The Bacillus Subtilis Ureabc Operon

The Bacillus subtilis ureABC Operon

1 1 2 2 HUGO CRUZ-RAMOS, PHILLIPE GLASER, LEWIS V. WRAY, JR., AND SUSAN H. FISHER * Unite´deRe´gulation de l’Expression Ge´ne´tique, Institut Pasteur, 75724 Paris Cedex 15, France,1 and Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 021182

Received 8 January 1997/Accepted 10 March 1997

The Bacillus subtilis ureABC operon encodes homologs of the three subunits of urease enzymes of the family Enterobacteriaceae. Disruption of ureC prevented utilization of urea as a nitrogen source and resulted in a partial growth defect in minimal medium containing limiting amounts of arginine or allantoin as the sole nitrogen source.

Urea is a nitrogenous compound that can be generated by (45% identity). UreC (569 residues) has 65 and 69% sequence the degradation of arginine and purines (5, 15). Many bacteria identity with the UreC proteins from K. aerogenes and Bacillus synthesize nickel-dependent ureases that are responsible for sp. strain TB-90, respectively. Crystallographic (10) and ge- the enzymatic step in the degradation of urea to ammonia and netic (14) analysis of K. aerogenes urease has identified an carbon dioxide (12). Urease is synthesized constitutively in aspartate, a carbamylated lysine, and four histidine residues in some bacteria, while its expression is regulated in response to UreC that function as nickel ligands. All of these amino acids urea or nitrogen availability in other microorganisms (12). In are conserved in the UreC proteins from B. subtilis and other Bacillus subtilis, urease is synthesized at high levels only during bacteria. The two histidine residues of the K. aerogenes urease nitrogen-limited growth (2). Structural genes for the urease implicated in substrate binding and catalysis (10) are also con- subunits, ureA, ureB, and ureC, are typically located adjacent to served in the B. subtilis UreC protein. These observations sug- several other genes that encode accessory proteins required for gest that the structure of the nickel metallocenter for B. subtilis assembly of the urease nickel metallocenter (12). Here we urease is similar to that of the K. aerogenes enzyme. report the isolation and analysis of the ureABC operon in the Genetic organization of the ureABC locus. The B. subtilis gram-positive sporulating soil bacterium B. subtilis. ureA, ureB, and ureC genes are most likely encoded within an Cloning and sequence analysis of ureABC. The ureABC operon. The ureA stop codon overlaps the first two nucleotides operon was discovered during the systematic sequencing of the of the start codon for ureB. This same overlap occurs between B. subtilis genome. Sequence analysis of the B. subtilis chro- the ureB stop codon and the ureC start codon. Immediately ␭ mosomal DNA cloned in p narA4 (8) indicated that a coding downstream of the ureC stop codon is a nucleotide sequence sequence (CDS) for a protein with similarity to the UreA with the potential to form a GϩC-rich stem-loop structure that subunit of bacterial ureases was located at one end of the DNA may act as a transcriptional terminator (Fig. 1). A second insert (Fig. 1). The