MICHIHISA MAEDA,t MAKOTO HIDAKA, AKIRA NAKAMURA, HARUHIKO MASAKI, ..... 900. S. F. S M. S. R K. Q Y A D. M F. G. P T. V. G D. A I. R L A D. S. E. L. F.
JOURNAL
OF
Vol. 176, No. 2
BACTERIOLOGY, Jan. 1994, p. 432-442
0021-9193/94/$04.00+0
Copyright X 1994, American Society for Microbiology
Cloning, Sequencing, and Expression of Thermophilic Bacillus sp. Strain TB-90 Urease Gene Complex in Escherichia coli MICHIHISA MAEDA,t MAKOTO HIDAKA, AKIRA NAKAMURA, HARUHIKO MASAKI, AND TAKESHI UOZUMI*
Department of Biotechnology, Faculty ofAgriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan Received 25 March 1993/Accepted 25 October 1993
The urease of thermophilic BaciUlls sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designated ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of KiebsieUla aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter. Urease (EC 3.5.1.5) is an enzyme which catalyzes the decomposition of urea to form ammonia and carbon dioxide and is found widely in plants and microorganisms. The urease of jack bean is a homohexamer of a 90,770-Da subunit, the amino acid sequence of which was revealed by Mamiya et al. (17), and contains nickel (5). On the other hand, the quaternary structures of ureases isolated from some gram-negative bacteria differ from that of jack bean urease. Helicobacterpylori urease consists of two subunits with molecular masses of 62 and 30 kDa, and the native urease molecule is supposed to contain 4 mol of the 62-kDa subunit and 4 mol of the 30-kDa subunit (6). The ureases of some members of the family Enterobacteriaceae, such as Klebsiella aerogenes and Proteus mirabilis, consist of three different subunits with molecular masses of approximately 70, 11, and 9 kDa, which were once designated subunits a, and y, respectively, and the composition of which is presumed to be a2P4Y4 (11, 19, 31). In spite of the difference in the subunit structures of plant and bacterial ureases, the amino acid sequences of bacterial urease subunits show significant homologies to distinct portions of the jack bean urease sequence (12, 13, 20). Recently, genetic and biochemical analyses of gram-negative bacterial ureases revealed that expression of structural genes is not sufficient for expression of catalytically active urease. Several additional genes were needed, and they were named accessory genes by Mulrooney and Hausinger (21). Their products, called accessory proteins, are thought to play important roles in the maturation of urease in a co- and/or posttranslational manner. At least one of these proteins is involved in nickel incorporation into the urease apoenzyme (14, 21). The accessory proteins of different bacteria show significant homol-
ogies. This indicates that some bacterial ureases may mature through very similar pathways. Bacillus sp. strain TB-90 (30) is a thermophilic bacterium screened as a potent producer of uricase at Sapporo Breweries, Ltd., Japan. The optimal temperature for its growth is between 50 and 60°C. This bacterium produces an intracellular thermostable urease with a molecular mass of approximately 240 kDa, and it has a high affinity for urea (Ki,m 0.3 mM). In this article, we report that TB-90 urease is composed of three subunits, like the enterobacteriaceal ureases, and that the structural genes of the three subunits are accompanied by several accessory genes. This is the first report on the subunit structure of a grampositive bacterial urease. MATERIALS AND METHODS
I,
Bacterial strains. Thermophilic Bacillus sp. strain TB-90 was obtained from Sapporo Breweries, Ltd. Escherichia coli HB101 (F- hsdR hsdM supE44 proA2 leuB6 rpsL20 recA13 lacYl galK2 thi-1 ara-14) and E. coli MV1184 [ara A(lac-proAB) rpsL thi (4801acZAM15) A(srl-recA)306::Tn1O(Tetr)/F'(traD36 pro AB' lacIq lacZAM15)] were used as the recipients for transformation with recombinant plasmids. E. coli MC1116 [Alac(IPOZYA)X74 galUgalK trpB9604(Am) SpcAr recA56] was used as the host for measuring ,-galactosidase activity. Measurement of urease activity. Urease activity was measured by quantitating the rate of ammonia release from urea in two ways. One is monitoring the pH change with phenol red (phenol red assay). The other is measuring the formation of glutamate from ammonia and a-ketoglutarate, which was evaluated as a decrease in NADPH by monitoring at 340 nm (coupled enzyme assay). The phenol red assay was used for rough measurement of urease activity as follows. Ten microliters of enzyme solution was added to 0.2 ml of reaction mixture (1 mM Tris-HCl [pH 6.8], 200 mM urea, 0.24 mg of phenol red per ml) and incubated at 37°C. A change in the color of the
* Corresponding author. t Present address: The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan.
