U/mg protein, where one unit is the amount of enzyme required to hydrolyze 1 mol of urea per min at 37°C in 50 mM HEPES buffer, pH 7.8, containing 50 mM urea) (38) was observed in cell extracts obtained by sonication followed by centrifugation (10,000 ϫ g, 20 min, 4°C). This level of activity is comparable to the level of 0.103 Ϯ 0.012 U/mg (after correction to the same units) described previously for extracts of these cells (3) and compares to ϳ2 U/mg for cell extracts of K. aerogenes (36) or 2,500 U/mg for the purified K. aerogenes urease (35). The addition of 100 M NiCl 2 to the culture had no effect on the urease activity (0.107 Ϯ 0.016 U/mg), which suggests that the trace levels of Ni 2ϩ in the minimal medium were sufficient for ...

Biosynthesis of the Urease Metallocenter

“…A dramatic example of this situation exists in Bacillus subtilis, where the genome reveals the presence of only the structural urease genes; nevertheless, the cells synthesize an active nickel urease, although with poor efficiency (80). In many other cases, however, the sequenced microorganisms were not examined for urease activity.…”

Characterization of the Urease Operon of Brucella abortus and Assessment of Its Role in Virulence of the Bacterium

“…At the same time, the metal transporters and chelating proteins involved in nickel homeostasis may affect urease activity. As a consequence, mutations in many genes outside the urease operon may result in urease-deficient phenotypes (17,18).…”

“…At the same time, the metal transporters and chelating proteins involved in nickel homeostasis may affect urease activity. As a consequence, mutations in many genes outside the urease operon may result in urease-deficient phenotypes (17,18).Bacteria belonging to the genus Brucella (except B. ovis) usually exhibit a characteristic strong urease activity (8). The properties of this enzyme, as well as its significance in Brucella metabolism or pathogenicity, remain basically unexplored.…”

Chaperones of Nickel Metabolism

“…For example, Bacillus subtilis synthesizes suffi cient levels of Ni-containing urease to allow for growth on urea as sole nitrogen source, even though its genome lacks homologs to ureDEFG [134]. The mechanism by which this organism generates active urease in the apparent absence of any accessory protein remains unknown.…”