remainder of the ure operon was cloned by potential factor-independent transcriptional terminator nucle- directed chromosomal DNA walking (9). Briefly, a 433-bp otide signal is located 950 bp upstream of the ureA start codon ␭ HincII-HindIII DNA fragment from the end of the p narA4 (Fig. 1). chromosomal DNA insert was cloned into pDIA5304 (9), Genes encoding the urease accessory proteins are tightly which had been digested with the same enzymes. The resulting linked with the ureABC genes in Bacillus sp. strain TB-90 and plasmid was integrated into the B. subtilis chromosome by a most other bacteria (12). A search of the translated GenBank Campbell-type recombination. After verification of the plas- sequences with the BLASTP program (1) revealed that the mid integration event by Southern blot analysis, SacI-digested CDSs located adjacent to the B. subtilis ureABC operon do not chromosomal DNA from this strain was ligated at a low con- ␮ encode homologs of any known urease accessory proteins and centration of DNA (5 ng per l) and transformed into Esch- that no B. subtilis 168 (trpC2) sequences encoding proteins with erichia coli TP611 (9). Four independent transformants were similarity to the urease accessory proteins from Bacillus sp. shown to contain plasmids with identical restriction maps. The strain TB-90 have been reported. It is possible that the B. nucleotide sequence of the 11-kb DNA insert from one of subtilis urease accessory genes are located in an unsequenced these plasmids, pDIA5366 (Fig. 1), was determined on both region of the chromosome. Alternatively, assembly of the B. DNA strands and analyzed as previously described (9). ␭ subtilis urease nickel metallocenter may be mediated by pro- Within the DNA region encompassed by the p narA4 and teins lacking significant sequence similarity to the known ure- pDIA5366 plasmids lie three CDSs with significant similarity ase accessory proteins. to the subunits of other bacterial ureases. The B. subtilis UreA B. subtilis ureC mutant. To determine whether ureABC en- protein (105 residues) shares 69 and 64% sequence identity codes a functional urease, the chromosomal ureC gene was with UreA of Klebsiella aerogenes (13) and Bacillus sp. strain inactivated. pURE3 was constructed by cloning the 2.4-kb TB-90 (11), respectively. The B. subtilis ureB gene encodes a NcoI-EcoRI DNA fragment from the ureABC region into protein of 124 amino acids with similarity to the UreB proteins pMTL21P (4) (Fig. 1). In pURE4, a spectinomycin resistance from K. aerogenes (47% identity) and Bacillus sp. strain TB-90 gene (spc) was inserted at the unique NsiI site within ureC (Fig. 1). Linearized pURE4 DNA was used to transform B. subtilis * Corresponding author. Mailing address: Department of Microbi- 168 (trpC2) to spectinomycin resistance as previously described ology, Boston University School of Medicine, 80 E. Concord St., Bos- (16). The resulting strain, SF168U (trpC2 ureC::spc), grew like ton, MA 02118. Phone: (617) 638-5498. Fax: (617) 638-4286. E-mail: wild-type 168 cells in liquid cultures with glucose minimal shfi[email protected]. medium (6) containing glutamine, NH4Cl, or glutamate as the