432
EXPRESSION OF BACILLUS UREASE GENE COMPLEX IN E. COLI
VOL. 176, 1994
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FIG. 1. Physical maps of three cloned fragments and the reconstructed fragments. (A) Alignment of cloned fragments in pUME, pUMS, and pUMH and positions of probes I and II are illustrated by boxes and thick bars. The positions of the 5.0-kb NheI fragment detected by Southern hybridization with probe I and the 2.3-kb EcoRI fragment detected with probe II are represented by double-headed arrows. In the lower part, the structures of four reconstructed fragments contained in pUMOO, pUM202, pUM002, and pUM011 are illustrated; the origin of each part of the fragments is indicated by open, dotted, and hatched boxes. The urease activity of E. coli cells harboring each plasmid is shown on the right. (B) Magnified physical map of the pUM011 fragment. Beneath it, the arrangement of the nine ure genes is illustrated. A double-headed arrow represents the portion corresponding to the NheI-EcoRI region of pUME which was sequenced prior to cloning of pUMS and pUMH. Abbreviations for restriction enzymes: B, BamHI; E, EcoRI; H, HindIII; N, NheI; Sa, Sacl; Sm, SmaI; Sp, SphI. A
mixture to red was judged as a positive reaction for urease. The coupled enzyme assay was used for measuring urease activity precisely. One hundred microliters of urease solution was added to 2.9 ml of reaction mixture (50 mM Tris-HCl [pH 8.0], 200 mM urea, 0.375 mM NADPH, 1.04 mM a-ketoglutarate, 50 U of glutamate dehydrogenase) in a cell settled in a spectrophotometer and incubated at 37°C. The decrease in the A340was measured continuously for 5 min. A decrease of 1 absorbance unit during 1 min was defined as 0.241 U of urease activity. Thus, 1 U corresponds to the decomposition of 1 pLmol of urea per min. Purification of the TB-90 urease. Bacillus sp. strain TB-90 was grown for 17 h at 52°C in 15 liters of a medium containing 0.2% uric acid, 1% glucose, 0.1% K2HPO4, 0.05% MgSO4 * 7H20, 0.05% Bacto-yeast extract, and 0.05% Bactotryptone with 0.1% metal solution, containing (per liter) 2.5 g of MgSO4. 7H20, 0.3 g of FeSO4 7H20, 0.5 g of CaCl2 * 2H20, 0.06 g of CoCl2 * 6H20, 0.5 g of CUSO4* 7H20, 1.7 g of MnSO4 * 7H20, 0.6 g of NaCl, 0.06 of ZnSO4* 7H20, 1 g of KCl, and 2.38 g of NiCl2 * 6H20. Cells were harvested and sonicated in PET buffer (20 mM potassium phosphate [pH 6.8], 1 mM EDTA, 0.1% Triton X-100) and then centrifuged at 20,000 x g for 10 min. The soluble fraction was recovered and applied to Q-Sepharose column for chromatography. Proteins
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FIG. 2. SDS-PAGE of TB-90 urease. (A) Urease partially purified from Bacillus sp. strain TB-90 was denatured in SDS gel sample buffer and electrophoresed on a 12 to 18% gradient polyacrylamide-SDS gel. The mobilities of molecular size markers are indicated on the right. (B) Extracts of E. coli cells harboring pUC19 (lane 2) or pUM31 (lane 3) were electrophoresed. A cell lysate of E. coli(pUM31) and the precipitate (inclusion body fraction) obtained by centrifugation of the lysate at 85 x g for 10 min were also electrophoresed in lanes 4 and 5, respectively. (C) Cell extracts of E. coli(pUM011) were electrophoresed (lane 1). (B and C) Partially purified TB-90 urease was electrophoresed in lanes 1 and 2, respectively, and the positions of UreA, UreB, and UreC are indicated.
434
J. BACTERIOL.
MAEDA ET AL.
-10 -35 GCTAGCGGGTTTGTAAAATGAGTGAGAATCTGTTGGATGTATTAGAAGGAAATTTGATATAATTATTAGACGAAAAATTCTAAATATTCGATCTGTGAAT
GGAGATTTACAAGATCAATTTCATAGTTTGGAACAAGCACTTAAAATAGGAGGTTATCCATGAAACTGACTTCACGTGAAATGGAAAAGCTCATGATTGT S K
