Appl Microbiol Biotechnol (1992) 37:211-215

App//ed Microbiology Biotechnology © Springer-Verlag 1992

The DNA sequence of ?,-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1 Toshio Hara 1, Shinichiro Nagatomo 1, Seiya Ogata 1, and Seinosuke Ueda 2 1 Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan 2 Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda, Kumamoto 860, Japan Received 5 July 1991/Accepted 5 December 1991

Summary. The y-glutamyltranspeptidase (y, GTP) gene o f Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate production, has been cloned and the nucleotide sequence determined. The sequence contains a single open-reading frame stretching for 1260 bp with a relative molecular mass of 49356. Putative - 3 5 and - 1 0 sequences, T T C A A A and TATTAT, were observed as the consensus sequence for the promoter recognized by the or43 R N A polymerase of B. subtilis, and the ribosome binding site, the sequence of which was AACGAG, was complementary to the binding sequence of B. subtilis 16S r R N A except for one base. The amino acid sequence of the gene with the segment o f putative protein C403 of staphylococcal plasmid pE194 indicates homology, whereas that with Escherichia coli and mammalian 7/GTPs does not show any similarity at all.

in B. subtilis (natto) strains isolated from a non-salty fermented soybean food, natto ( H a r a et aI. 1983; unpublished results). y-Glutamyltranspeptidase (EC 2.3.2.2) catalyses the transfer o f the y-glutamyl residue from y-glutamyl compounds, such as glutathione, to amino acids and peptides, and the hydrolysis of y-glutamyl compounds (Tate and Meister 1981), but its physiological role remains controversial. The cDNAs of rat renal (Laperche et al. 1986) and human hepatic (Sakamuro et al. 1988) y-GTPs have been cloned, and the nucleotide sequences determined. Recently, Suzuki et al. (1989) reported the nucleotide sequence of Escherichia coli K-12 y-GTP. However, the percentage match o f the amino acid sequence between the E. coli and mammalian 7/. GTPs was low. In the present work, we have cloned and sequenced the 7/-GTP gene that is responsible for ?'-PGA production o f B. subtilis (natto).

Introduction

Materials and methods

"Natto" is a Japanese traditional fermented food, manufactured by growing Bacillus subtilis (natto) on steamed soybeans. It is an adhesive, and consists o f polysaccharide (levan-form fructan) and y-polyglutamate (y-PGA). The adhesive material is mainly composed of 2'-PGA containing D- and L-glutamate in varying proportions (Fujii 1963). A plausible mechanism of the biosynthetic pathway of 7/-PGA has been proposed by Thorne et al. (1955) for one of the y-PGA-producing strains (B. licheniformis ATCC 9945A). However, since it is known that various strains differ a great deal in the basic requirements for y : P G A production as a capsule, it is to be assumed that there is more than one biosynthetic pathway. We reported that a 5.7-kb plasmid designated pUH1, which encodes the y-glutamyltranspeptidase (y-GTP) gene responsible for y-PGA production, is distributed widely

Bacterial strains and plasmids. Eo coli JM101 (supE44, thi, A(lacproAB)[P, proAB, laclqZAM15, traD36], mcrA +) and B. subtilis MIll2 (arg-15, leuB8, thr-5, recE4) were used as a cloning host. Plasmid pUH1 has been previously described (Hara et al. 1983). Plasmid pUB110 was used as a vector for B. subtilis, and plasmids pUC18 and pUC19 were for E. coil

Correspondence to: T. Hara

Media. LB broth and Panassary broth (Difco, Detroit, Mich., USA) for both B. subtilis and E. coli, Spizizen minimal medium for B. subtilis, and M9 minimal medium for E. coli were the same as described previously (Hara et al. 1991). The cells carrying Kanamycin-resistant (Kmr) plasmids were grown in AA medium (Tanaka and Sakaguchi 1978) containing kanamycin (50 Ixg/ml). DNA manipulations. The plasmid pUH1 from B. subtilis (natto) strain Asahikawa and its derived plasmids from Km r transform~ints were prepared and purified as previously described (Hara et al. 1983). The pUH1 was digested with BstEII, and then the ends were filled in with Klenow fragment to generate blunt ends. The DNA fragments were ligated with BamHI linker, inserted at the BamHI site of pUB110 with T4 ligase and then added to B. subtilis MIll2 protoplasts. Restriction enzymes, T4 DNA ligase and bacterial alkaline phosphatase were purchased from Takara

