Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis Nobuyuki Sumitomo, Katsuya Ozaki, Shuji Kawai & Susumu Ito To cite this article: Nobuyuki Sumitomo, Katsuya Ozaki, Shuji Kawai & Susumu Ito (1992) Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis, Bioscience, Biotechnology, and Biochemistry, 56:6, 872-877, DOI: 10.1271/bbb.56.872 To link to this article: http://dx.doi.org/10.1271/bbb.56.872
Published online: 12 Jun 2014.
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Biosci. Biotech. Biochem., 56 (6), 872-877, 1992
Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus suhtilis Nobuyuki SUMITOMO, Katsuya OZAKI, Shuji KAWAI, and Susumu ITot Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-34, Japan Received October 21, 1991
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The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HBIOI. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-l and ORF-2. The protein deduced from ORF-l was composed of 244 amino acids with an Mr of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an Mr of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGT AAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.
Numerous strains of the genus Bacillus produce extracellular endo-l,4-f3-glucanases (EG; Ee 3.2.l.4).1,2) The EGs of Bacillus have been the focus of some attention because of their potential use in the bioconversion of agricultural wastes into useful products 3) and in the brewing industry.4) For the latter purpose, an EG gene from Bacillus has been cloned and expressed in Saccharomyces cerevisiae. 5 ) Recently, we have isolated some alkalophilic and neutrophilic strains of Bacillus that produce alkaline EGs, the properties of which fulfill the essential requirements for enzymes to be used in laundry detergents. 6 - 8 ) We have purified and characterized some of these alkaline EGS. 9 ,lO) A mutant of one of our isolates, Bacillus sp. KSM-635,7,lO) is currently exploited for the large-scale industrial production of an alkaline EG for use in laundry detergents. ll ,12) To clarify the mechanism of action of EGs, with a special focus on differences in pH optima, we cloned and sequenced the gene for alkaline EG from Bacillus sp. KSM-635. The deduced amino acid sequence of the alkaline EG protein had significant homology to the amino acid sequences of other alkaline and neutral EGs produced by various strains of Bacillus, with some amino acid residues conserved in either the alkaline enzymes or the neutral enzymes, or in both types of enzyme. 13) Since the major factor limiting the inclusion of alkaline EG in detergents is the cost of the enzyme, cloning the gene for a suitable EG may also make possible a more detailed study of some aspects of enhanced production of the enzyme. In this paper, we describe the nucleotide sequence of the gene for an alkaline EG, suitable for use in laundry detergents, from Bacillus sp. KSM-64. 8 ) We also describe the construction of a recombinant plasmid from a shuttle vector and the alkaline EG-coding fragment and demonstrate that the EG gene in the plasmid can be expressed at high levels both in Escherichia coli and in
Bacillus subtilis.
Materials and Methods Bacterial strains, plasmids, and bacteriophages. Bacillus sp. KSM-64 and Bacillus sp. KSM-64C were used as sources of the EG gene. Bacillus sp. KSM-64C was derived from Bacillus sp. KSM-64 as a mutant that was insensitive to catabolite repression (S. Ito and K. Saeki, unpublished data). Escherichia coli HB 10 1 (F - hsdS20 recA 13 ara-14 proA2 lac Y 1 galK2 rpsL20 xyl-S mtl-l supE44 leuB6 thi-I), E .. coli JMI09 (recAl LJ(lac-pro) endAl gyrA96 thi-l hsdR17 supE44 relAl F'traD36 proAB lacJQZ LJMlS), and Bacillus subtilis ISW1214 (leuA8 metES hsdMl) were used as hosts for cloning and nucleotide sequencing. Plasmids pBR322 and pHY300PLK, and bacteriophages Ml3mp18 and M13mpl9 were used as the vectors. Culture media. E. coli strains carrying plasmids were grown on LB broth. 