M K L T R E M E L M I V ureA AGTGGCGGCTGACTTGGCCCGCCGTCGTAAAGAGCGGGGCTTAAAATTAAATTATCCTGAAGCTGTCGCAATGATTACATATGAAGTGCTGGAGGGGGCG V A A D L A R R R K E R G L K L N Y P E A V A M I T Y E V L E G A CGGGATGGAAAAACGGTAGCTCAGTTAATGCAATACGGTGCAACGATTCTTACAAAAGAAGATGTAATGGAAGGGGTTGCCGAAATGATCCCGGATATTC R D G K T V A Q L M Q Y G A T I L T K E D V M E G V A E M I P D I Q AAATTGAGGCAACCTTTCCTGATGGAACAAAGCTTGTCACGGTTCATGACCCGATCCGTTAAATGAGGAGG~ACGTACGATGATACCAGGGGAGTATGTAT ureB M I P G E Y V L I E A T F P D G T K L V T V H D P I R * TAAAAAAAGAACCTATTTTATGCAATCAAAATAAGCAGACGATCAAGATTCGCGTGTTAAACCGGGGCGATCGACCTGTTCAGGTTGGTTCCCATTTTCA KK E P I L C N Q N K Q T I K I R V L N R G D R P V Q V G S H F H TTTTTTTGAAGTGAATCAATCGCTTCAATTTCATCGTGAAAAAGCATTTGGCATGCGTTTGAATATTCCGGCTGGAACGGCGGTTCGCTTCGAGCCCGGA F F E V N Q S L Q F H R E K A F G M R L N I P A G T A V R F E P G
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300 400 500 600
700
GATGCGAAAGAAGTAGAAATAATTCCATTTTCAGGTGAACGCAAAGTGTATGGTTTAAATAATGTAACGAATGGATCAGTTGAAATGGGGAAAAGAAAAT V K R K
800
GAGTTTTTCGATGTCTCGAAAGCAATATGCGGATATGTTTGGACCAACTGTCGGCGACGCCATTCGTTTGGCAGATTCAGAATTGTTTATCGAAATTGAA S F S M S R K Q Y A D M F G P T V G D A I R L A D S E L F I E I E AAGGACTATACAACGTATGGAGATGAGGTAAAGTTTGGCGGCGGCAAGGTGATCCGAGATGGAATGGGGCAGCATCCTTTGGCGACAAGCGATGAATGCG K D Y T T Y G D E V K F G G G K V I R D G M G Q H P L A T S D E C V TCGATCTCGTATTAACAAATGCGATTATTGTTGATTACACAGGTATTTATAAAGCAGATATCGGCATAAAAGATGGAATGATTGCCTCCATAGGAAAAGC D L V L T N A I I V D Y T G I Y K A D I G I K D G M I A S I G K A GGGGAACCCGTTGTTAATGGACGGGGTCGATATGGTGATTGGAGCAGCAACAGAAGTCATAGCCGCAGAAGGGATGATTGTGACAGCCGGAGGAATAGAT G N P L L M D G V D M V I G A A T E V I A A E G M I V T A G G I D GCTCATATTCACTTTATTTGCCCTCAGCAAATCGAAACCGCTCTTGCATCGGGTGTGACCACTATGATTGGCGGAGGAACAGGACCCGCTACAGGCACAA A H I H F I C P Q Q I E T A L A S G V T T M I G G G T G P A T G T N ATGCCACTACTTGTACACCGGGGCCTTGGAATATCCATCGTATGCTTCAAGCAGCCGAAGAATTCCCGATAAACTTGGGCTTTTTAGGAAAGGGAAACTG A T T C T P G P W N I H R M L Q A A E E F P I N L G F L G K G N C TTCAGATGAGGCTCCTTTAAAGGAACAAATTGAAGCGGGAGCGGTGGGATTAAAGCTTCACGAAGATTGGGGATCGACGGCGGCGGCTATTGATACATGT S D E A P L K E Q I E A G A V G L K L H E D W G S T A A A I D T C TTGAAAGTGGCGGATCGATATGATGTGCAAGTAGCGATTCATACAGACACTTTAAATGAAGGCGGATTTGTCGAGGATACTTTGAAAGCCATAGACGGTC L K V A D R Y D V Q V A I H T D T L N E G G F V E D T L K A I D G R GAGTGATTCATACCTATCATACAGAAGGGGCTGGCGGGGGACATGCTCCGGATATTATAAAAGCGGCCGGCTTCCCGAATATTTTGCCTTCTTCCACGAA V I H T Y H T E G A G G G H A P D I I K A A G F P N I L P S S T N
900
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TCCAACTCGACCTTATACTATCAATACTTTGGAAGAGCATTTAGATATGTTAATGGTTTGCCACCACCTAGACGCTAATATTCCAGAGGATATTGCTTTT E
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F GCCGATTCACGCATACGGAAAGAGACCATCGCGGCGGAAGATGTTTTACATGATTTAGGCGTTTTCAGCATGATTTCGTCTGATTCACAGGCGATGGGGC A D S R I R K E T I A A E D V L H D L G V F S M I S S D S Q A M G R P
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GAGTAGGAGAAGTGATCATTCGTACGTGGCAAACGGCTGACAAGATGAAAAAGCAAAGAGGGAAGTTACAAGAAGACAATGGTGTGGGAGACAACTTTCG V G E V I I R T W Q T A D K M K K Q R G K L Q E D N G V G D N F R TGTGAAACGTTATATTGCCAAATATACGATCAATCCGGCCATTGCTCATGGTATTGCGGATTATGTGGGTTCTGTTGAAGTGGGAAAATTAGCTGATTTA V K R Y I A K Y T I N P A I A H G I A D Y V G S V E V G K L A D L GTGGTGTGGAATCCTGCTTTTTTTGGTGTGAAACCTGAACTGGTCTTAAAAGGAGGAATGATTGCTTACAGCACTATGGGAGATCCCAATGCCAGCATTC
I A Y S T M G D P N A S I P CGACACCGCAGCCGGTTTTATATCGTCCGATGTTTGCAGCGAAAGGAGATGCCAAATATCAAACGTCTATCACCTTTGTTTCGAAAGCAGCGTATGAAAA T P Q P V L Y R P M F A A K G D A K Y Q T S I T F V S K A A Y E K
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1000 1100
1200 1300 1400 1500
1600 1700
1800 1900
2000 2100 2200
2300
AGGCATTCATGAACAGTTGGGTTTGAAGAAAAAGGTGAAACCAGTCCATGGAATTCGAAAATTGACGAAAAAAGATTTAATTTTGAACGATAAAACCCCA G I H E Q L G L K K K V K P V H G I R K L T K K D L I L N D K T P AAAATTGACGTCGATCCTCAGACATATGAAGTAAAGGTAGACGGTCAATTAGTGACATGTGAACCGGCAGAAATCGTCCCTATGGCACAACGGTATTTCT K I D V D P Q T Y E V K V D G Q L V T C E P A E I V P M A Q R Y F L
2400