212 BS

Shuzo (Kyoto, Japan), and used as recommendedby the manufacturer. Degradation of DNA with exonuclease Bal31 (Takara Shuzu) was followed by the procedure of Legerski et al. (1978). Methods for transformation. E. coli JM101 was transformed by the method of Morrison (1977), and B. subtilis MIll2 was transformed by using protoplasted cells (Chang and Cohen 1979). Assay of 7/-GTP activity. The y-GTP activity was assayed as previously described (Aumayr et al. 1981). DNA sequencing. DNA fragments were subcloned into plasmids pUC18 and pUC19, and then, if needed, the Bal31-deletedderivatives were obtained by the stepwise deletion method of McCutchan et al. (1986). DNA sequencing was carried out by means of the dideoxy chain termination method (Sanger et al. 1977) with Sequenase Version 2.0 DNA Sequencing Kit (United States Biochemical Corporation, Cleveland, Ohio, USA). Nucleotide and amino acid sequences were analysed by the Hitachi (Tokyo, Japan) DNASIS system. Chemicals. Restriction enzymes, T4 DNA ligase and bacterial alkaline phosphatase were purchased from Takara Shuzo, and used as recommended by the manufacturer. Degradation of DNA with exonuclease Bal31 (Takara Shuzo) followed the procedure of Legerski et al. (1978).

Results and discussion As reported previously (Hara et al. 1981), the y-GTP gene is encoded on an endogenous plasmid, pUH1, in B. subtilis (natto). We constructed a set of derived p!asmids using p U B l l 0 as a vector, in which a series of pUH1 fragments were inserted. Figure 1 shows the subclones of B. subtilis (natto) plasmid pUH1 and their expression of y-GTP in B. subtilis MIll2. The y-GTP activity was detected in several Km r transformants that carried plasmid pPB1 (column BsL in Fig. 1) with a size of 8.2 kb, including the 3.7-kb BstEII fragment of pUH1. To define the boundaries of a functional unit of inserted DNA, a 3.7-kb BstEII fragment of pUH1 was digested with selected restriction endonucleases to obtain a set of overlapping DNA fragments. The plasmid DNA preparations containing each generated fragment were tested for the enzyme activity in B. subtilis. The deletion experiments revealed that a 1.7-kb TaqI fragment (column T in Fig. 1) is necessary for the expression of the y-GPT gene in B. subtilis M I l l 2 . The nucleotide sequences of both strands of the 1.7kb TaqI fragment were determined by the dideoxynucleotide-chain-termination method (Sanger et al. 1977) after successive Bal31 exonuclease deletions (Davis et al. 1986). Although the strategy is not shown, the nucleotide sequence was determined for both strands by using numerous restriction fragments to give enough overlapping regions. Figure 2 shows the nucleotide sequence of the 1.7-kb TaqI fragment. By examination of possible open readings frames (ORFs), we found only one large frame, which consists of 1260 bp and encodes a protein molecule with 420 amino acids and has a relative molecular mass of 49356. The y-GTP ORF was found to be preceded by a putative o-43 RNA polymerase promoter. The 5' upstream region of the y-GTP

0

H

1

Bs

Ba

2

T

3

Re H T H

4

5 kb

pUH1

H Bs

~'-GTP "I-

BsS . BsL BaL

I

"t"

I

BaS HL T

II

I

"t"