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FIG. 1. Physical structure of the ureABC region. The physical maps of the ureABC DNA inserts cloned in various plasmids are shown below the ureABC operon map. The location of the spc gene insertion in ureC is indicated. The stem-loop structure indicates a putative transcriptional terminator.

FIG. 2. Arginine and allantoin degradative pathways. (A) The arginine deg- sole nitrogen source with previously described culture condi- radative enzymes in B. subtilis (5) are as follows: 1, arginase; 2, ornithine tions (3). transaminase; 3, pyrroline-5-carboxylate dehydrogenase. (B) The allantoin deg- Ϫ radative enzymes (16) are as follows: 4, allantoinase; 5, allantoicase; 6, allantoate To determine whether strain SF168U (UreC ) could utilize amidohydrolase; 7, ureidoglycolase. urea as a source of nitrogen, wild-type (strain 168) and SF168U cells growing exponentially in glucose minimal medium containing the limiting nitrogen source glutamate were pelleted, coded urease could be involved in the utilization of urea in washed with glucose minimal medium lacking any nitrogen 8 arginine-grown cells. source (MOPSG), and resuspended at 10 cells per ml in To determine whether ornithine is preferentially utilized in glucose minimal medium containing 0.2% urea (International arginine-grown cultures, growth of 168 and SF168U cultures Biotechnologies, Inc.) (99.9% pure) as the only nitrogen Ϫ was examined on limiting arginine. Wild-type and UreC cells source. In the urea medium, the doubling time of the 168 growing exponentially in glucose medium containing excess culture was 130 min, but no increase in the turbidity of the arginine (0.2%) were pelleted, washed with MOPSG, and re- SF168U culture was observed. When cells grown in glucose suspended in glucose medium containing 0.025% arginine minimal medium containing either arginine, glutamate, or al- (Calbiochem; 99% pure). The 168 culture exhibited a biphasic lantoin as the sole nitrogen source were used, urease activity growth pattern in limiting arginine (Fig. 3). During the first was detected in extracts of the wild-type strain but not in growth phase, the doubling time of the 168 culture was 60 min, SF168U (Table 1). These results indicate that ureABC encodes and urease was expressed at low levels. After a growth plateau, the only urease enzyme in B. subtilis. urease levels increased five- to sixfold, and growth resumed at Growth of the UreC mutant on arginine. Arginine is con- a slower rate. The SF168U culture grew at wild-type rates verted to urea and ornithine by arginase (Fig. 2) in B. subtilis during the initial growth phase but had no significant second (5). Although urea is a primary degradative product of arginine, 168 and SF168U cultures both grew rapidly (55 min doubling time) in glucose minimal medium containing excess (0.2%) arginine. Urease is expressed at high levels only in cultures whose growth is nitrogen limited (2). During exponen- tial growth on excess arginine, one of the best nitrogen sources for B. subtilis (3), 168 cells synthesize low levels of urease (2) (Table 1). This suggests that ornithine degradation produces sufficient nitrogen catabolites to permit rapid growth and re- duce ureABC expression. The previously published observation that 168 cultures have similar doubling times in glucose minimal medium containing either arginine or ornithine as the sole nitrogen source (7) is consistent with this hypothesis. Alterna- tively, a urea-degrading enzyme other than the ureABC-en-

TABLE 1. Urease levels in wild-type and mutant strains

Urease sp act (U/mg of protein) a Relevant in cells grown on : Strain genotype Glutamate Arginine Glutamate Allantoin plus NH4Cl 168 Wild type 12 Ͻ8 138 235 Ͻ Ͻ Ͻ Ͻ SF168U ureC::spc 8 10 9 8 Ϫ FIG. 3. Growth and urease levels in strains 168 and SF168U (UreC )in a Urease activity was determined in cell extracts as previously described (2). glucose minimal medium containing limited (0.025%) arginine as the sole nitro- Cultures were grown in minimal medium containing glucose as the carbon source gen source. Samples were removed periodically; data from a typical experiment and 0.2% of each nitrogen source indicated. Data are averages of two to four are shown. Symbols: E, Klett units of the 168 culture; F, Klett units of the determinations which did not vary by more than 30%. SF168U culture; ᮀ, urease activity assayed in wild-type cell extracts. VOL. 179, 1997 NOTES 3373

minimal medium containing both 0.2% NH4Cl and 0.2% allantoin (Sigma) were pelleted, washed with MOPSG, and resuspended in glucose minimal medium containing as the nitrogen source either excess (0.2%) or limited (0.012%) Ϫ allantoin. The wild-type and UreC strains had similar growth patterns in medium containing excess allantoin (Fig. 4). In contrast, the wild-type culture grew to a higher ﬁnal turbidity Ϫ than the UreC culture in medium containing limiting allantoin (Fig. 4). This suggests that allantoate amidohydrolase is involved in allantoin degradation in B. subtilis. Nucleotide sequence accession number. The nucleotide sequences for B. subtilis DNAs in plasmids p␭narA4 and pDIA5366 have been assigned GenBank-EMBL-DDBJ accession no. Z81356 and YO8559, respectively.

We thank R. P. Hausinger for helpful discussions. Work performed at the Pasteur Institute was supported by the Eu- ropean Commission (Biotechnology contract B102-CT-930272) and the Groupement de Recherche et d’Etude sur less Genomes (GREG) (grant 1993-N120). The work at Boston University Medical School was supported by NIH grant GM51127. H.C.-R. was supported by a fel- lowship from the Universidad National Autonoma de Mexico.

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