TATTTTGAGGTGAGAAAAACATGATGGTTGAAAAAGTAGTCGGAAACATCACGACATTAGAGAAAAGAGTTCCACATATAGAACGGGTTTATATGCGAAG * V P H I E R V Y M R S K
2600
CGATGATTTAGTCAAACGGGTGAAACGGGTTGTGACAGATCATGGCAAAGAGATTGGCATTCGCTTAAAAGAACATCAGGAATTACAAGATGGAGATATT D D L V K R V K R V V T D H G K E I G I R L K E H Q E L Q D G D I TTATACATGGACGATCACAATATGATTGTGATATCGGTCTTAGAAGATGATGTATTAACGATCAAACCAACATCCATGCAGCAAATGGGGGAAATTGCGC L Y M D D H N M I V I S V L E D D V L T I K P T S M Q Q M G E I A H ATCAACTTGGCAATCGGCATTTGCCGGCGCAGTTTGAAGGAAATGAAATGATTGTGCAATATGACTATTTAGTAGAAGAACTGCTTCAGAAGCTATCGAT Q L G N R H L P A Q F E G N E M I V Q Y D Y L V E E L L Q K L S I
2700 2800
TCCATTTACACGTGAAAACAGAAAAATGAAACAAGCATTTCGCCCAATAGGACATCGTCATGAATAGATTACTTTCACTTTTTCAGTTGTGTGATTCCAA A F R P I G H R H E * R N R K M K
3000
ureE
F
P
F
M
M
V
V
V
G
N
I
T
T
L
R
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ureF M N R L L S L F Q L C D S N CTTTCCATCGGGATCGTTTTCTCACTCTTTTGGATTGGAAACATACATTCAAGAAAAAGTGATCACGGATAAAGAAAGCTTTAAGAATGCTATTTCCGTT F P S G S F S H S F G L E T Y I Q E K V I T D K E S F K N A I S V TATATTCGGAAACAGCTGTTTTTCACAGAAGGCCTTGCTTGCATACTGGCTTATGAAGCAATGGAAAAAAATGAACCATCCGCGTTGGTAGAGCTTGATC Y I R K Q L F F T E G L A C I L A Y E A M E K N E P S A L V E L D H ATATTTTATTTGCATCGAACGTTGCGCAAGAAACAAGAAGCGGGAATCAAAGGATGGGGGAACGGATGGCCAAGCTATGTGTGGATCTGTATCCATCTCC I L F A S N V A Q E T R S G N Q R M G E R M A K L C V D L Y P S P TATTTTAATAGAATATACAAACCGGATAAAAGAAAAGAAAGCGTATGGTCATTCTGCCATAGTGTTTGCGATTGTCGCTTACCACTTAAAGGTGACAAAG I L I E Y T N R I K E K K A Y G H S A I V F A I V A Y H L K V T K -35 -10 GAAACGGCAGTCGGAGCCTATTTATTTGCCAATGTATCTGCTCTTGTGCAAAATGCGGTGCGTGGAATTCCGATTGGTCAAACAGACGGTCAAAGGATCT E T A V G A Y L F A N V S A L V Q N A V R G I P I G Q T D G Q R I L TAGTTGAAATTCAACCTCTTTTAGAGGAAGGGGTAAGAACGATTTCACAATTGCCTAAAGAGGATTTAGGAGCGGTAAGTCCCGGGATGGAAATTGCTCA V E I Q P L L E E G V R T I S Q L P K E D L G A V S P G M E I A Q AATGCGCCATGAACGTTTAAATGTGCGCTTATTTATGTCTTAAAATTCGGAATATAAGGAG~TGAGAAACGTGGAACCAATACGTATTGGAATCGGAGGGC M E P I R I G I G G P M R H E R L N V R L F M S * ureG CTGTTGGAGCGGGAAAAACGATGCTTGTGGAAAAATTGACGAGAGCCATGCATAAAGAATTGAGCATCGCCGTTGTGACAAATGATATCTATACGAAAGA V
G
A
G
2900
Q
E
T
K
E
2500
K
T
M
L
V
E
K
L
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A
M
H
K
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L
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I
A
V
V
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3100 3200 3300 3400
3500 3600
3700 3800
EXPRESSION OF BACILLUS UREASE GENE COMPLEX IN E. COLI
VOL. 176, 1994
435
AGATGCTCAATTTCTTCTGAAGCATGGTGTGCTGCCGGCAGATCGCGTCATTGGTGTGGAAACAGGAGGGTGTCCGCATACAGCTATTCGTGAAGATGCA
3900
TCCATGAATTTTCCCGCCATTGATGAACTAAAAGAAAGGCATCCCGATCTTGAGTTGATATTTATCGAAAGCGGCGGGGATAATCTAGCCGCTACATTTA
4000
GTCCGGAACTAGTCGATTTCTCTATTTACATTATTGATGTAGCGCAAGGGGAAAAAATTCCGCGTAAAGGTGGACAAGGGATGATTAAATCTGTACTTTT
4100
Q
A
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4200 TATCATCAATAAAATTGATCT4CGCTCCGTACGTTGGAGCCAGTTTAGAGGTTATGGAGCGCGATACTCTGGCAGCGAGAGGGGATAAGCCATATATTTTT N
I
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ACCAATTTAAAAGATGAAATCGGTCTTGCAGAAGTATTGGAATGGATCAAAACCAATGCACTATTGTACGGATTGGAATCATGAAATGTACAGGAGTATT *
4300
GCAGCTATCTGCCGCTAAAAAGCGGCAAAAAACCATTATCTCATCTTGCTATCATGAAGGGGCCTTAAAAGTGAGCCGTCCTATTTATCTTGAAAAGGAT
4400
CTACCTTTCTTATATCTCATTCATGTTGGCGGAGGTTATGTAGACGGGGATGTTTATTTAACCAATCTTGATGTGGAAGAAGAAGCTGAGCTGGCGGTTA
4500
CAACGCAATCCGCTACAAAGGTATATAAAACACCAAAGAAGCCTGTAGTTCAGCAAACGAACATTCATTTAAAGAAAGGAAGCGTTCTTGAATACTTGCT
4600
GGATCCATTAATTTCTTATAAAGGAGCACGTTTTATACAAGAAACGACCGTTCATATAGAAGAGGATTCCGGTTTTTTTTACAGCGATGTCATTACACCG
4700
GGTTGGGCGGAAGATGGGAGTTTGTTTCCTTATGATTGGATTCGTTCGAAATTAAAAGTATATAAAAAGGACCGGCTTGTGTTGTTTGATCATTTACGGC
4800
TTGAGCCGGATGAAGATATGTCCGGAATGCTGCAAATGGACGGATATACTCATATCGGCACTTTTTTGATCTTTCATCAGAAAGCAGATAAAACATTTCT
4900
TGACCGTTTATATGATGAAATGGAGGCTTTTGATTCCGACGTTCGATTTGGTATGACATCATTGCCGGCTAGCGGCATTATTTTACGTATACTTGCACGT