Fig. 1. Subclones of Bacillus subtilis (natto) plasmid pUH1 and their expression of 7~-glutamyltranspeptidase(y-GTP)in B. subtilis MIll2. The open arrow in the BstEII fragment indicates an openreading frame found in the sequence data (see Fig. 2). The expression of y-GTP was assayed as previouslydescribed (Aumayret al. 1981). Abbreviations for restriction sites are: Ba, BamHI; Bs, BstEII; H, HindlII; T, TaqI

gene contains a 5'-AACGAG-3' sequence (indicated as SD in Fig. 3) complementary to the 3' end of the 16S rRNA (3'-OH-UCUUUCCUCCAGUAG-5') of B. subtilis (McLaughlin et al. 1981) at nucleotides 295-300 for translation initiation. There is a 5'-TATrAT-3' sequence ( - 10 in Fig. 2) resembling a Pribnow box (Moran et al. 1982) at nucleotides 257 to 262, and at a site 23 bp upstream of this - 1 0 sequence, there is a 5'TTCAAA-3'-sequence resembling the - 3 5 sequence (Moran et al. 1982) of the B. subtilis gene. The observed distance (17 bp) between the - 1 0 and - 3 5 sequences accords well with that observed generally in B. subtilis genes (17-18 bp) (Moran et al. 1982). The amino acid sequence of the predicted protein 7/GTP encoded on pUH1 was compared with a number of protein sequences registered in GenBank with use of the homology search system GENAS (Kuhara et al. 1984). As shown in Fig. 3, the sequence homologous to the pUH1 y-GTP were found also in putative protein C403 (420 residues; Horinouchi and Weisblum 1982) with 48 300 daltons encoded in staphylococcal plasmid pE194. Approximately 51.2% amino acid homologies with pUH1 y-GTP was observed in a segment of 86 residues of C403, but the percentage match of both proteins overall was quite a low value, 11.9%. The cDNAs of rat renal (Laperche et al. 1986), human hepatic (Sakamuro et al. 1988), hepatoma (Goodspeed et al. 1989), and placental (Meyts et al. 1988) y-GTPs were cloned, and the nucleotide sequences were determined. The mammalian y-GTPs, the amino acid sequences of which were essentially the same, are synthesized as single polypeptides and then processed into large and small subunits (Matsuda and Katsunuma 1984), but did not show any similarity at all with the pUH1 y-GTP of B. subtilis (natto). The percentage match of the amino acid sequence of pUH1 y-GTP for human hepatic 7/GTP was 8.4%. Recently, Suzuki et al. (1989) performed DNA sequencing of E. coli y-GTP, and they suggested that the E. coli y-GTP might be also processed posttranslationally. The homology of amino acid sequences between the E. coli and pUH1 y-GTPs is relatively low,

213 Taql 50 TCGATGCGACAGCGAGAATGAGAGAC GCACCAGCACCGCAGGTCGCACGTCCAAATTT

i00 GC~TGGCATAATTTGGTGTAGTGCGTTACAC2AAAGATAAACTTTGTGTTACCATAACCCCTATACAGTGGTCTGAATC~

5O 2OO CTCA~TTATGCAGTCATCAGGATGGAAAAATACAAAAAAGATAGATTGAAT

(-35)

.



250

1

G G A A ~ C A C A A T C A ~ ~

(-i0)

(SD)