5000
AGTACGGGTATCATTGAGAACATGATTTCTCGCGCTCATTCATTTGCCAGACGAGAGTTATTAGGCAAGAATGGTGTCACTTGGCGAAAGTATTGAAAGA
5100
TAGGGTGAGGAATAAATGATATTA'G'A'GGCTTATAGATGGACCAAGTAGACTTGCTTTCCATTTTAACTTTAGGATTCGTTCTTGGAATAAAACATGCTA M D Q V D L L S I L T L G F V L G I K H A M ureH
5200
TGGAGCCGGATCATGTGATAGCTGTATCAACGATTGTTTGTCAAAGTAAAAAACTTTGGAGATCCTCTTTAGCTGGAGTCTTTTGGGGGATCGGGCATAC
5300
ATCCACATTACTTATATTTGGAATGACTATTATTTTGATGAAGAAAAAAATTTCACAAGAATGGTCGATGTCATTAGAGTTTTTAGTCGGAATCATACTC
5400
GTTTATTTTGGAATTTCAGCCATTCTTTCTCTTAAGAAAACGCATGAACATTCCCATTCACGTCTTCATCTTCATACAGATCATCCTATTTATACATATA
5500
AAGGGATTCCCTATGTTAAATCTCTTTTTATTGGGATCATTCATGGACTTGCCGGAAGTGCAGCCATGGTATTGTTGACAATGAGTACAGTAGAAAAGGC
5600
ATGGGAAGGTCTTCTTTATATTCTGTTTTTTGGCGCCGGAACGGTTTTAGGCATGCTTTCGTTTACGACTTTAATTGGAATCCCATTTACACTGAGTGCC
5700
AGAAAGATTCGTATTCACAATGCCTTCATTCAAATAACGGGATTCATCAGCACGGTATTCGGAATTCATTATATGTATAATTTAGGTGTAACCGAAGGCT
5800
TATTTAAACTTTGGATACGGTAAATGGGAACAAGAAAATTTTTTTTGCTGCATCATGATAAGATCATTCGTTCCATCATAATGGCGAACGGTATTAAAAA *
5900
N
T
L
Q
L
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I R M G T R K F F L L H H D K I I R S I I M A N G I K N urel CTATTGGCAGGATGATGGGCGGTTTGACAGATGCTCGGAGATCTTTTTTCTAAGCCAAAAGCAAGGGCGGGGTCTTTACCGTTTTTGGTTAGGAAATAAA Y W Q D D G R F D R C S E I F F L S Q K Q G R G L Y R F W L G N K AAACAAGGGAATCGTGATTAGACGAAAGATTCCCCTGTTTTCATTTCTGCGCTTGCTAAGTGTTCCTAAACTCTTTTCTTTTGAAAGGCTGCAATACTTT K Q G N RD * CATATTCTTCTTTTAACAGTCGAGAAAGACGAATGTGATAGGAATTAATTCTTTATAGACGGATACATGTTCTGGATTCGGTACATGGTGGTGGGTAGGC ACCTACCATTTAGATACTACGTGTAATGAATGAATTTCACCTAGACTATATAAGCCAAGGATAGCGGCTCCCAAACAAGAACTTTCAAAACTTTCCGGTA TATATACCTCTTGATCGAAGATGTCCGCCATCATTTGACGCCAGAGTGGGGATCTGGCAAAGCCTCCGGTGGCTTGAATTTTTTTCGGCTCACCGATTAA TTCTTCTAAGGCAAGCAGAACGGTGTATAAGTTGAAAATAACCCCTTCCAAAACAGCACGAATTAGATGTTCTTTTTTATGATCTAAACCTAATCCAAAG AAAGAGCCTCTTGCATTGGCATCCCACAATGGAGCTC F
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6200 6300 6400 6500 6537
FIG. 3. Nucleotide and deduced amino acid sequence of TB-90 urease gene complex. Two putative promoter structures are indicated by -35 and -10 above the nucleotides. Probable Shine-Dalgarno sequences are marked with dots. Two inverted repeat structures are indicated by solid and dashed underlines. Amino acid sequences of the urease subunit polypeptides, determined by chemical degradation with an amino acid sequencer, are represented in boldface type. were eluted with a linear KCI gradient from 0 to 1 M in S buffer
(20 mM potassium phosphate [pH 6.8], 1 mM EDTA, 15 mM 2-mercaptoethanol, 0.l1o Triton X-100). Protein elution was monitored at 280 nm. All separated fractions were screened for urease activity by the phenol red assay. Further purification was achieved on a Butyl-Toyopearl column, then on a Mono-Q column, and finally on a Superdex 200 column with the high-pressure liquid chromatography (HPLC) system. The Butyl-Toyopearl column was eluted initially with S buffer containing 1 M KCI and then with S buffer. The Mono-Q
column was first washed with S buffer and then eluted with a linear gradient of KCI from 0.1 to 0.5 M. Chromatography on the Superdex 200 column was performed with S buffer con-
taining 0.2 M KCI. The urease fractions obtained were pooled and used for the following experiments.
Determination of N-terminal amino acid sequence of each subunit. The purified urease was boiled in 100 mM Tris-HCI (pH 8.5) containing 2% sodium dodecyl sulfate (SDS) and 50 mM 2-mercaptoethanol and then incubated with sodium monoiodoacetate at 30°C for 30 min. The carboxymethylated subunits were isolated by a combination of TSK G3000SW (Tosoh) gel filtration and FP-308-1251 (Tosoh) reverse-phase column chromatographies performed with the HPLC system. The N-terminal amino acid sequence of each subunit was determined with an amino acid sequencer (model 477A;
Applied Biosystems).
436
MAEDA ET AL. UreH HoxN UreH HoxN
UreH HoxN
J. BAcrIERIOL. M------------------------------------------ DQVDLLSILTLGFVLGI
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UreH HoxN
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228 301
FIG. 4. Comparison of amino acid sequences of TB-90 UreH and A. eutrophus HoxN. Identical amino acids are marked with colons. Amino acids with similar properties are marked with dots.
Synthetic oligonucleotides. Three kinds of oligonucleotides synthesized with a DNA synthesizer (model 380B; Applied Biosystems) based on the N-terminal amino acid sequence of each subunit. The nucleotide sequence of each mixture was as follows. CARTAYGCNGAYATGTTYGG (where R is an A or G, Y is a C or T, and N is an A, G, C, or T) was designed from the Gln-Tyr-Ala-Asp-Met-Phe-Gly sequence of the 66-kDa subunit (UreC); AARAARGARCC NATNYT was designed from the Lys-Lys-Glu-Pro-Ile-Leu sequence of the 12-kDa subunit (UreB); and GARATGGA RAARYTNATGAT was designed from the Glu-Met-Glu-LysLeu-Met-lie sequence of the 8-kDa subunit (UreA). Each mixture was labeled with [_y-32P]ATP and T4 polynucleotide kinase and used as a probe for hybridization. Southern hybridization. Ten micrograms of chromosomal DNA isolated from TB-90 by the method of Saito and Miura (24) was digested with several restriction enzymes and electrophoresed in a 0.8% agarose gel. The fractionated DNA was transferred to a nitrocellulose filter and hybridized with the 32P-labeled probes as described by Sambrook et al. (25). Determination of nucleotide sequence. The cloned DNA fragment was subcloned into pUCt18 and pUC119 (32). The resulting plasmids were introduced into E. coli MV1184, which was then infected with bacteriophage M13KO7 (32), and single-stranded DNAs derived from the plasmids were recovered. The nucleotide sequence of each single-stranded DNA was determined by the dideoxy chain termination method of Sanger et al. (26). Plasmid construction. The complete urease gene complex and several deletion and frameshift mutations in the urease locus were constructed by linking some portions of the cloned 3.2-kb EcoRI, 5.0-kb SphlI, and 6.4-kb HindIII fragments (pUME, pUMS, and pUMH, respectively) on pUC18 or pUC19 (Fig. 1A). By combining a 2.5-kb EcoRI-SphI fragment or a 0.7-kb NheI-SphI fragment of pUME with the 5.0-kb pUMS fragment, pUM00 and pUM202 were constructed, respectively. By replacing a 2.1-kb SmaI-SphlI portion of pUM202 with a 5.9-kb SmaI-HindIII fragment or a 3.0-kb SmaI-SacI fragment of pUMH, pUM002 and pUM011 were also constructed, respectively. In the following, the position of each restriction site is indicated by the nucleotide (nt) position of the pUM011 fragment (see Fig. 3). As summarized in Fig. 5, pUM03, pUM34, and pUM31 were made by deleting the 1.1-kb were
BamHI-SphI (nt 4601 to 5651) portion, the 2.0-kb SnaBI-SphI (nt 3680 to 5651) portion, or the 2.5-kb StuI-SphI (nt 3130 to 5651) portion of pUM202, respectively. An 8-bp Sall linker (GGTCGACC) was inserted at the BalI site (nt 3267) of pUM202 to generate pUM212. pUM202 was digested with Spel (nt 4008) or NheI (nt 4968), filled in with Klenow fragment, and then self-ligated, giving rise to pUM222 and pUM242, respectively. Likewise, pUM011 was digested with AflII (nt 5431) or BglII (nt 5939), filled in with Klenow fragment, and then self-ligated to construct pUM051 and pUM061, respectively. pUM014 was constructed from pUM011 as follows. The AGATCA (nt 2637 to 2642) of pUM011 was mutated to AGATCT (BglII site). The resulting plasmid was digested with BglII, filled in with Klenow fragment, and self-ligated. Expression of urease genes in E. coli. E. coli MV1184 harboring each constructed plasmid was inoculated into a medium (ammonia-free M9 medium [25] supplemented with 0.1% Bacto-tryptone, 0.05% Bacto-yeast extract, 0.5% sodium succinate, and 10 ,uM NiCl2) and cultivated at 37°C until the optical density at 660 nm reached 1.0. The cells were harvested and washed with 20 mM potassium phosphate (pH 6.8). The cells were suspended in PET buffer, sonicated, and centrifuged at 10,000 x g for 5 min. The recovered supemratant was used directly for the coupled enzyme urease assay. The total amount of protein in the extract was determined by the method of Bradford (1). Nickel dependence studies. The effect of the nickel concentration on the urease activity expressed from recombinant urease genes in E. coli was determined by growing E. coli to the late exponential phase in a medium (ammonia-free M9 medium supplemented with 0.1% Bacto-tryptone, 0.05% Bactoyeast extract, and 0.5% sodium succinate) containing defined levels of nickel. Construction of ureA::*acZ gene fusion and plasmids pBAD and pADG. To construct pBF101, pUM202 was digested with PmaCI (nt 173), treated with alkaline phosphatase, and ligated to a lacZ fragment, an XmaI-SalI fragment derived from pMC1871 (2), both ends of which were filled in with Klenow fragment. In this construction, an open reading frame (ORF) encoding ,-galactosidase was fused in-frame to that coding for the N-terminal five amino acids of UreA. Then, a 5.0-kb SphI fragment (nt 651 to 5651) of pBF101 was replaced with the corresponding SphlI fragment derived from pUM212,
75
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between 13.4 U/mg of protein (pUM014) and 21.1 U/mg of protein (pUM011). Effects of nickel concentration on urease activity. As described above, the lack of ureD, ureF, and ureG resulted in the loss of urease activity. On the other hand, the lack of ureH and ureI did not abolish urease activity in E. coli. The similarity of UreH to A. eutrophus HoxN, a high-affinity nickel-specific transporter, reminded us that the E. coli high-affinity nickelspecific transport system encoded by hydC (35, 36) partially suppressed the effects of the lack of ureH and ureL. To investigate whether these abolished and reduced urease activities would be restored by a higher extracellular concentration of nickel ions, urease activities in the E. coli transformants described above were examined after cultivation in medium containing nickel ions at various concentrations. As shown in Fig. 5, the loss of urease activity caused by the lack of ureD, ureF, and ureG was not restored even