~AAAGCAAAAATGAAAAGTATTAT~GCGGACGACTTAAATTATGATCTAGT

~CCGATTAGCTATTCAAAAGCGATTC

350 400 ATGAAAAAATTGAGGGGC2~GTCAAACGGAAGGTCCGAGCGGATGCATGTTTTGGT CAGCGAATTTTTGATCA~GTCCTGACTAT Met Lys Ly sLeuArgGlyGluSe rAsnGlyArgSe rGluArgMetHi sValLeuValSerGluPheLeu I leThrAlaSerProAspTyr 450 500 ATGAATGC~?4~TGAG~GATGAGGA~GCTATTTTGAAACAGCGGTTGATCATTTGAAAGAGAAATACAGCGCTGAAAACATGCTT MetAsnGlyLeuSe rAspGluGl uGlnArgArgTyrPheGl uTh rAl aValAs phi sLeuLy sGlu Ly sTyrSe rAlaGl uAs nMet Leu 550 6 TATGCTACAGTCCATATGGATGAAGCGACT CCTCATATGCATGTT GGTATTGTACCGATCACAGAGGACGGCCGACTCTCTGCGAAAC~T TyrAlaThrValHisMetAspGluAlaThrP roHisMetHi sVa iGly I i eVa IP ro I i eThrGluAspGlyArgLeuSerAlaLysAsp 00 650 TFFI-I~fAATGGCAAATTGAAGATGAAAGCCATT CAAGAT GATTT TCAT ~ A C A T G G T T G A A A A C G G T T T T G A C C T G G T ~ G G C G A A PhePheAs nGlyLy sLeuLy sMet LysAl a I leGlnAspAspPheHi sArgHi sMetVa IG luAsnGlyPheAspLeuVa iArgGlyGlu 70O 75O CCAAGCC/%AAAGAAGCATGAGAATGTTCACCAGTATAAAATAAATCAGCGC~AACCGGAGCTT GAGC~AATGCTGAAATT GCTTTA ProSerGluLysLysHi sGluAsnValHisGlnTyrLys I leAsnGlnArgGluP roGluLeuGluArgLeuAsnAlaGlu I leAlaLeu 800 850 AAGGAAAAGCAGAGAGAGGAACTGGAAAAC~AAAACAAAGCT GTTCAACqlAGTTATAGAAGTGAAAAAAGAATCGCT GACACgDTAAGGCT Ly sGluLy s GlnArgGluGluLeuGluLys GlnAsnLysAlaValGl nAlaVa i I i eGluVa iLys Ly sGluSerLeuThrAlaLy sAl a ". 950 900 GAAGAGTTGAAAATGCCGACTATTGAACATGAAAAAGOGTGGCT CAAAAAGGATAAAGTCATTGTGCCAGAGCGGGAACTCCAT ~ G GluGluLeuLysMetProThrI leGluHi sGluLysAlaTrpLeuLysLy sAspLysVal I leValP roGluArgGluLeuHi sAlaLeu • o . . . I000 . . . i0 TATGCCTATGCGGAGCAGAAAACTAAAACGGCAGCCGAGTTGGC~TTGAAGTCGGAAACGCAGGAAAAGC~~GTCT TyrAlaTyrAlaGluGlnLysThrLysThrAlaAlaGluLeuAlaGlyGlnLeuLy s Se rGluThrGlnGluLysGluArgTrpGlnSer . • . . . . .,. 50 ii00 ATCGCCCGCg?AGAAGCAGATCGGGCGGATGAAAAAGACCAACGGCr TCAGGAACTGCAGAGTAGGATCCATTCAGAAGTTGAAGCGTCCA I leAlaArgGlnLysGlnI leGlyArgMet LysLysThrAsnGlyPheArgAsnCysArgValGlySer I leGlnLysLeuLysArgPro 1150 1200 AAAA ~ G G A A A ~ G C T T G C A A A G G A A T T T A C G G A A G A A C A A G C G T C A G G A T C T T C G G C A G G A A G T G A A A G A G G A A C T G A C G A C T LysArgLy sCysGlyAl a SerLeuGlnArgAsnLeuArgLysAsn Ly sArgGl nAs pLeuArgGlnGluVal LysGluGluLeuThrThr 1250 1300 TTACGAACGGAAAACGAGGAACTGTCAGCT G A A A A T A A A G T T T T G A T C A T T C A A A G A A A T A G C G A A G C T ~ G A G C C T A A A A C T A A A A LeuArgThrGluAsnGluGluLeuSerAlaGluAsnLysVa 1 Leu I le I leGlnArgAsnSerGluAlaAlaGluSerLeuLysLeuLys 1350 1400 CAGGAACTTC~TAAGAGAAA~GTATGCf GAGGTTTTGAGTTTCGCCAAGAAGCAC~ATCAAACGCTTGAAAAAGTGGCTGGAGAA GlnGluLeuAspLysArgAsnGlyGlnTyrAlaGluVal Leu Se rPheAl aLy sLys GlnAsnGlnThrLeuGluLysVa iAlaGlyGlu 1450 15 ~C.~GGCGTT~k~GAA~T~kC4~CACT~kAAC_~kGAGAGTTGCC GTACT G C 4 ~ k C ~ T ~ ~ r ~ G ~ ~ AsnLysAla~euLysLys~luAsnLysThrLeuLysGluArgVa iAlaVa iLeuGluGlnTrpLysAspLysMetValGlnTrpAlaLys • ° . . . . 1550 . . 00 GAAAAATTACCAAAGATGCGGAKATTAC42GGCATCGTTTTTCGTACGC4Yf G G A A T G C C T A G A G A A G C C A A T ~ T A ~ ~ T ~ T GluLysLeuProLysMetArgLys LeuAlaAlaSe rPhePheVa iArg.LeuGluCys LeuGluLysP ro I leAsnThrArgThrMetAsn 1600 1650 TAGAGCC/YfGAAAA~TCAATTGCCCTTCCAAAATTT CCACCACTTTTTTTGTTTGGTGGCTGCGATCATTTTTTGTGT~G ***