if the cultivation medium was supplemented with 100 ,uM nickel ions. In contrast, addition of 100 ,uM nickel ions restored urease activity in E. coli cells containing the ureH- or ureI-defective urease gene complexes, implying that UreH and Urel are not necessary for the expression of catalytically active urease in E. coli when the cells are surrounded with a high concentration of nickel ions. A detailed analysis of the relationship between the nickel concentration and defects in ureH or ureI was carried out. In the case of E. coli(pUM011), urease activity reached the maximum level even at a concentration of 2 jiM nickel ions (Fig. 6). On the other hand, the urease activities of E. coli harboring either pUM051 or pUM061 increased in proportion to the nickel concentration in the medium and reached the maximum level at a nickel concentration of 50 ,uM, although the activity of E. coli(pUM051) was lower than that of E. coli(pUM061). Abundant production of the urease subunit polypeptides in all three transformants was observed in SDSPAGE irrespective of the nickel concentration (data not shown). The decrease in specific activities at a nickel concentration of 200 ,uM may be caused by the toxicity of nickel for E. coli cells. UreD, UreF, and UreG activate urease at a co- and/or posttranslational stage. As a clue to reveal the role of the accessory genes, Lee et al. demonstrated that ureD, ureE, ureF, and ureG of K aerogenes are necessary for the functional incorporation of the urease metallocenter (14). One possibility
439
is that one or several of the accessory proteins fulfill the role of nickel incorporation into apourease. We observed that the deficiency of TB-90 UreD, UreF, and UreG did not affect the production of the urease subunit polypeptides. To confirm this event quantitatively, a ureA-lacZ fusion gene was constructed, and the levels of its transcription from the urease gene complexes defective in ureD, ureF, or ureG (pBF104, pBF102, and pBF103, respectively) were tested by measuring J-galactosidase activities. The activity in an extract of E. coli harboring pBF101, which contains intact ureD, ureE, ureF, and ureG, was 182 Miller units, and extracts of E. coli harboring pBF102, pBF103, or pBF104 showed almost equal activities (219, 116, and 249 Miller units, respectively). From this, the independence of ureA transcription from the functions of UreD, UreF, and UreG was confirmed, supporting the idea that these accessory proteins function at a co- and/or posttranslational stage. Whether the catalytic activity of urease protein translated without accessory proteins is restored in vitro by the addition of accessory proteins was then tested. To do this, pBAD, which contains ureA to ureE under the control of the tetracycline resistance gene promoter on pBR322, and pADG, which contains ureE to ureD under the control of the tetracycline resistance gene promoter on pACYC184, were constructed. Extracts of E. coli(pBAD) and E. coli(pADG) showed no urease activity, as expected. On the other hand, E. coli transformed with both plasmids showed a urease activity of 19.8 U/mg of protein when the cells were cultivated in a medium containing 100 ,uM nickel ions. However, no activity was observed when an extract of E. coli(pADG) cultivated with 100 p,M nickel ions was mixed with an equal amount of an extract of E. coli(pBAD) and incubated at 37°C for 1 h, indicating that reconstitution of urease activity in vitro is not possible. This is consistent with the result observed with the K aerogenes urease gene complex (21). DISCUSSION We purified the urease from the thermophilic Bacillus sp. strain TB-90 and revealed that this urease consists of three distinct subunits. We also reconstructed a urease gene complex which comprises nine genes and expresses catalytically active TB-90 urease in E. coli. The urease of Bacillus pasteurii has been well studied, and this enzyme is a tetramer of identical subunits (3). In contrast, TB-90 urease possesses two small subunits (UreA and UreB) and a large subunit (UreC), the molecular masses of which were estimated to be 8, 12, and 66 kDa by SDS-PAGE of the purified urease and 11, 12, and 61 kDa from the deduced amino acid sequences, like the ureases of K aerogenes (21, 31) and P. mirabilis (11, 12). We have not yet analyzed the subunit composition of TB-90 urease experimentally. When we assumed that native TB-90 urease was composed of 2 mol of the large subunit and 4 mol of each small subunit, the calculated molecular mass became 212 kDa (by SDS-PAGE) or 214 kDa (deduced from the sequence), which are in good agreement with the experimentally determined molecular mass of native TB-90 urease, 240 kDa. From this, we suppose that not only the subunit structure but also the subunit composition may be the same as that of the enterobacteriaceal urease. In the GenBank DNA sequence data base, we found a nucleotide sequence encoding the three subunits of Lactobacillus fermentum (a gram-positive bacterium) urease (29). The sequence similarities between UreA, UreB, and UreC of TB-90 urease and those of K aerogenes, P. mirabilis, and L. fermentum and UreA and UreB of H. pylori (13) are shown in
440
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MAEDA ET AL. Amino Acid Sequence Identity 50
required for the expression of catalytically active ureases, and
1 00
we also confirmed this property by using ureD-, ureF-, and ureG-defective TB-90 urease gene complexes. On the other hand, TB-90 ureE was not essential for generating active urease in E.. coli. A lack of K aerogenes ureE did not affect urease activity expressed in E. coli, either (14). One possibility
(%)
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L. fermentum UreC K aerogenes UreD P. mirabilis UreD TB-90 UreD H. pylon UreH K aerogenes UreE P. mirabilis UreE
TB-90 UreE H. pylon UreE K. aerogenes UreF P. mirabilis UreF
TB-90 UreF H. pylon UreF K. aerogenes UreG P. mirabilis UreG
TB-90 UreG H.
pylon UreG
FIG. 7. Comparison of TB-90 Ure proteins with those of L. fermentum and several gram-negative bacteria. The similarities are indicated as dendrograms, drawn by the UPGMA method (27) with CLUSTAL V (9). The scale represents the percent amino acid identity between each Ure protein.
Fig. 7 as dendrograms drawn by the UPGMA method (27). Every polypeptide exhibited significant similarity with the others, and we can point out two characteristics. (i) TB-90 UreA and UreB are similar to the N- and C-terminal halves of H. pylori UreA, respectively, at levels of 61 and 51% identity, as indicated for the enterobacteriaceal ureases (13). (ii) The TB-90 and L. fermentum subunits are not so similar, resembling the similarities of TB-90 urease subunits with those of ureases from gram-negative bacteria. These characteristics interested us in the origin and evolutionary pathway of the enterobacteriaceal urease genes. Another feature of the TB-90 urease gene complex is that in this complex, a complete set of accessory genes existing in the gram-negative bacterial urease gene complexes is conserved. Until now, four genes were characterized as urease-related accessory genes, i.e., ureD, ureE, ureF, and ureG of K aerogenes (14, 21) and P. mirabilis (12, 28), and all four genes are conserved in the TB-90 urease gene complex. In the case of H. pylori, Lee et al. pointed out that not ureD but ureH is homologous to ureD of K aerogenes (14), and we also found that TB-90 UreD was 24% identical to H. pylori UreH. It has already been demonstrated that ureD, ureF, and ureG of K aerogenes (14) and ureF, ureG, and ureH of H. pylori (4) are
is that the lack of UreE was complemented by one or more E. coli proteins. One remarkable character of TB-90 UreE is the loss of a histidine cluster which is predicted to exist at the carboxyl terminus of UreE from K aerogenes and P. mirabilis and
expected to function as a nickel-binding site (21). In Fig. 7, the similarities of the accessory proteins of TB-90 and gramnegative bacteria are shown. Although UreD, UreE, and UreF have diverged at rather high levels, UreG was conserved among the four bacteria. UreG may be the key enzyme of the urease maturation pathway, i.e., nickel incorporation of the urease metallocenter. A significant similarity (26% identity) of TB-90 UreG to the E. coli HypB protein, which is supposed to be involved in nickel donation to the hydrogenase apoprotein (15, 16), supports this idea. The most remarkable feature of the TB-90 urease gene complex is the presence of the genes designated ureH and ureL. Urease activities in E. coli containing the ureH- or ureldefective TB-90 urease gene complex were not abolished and increased in proportion to the nickel concentration in the cultivation medium. This event resembles the alteration of hydrogenase activity in hoxN-defective A. eutrophus, in which HoxN functions as a high-affinity nickel-specific transporter (7). In this case, nickel ions at a high extracellular concentration were taken up via a second transport system which normally mediates the uptake of magnesium. Similar nickel transport systems are present in E. coli. Five proteins encoded by the hydC locus constitute a high-affinity nickel-specific transporter (34, 37). Nickel transport via a high-capacity magnesium transport system was also demonstrated (10, 33). Mutation of hydC caused a reduction in hydrogenase activity, which was restored by addition of 500 ,uM nickel ions to the cultivation medium (35). Moreover, TB-90 UreH shows significant similarity to A. eutrophus HoxN, and the hydropathy profile of TB-90 UreH indicated that it may be a transmembrane protein. This situation strongly suggests that TB-90 UreH and UreI may constitute a multicomponent high-affinity nickel transporter, which can function even in E. coli cells. A deficiency in this transporter system must be partially suppressed by the function of the E. coli high-affinity nickel transporter at a low extracellular nickel concentration and completely by the function of the E. coli magnesium transporter at a high extracellular nickel concentration. We conclude that the TB-90 urease gene complex is an enterobacteriaceal-type gene complex. From this viewpoint, we compared the structures of the TB-90, K aerogenes, and H. pylori urease gene complexes. The results are summarized in Fig. 8. In the case of TB-90, all the accessory genes are located downstream of the structural genes, although they are located on both sides of the structural genes in K aerogenes and H. pylori. However, the alignment order of ureE, ureF, and ureG is strictly conserved. Moreover, the position of ureD (ureH in H. pylori) is fixed immediately downstream of ureG in the gene complexes of TB-90 and H. pylori. From this, and considering the sequence similarity of each ure gene product among the three bacteria (Fig. 7), it may be concluded that the TB-90 urease gene complex more closely resembles that of H. pylori than that of K aerogenes. It is very interesting to propose that an original urease gene complex, which consisted of three structural genes and four accessory genes, was spread widely
EXPRESSION OF BACILLUS UREASE GENE COMPLEX IN E. COLI
VOL. 176, 1994 1 2 I_ __I
0
3
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Accessory Genes
FIG. 8.; Structures of urease gene complexes. The urease gene complexes of TB-90, H. pylori (4, 13), and K aerogenes (14, 21) are aligned on the basis of the position of structural genes. Boxes represent the sizes of ORFs, and the molecular masses of the encoded polypeptides are indicated beneath the boxes. Homologous ORFs or regions are marked with the same patterns. Open boxes indicate ORFs unique to each complex.
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