. . . . . . . . .

>

< . . . . . . . .

1700 .TaqI GGACTCACGTAATGCAGCGGTGAGTGTCT CGTGCCTTT CTTGCTGTCT TTTrT CGA

Fig. 2. Nucleotide sequence of z-GTP gene of B. subtilis (natto) plasmid pUH1 and the deduced amino acid sequence of the enzyme. The putative -35, - 1 0 and Shine-Dalgarno (SD) sequences are underlined an the possible terminator sequences are

shown by arrows. Nucleotides are numbered from the 5' end of TaqI site of the 1.7-kb DNA strand with the same polarity as mRNA. Amino acid sequences are shown below the coding frame

and the percentage match was only 7.8%. Furthermore, the percentage match of the amino acid sequence among mammalian and E. coli y-GTPs was not so high (approx. 30%). Although mammalian 7-GTPs are connected with the metabolism of glutathione-related compounds, the organisms cannot produce 7-PGA, like B. subtilis (natto) strains. It is, therefore, to be assumed

that there is more than one enzyme that catalyses the transfer of the y-glutamyl residue from z-glutamyl compounds, and that one of them plays a important role in 7-PGA production in B. subtilis (natto).

214 i0 20 30 40 50 MSHSILRVARVKGSSNTNGIQRHNQRENKNYNNKDINHEETYKNYDLINA

S. aureus pE194 C403 B. subtilis (natto) pUHI E. coli K-12 Human hepatic 60

MIKPTFLRRVAIAALLSGSCFSAAAAPPAPPVSYGVEEDVFHPVRAKQ VKKKLWLGLLAVVLVLVIVGLCLWLPSASKEPDNHVYTR 80

70

90

i00

ll0

120

13

QNIKYKDKIDETIDENYSGKRKIRSD-AIRHVDGLVTSDKDFFDDLSGEEIERFFKDSLEFLENEYGKENMY-LAT--MKKLRGESNGRSERMHVLVSEFLITASPDYMNGLSDEEQRRYFETAVDHLKEKYSAEMMY-LAT--GMVASVDATATQVGVDILKEGGNAVDAAVAVGYALAVTHPQAGNLGGGGFMLI-RSKNGNTTAIDFR-EMAPAKATRDM AAVAADAKQCSKIGRDALRDDDSAVDAAIAALLCVGLMNAHSMGTGGGLFLTIYNSTTRKAEVINAR-EVAPRLAFATM 0 140 150 160 170 180 190 200 ....... V H L D E R V P H M H F G F V P L T E D G R L S A K E Q L G N K K D F T Q L Q D R F N - - E Y V N E K G Y E L E R G T S K E V T - E R E H K A M ....... V H M D E A T P H M H V G I V P I T E D G R L S A K D F F N G K L K M K A I Q D D F H - - R H M V E N G F D L V R G E P S E K K H E N V H Q Y K FLDDQGNPDSKKSLTSHLASGTPGTVAGFSLALDKYGTMPLNKWQPAFKLARDGFIVNDALADDLKTYGSEVLPNHEN F-NS-SE-QSQKG--G-LSVAVPGEIRGYELAHQRHGRLPWARLFQPSIQLARQGFPVGKGLAAALEN-KRTVIEQQPV

2

10 220 230 240 250 260 270 280 DQYKKDTVFHKQELQEVKDELQKANKQLQSGIEHMRSTKPFDYENERTGLFSGREETGRKILTADEFERLQETISSAER -INQREPELERLNAEIALKEKQREELEKQNKAVQAVIEVKKESLTAKAEELKMPTIEHEKAWLKKDKVIVPERELHALY SKAIFWKEGEPLKKGDTLVQANLAKSLEMIAENGPDEFYKGTIAEQIAQEMQKNGGLITKEDLAAyKA-VERTPISGDY LCEVFCRDRKVLREGERLTLPQLADTYETLAIEGAQAFYNGSLTAQIVKDIQAAGGIVTAEDLNNYRAELIEHPLNISL 290 300 310 320 330 340 350 360 IVDDYENIKSTDYYTENQELKKRRESLKEVVNTWKEGYHEKSKEVNKLKRENDSLNEQLNVSEKFQASTVTLYRAARAN AYAEQKTKTAAELAGQ-LKSETQE-KERWQSIARQEADR-ADEKDQRLQELQSRIHSEVEASKKEMRRKLAKEFD---T GYQVYSMPPPSSGGIHIVQILNILENFDMKKYGFGSAD--AM--QIMAEAEKY~YADRSEYLGDPDFVKVP-W-~QALT GDAVLY-MPSAPLSGPVLALILNILKGYNFSRESVESPEQKGLTYHRIVEAFRFAYAKRT-LLGDPKFVDVTEVVRNMT 370 380 390 400 410 420 430 440 FPGFEKGFNRLKEKFFNDSKFERVGQFMDVVQDNVQKVDRKREKQRTDDLEM ---EEQDLRQEVKEELTTLRTENE-ELSAENKVLIIQRNSEAAESLKLKQELDKRNGQYAE-VLSFAKKQNQTLEKVAG NKAYAKSIADQIDINKAKPSSEIRPGKLAPYESNQTTHYSVVDKDGNAVAVTYTLNITFGTGIVAGESGILLNNQMDDF SEFFAAQLRAQISDDTTHPISYYKPEFYTP-DDGGTAHLSVVAEDGSAVASTSTINLYFGSKVRSPVSGILFNNEMDDF 450

460

470

480

490

500

510

520

ENKALKK-ENQRKTLKERVAVL-EQWKDKHVQWA---KEKLPKMRKLAASFFVRLEC--LEKPINTRTMN SAKPGVPNVYGLVGGDANAVGPNKRPLSSMSPTIW-KDGKTWLVTGSPGGSRIITTVLQMWNSIDYGLNVAEATNAP SS-•SITNEFGV•P•PANFIQPGKQPLSSMCPT•M•GQDGQ•Rb•VVGAAGGTQ•TTATALAI•YNLWFGYDVKRAVEEP 530

540

550

560

570

580

590

RFHHQWLFDELRVEKGFSPDTLKLLEAKGQKVALKEA-MGSTQSIMVGPDGELYGASDPRSVDDLTAGY RLHNQLLFNVTTVERNIDQAVTAALETRHHHTQIASTFIAVVQAIVRTAGG-WAAASDSRKGGE-PAG¥

Fig. 3. Comparison of the amino acid sequences of the pUH1 7,GTP, the putative protein C403 of pE194 and the Escherichia coli and human hepatic z-GTPs. The putative C403 encoded on staphylococcal plasmid pE194 is from Horinouehi and Weisblum (1982). The E. coli y-GTP is from Suzuki et al. (1989). Gaps have been inserted to gain maximum matching. The one-letter amino

acid code has been used. The human hepatic y-GTP sequence is from Sakamuro et al. (1988). The percentage match of the amino acid sequence of pUH1 y-GTP for C403 on plasmid pE194 was 11.9%, and for E. coli and human hepatic y-GTPs were 7.8% and 8,4%, respectively

References

Hara T, Nagatomo S, Ogata S, Ueda S (1991) Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1. Appl Environ Microbiol 57:1838-1841 Horinouehi S, Weisblum B (1982) Nueleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J Bacteriol 150:804-814 Kuhara S, Matsuo F, Futamura S, Fujita A, Shinohara T, Takagi T, Sakaki Y (1984) GENAS: a database system for nucleic acid sequence analysis. Nucleic Acids Res 12:89-99 Laperehe Y, Bulle F, Aissani T, Chobert MN, Aggerbeck M, Hanoune J, Guellaen G (1986) Molecular cloning of rat kidney gamma-glutamyl transpeptidase eDNA. Proc Natl Acad Sci USA 83:937-941 Legerski RJ, Hodnett JL, Gray HB Jr (1978) Extracellular nucleases of Pseudomonas Ba131. III. Use of the double-strand deoxyribonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes. Nucleic Acids Res 5:14451464

Aumayr A, Hara T, Ueda S (1981) Transformation of Bacillus subtilis in polyglutamate production by deoxyribonucleic acid from B. natto. J Gen Appl Microbiol 27:115-123 Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168:111-115 Fujii H (1963) On the formation of mucilage by Bacillus natto. Part III. Chemical constituents of mucilage in natto. Nippon Nogeikagaku Kaishi 37:407-411 Goodspeed DC, Dunn TJ, Miller CD, Pilot HC (1989) Human y-glutamyl transpeptidase cDNA: comparison of hepatoma and kidney mRNA in the human and rat. Gene 76:1-9 Hara T, Aumayr, Ueda S (1981) Characterization of plasmid deoxyribonucleic acid in Bacillus natto: evidence for plasmidlinked PGA production. J Gen Appl Microbiol 29:299-305 Hara T, Zhang JR, Ueda S (1983) Identification of plasmids linked with polyglutamate production in Bacillus subtilis (natto). J Gen Appl Microbiol 29:345-354

215 Matsuda Y, Katsunuma N (1984) Biosynthesis and processing of .... y-glutamyl transpeptidase. Seikagaku 56:1389-1403 McCutchan TF, Hansen JL, Dame JB, Mullins JA (1984) Mung bean nucleases Plasmodium genomic DNA at sites before and after genes. Science 225:626-628 McLaughlin JR, Murray CL, Rabinowitz JC (1981) Unique features in the ribosome binding site sequence of the Gram-positive Staphylococcus aureus fl-lactamase gene. J Biol Chem 256:11283-11291 Meyts ERD, Heisterkamp N, Groffen J (1988) Cloning and nucleotide sequence of human y-glutamyl transpeptidase. Proc Natl Acad Sci USA 85:8840-8844 Moran CP Jr, Lang N, LeGrice SFJ, Lee G, Stephans M, Sonenshein AL, Pero J, Losick R (1982) Nucleotide sequence that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet 202:169-171 Morrison DA (1977) Transformation in Escherichia coli: cryogenic preservation of competent ceils. J Bacteriol 132:349-351 Sakamuro D, Yamazoe M, Matsuda Y, Kanagawa K, Taniguehi

N, Matsuo H, Yoshikawa H, Ogasawara N (1988) The primary . structure o f human gamma-glutamyl transpeptidase. Gene 73:1-9 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 Suzuki H, Kumagai H, Echigo T, Tochikura T (1989) DNA sequence of the Escherichia coli K-12 y-glutamyltranspeptidase gene, ggt. J Bacteriol 171:5169-5172 Tanaka T, Sakaguchi K (1978) Construction of a recombinant of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis. Mol Gen Genet 165:269276 Tate SS, Meister A (1981) y-Glutamyl transpeptidase: catalytic structural and functional aspects. Mol Cell Biochem 39:357368 Thorne CB, Gomez CG, Housewright RD (1955) Further studies on the biosynthesis of y-glutamyl peptides by transfer reactions. J Biol Chem 212:427-438

The DNA sequence of gamma-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1.

The gamma-glutamyltranspeptidase (gamma-GTP) gene of Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate product...
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