2 x YT broth was used for cultivation of E. coli JM 109 infected with M 13 bacteriophage. 14) B. subtilis ISWl2l4 was grown on PY broth that contained (w/v %) 2.0% polypepton (Nihon Pharmaceutical), 0.1 % Bacto yeast extract (Difco), 0.1 % KH 2 P0 4 , and O.S% NaC!. PYM medium that contained (w /v %) O.S% polypepton, 0.1 % Bacto yeast extract, 0.1 % KH 2 P0 4 , 0.02% MgS0 4 ' 7H 2 0, and 1.0% Na 2 C0 3 was used for the propagation of Bacillus sp. KSM-64 and Bacillus sp. KSM-64C. Solidified media contained 1.S% (w/v) Bacto agar (Difco). DM-3 agar plates, composed of O.S M disodium succinate (pH 7.0), O.S% casamino acid (Difco), O.S% yeast extract, 0.35% KH 2 P0 4 , O.lS% K 2 HP0 4 , O.S% glucose, 20mM MgCI 2 , 0.01 % bovine serum albumin (Sigma), and 0.8% Bacto agar, were used for the regeneration of protoplasts of B. subtilis ISWI214. 15 ) Preparation of DNA. The test bacilli were grown in PYM medium at 30°C overnight, with shaking, and pelleted by centrifugation (12,000 x g 20 min). Genomic DNA was prepared as described by Saito and Miura. 16 ) Plasmid DNA and the replicative form of M 13 bacteriophage DNA were isolated by the alkaline extraction procedure described by Birnboim and Doly. 1 7) Covalently closed circular DNA was purified by CsCl/ethidium bromide equilibrium density gradient centrifugation. Cloning of the EG gene. Genomic DNA from Bacillus and plasmid pBR322 were both digested with HindIII (Boehringer Mannheim) and ligated with T 4 DNA ligase (Boehringer Mannheim). The ligation mixture was used for transformation of competent E. coli HBI01 cells (purchased from Takara Shuzo). LB plates supplemented with SO Jlg/mi ampicillin
t To whom all correspondence should be addressed. The nucleotide sequence data reported in this paper have been submitted to the GenBank/EMBL Data Bank with the accession number M84963.
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Alkaline Endoglucanase Gene from Bacillus (sodium salt; Sigma), on which the transformants appeared, were overlaid with 1.0% (w/v) agar that contained O.S% (w/v) carboxymethy1cellulose (CMC; degree of substitution 0.68; Sanyo Kokusaku Pulp), 1.0% (w/v) NaCl, 0.1 mg/mllysozyme and SO mM glycine/NaOH buffer (pH 9.0). After incubation at 37°C for 6 hr, the activity of the EG produced by the transformants was detected by staining the overlaid CMC with Congo red dye, essentially as described by Teather and Wood. 18 ) After screening for ampicillin-resistant and EG-positive transformants of E. coli HB101, the plasmid, named pCL64, was obtained. B. subtilis ISW1214 was transformed by plasmid pHY300PLK or its derivatives by the protoplast method of Chang and Cohen. 1S ) Transformed cells were selected on DM-3 agar plates that contained tetracycline at lS/lg/ml (Sigma).
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Hybridization analysis of DNA digests. HindIII digests of the genomic DNA (S/lg) of Bacillus sp. KSM-64 were electrophoresed on a 0.8% (w/v) agarose gel and then fragments were electrophoretically transferred to a nylon membrane (Zeta-Probe Blotting Membrane, Bio-Rad) after denaturation by soaking the gel in 0.2 M NaOH/O.S M NaCI for 30 min at room temperature. Approximately 3/lg of the HindIII insert, isolated from pCL64, was labelled with digoxigenin-ll-dUTP and used as a probe for hybridization analysis of the HindIII digests of the genomic DNA on the membrane, by use of a DNA-Labelling and Detection Kit (Boehringer Mannheim). Nucleotide sequencing. The nucleotides of the 4.1-kb XhoI-HindIII region of pCL64 (see Fig. I) were sequenced by the dideoxy method of Sanger et al.,19) using an M13 Sequencing Kit (Takara Shuzo) and [1X- 32 PJdCTP (Amersham). Parts of the nucleotide sequence were identified by the dideoxy method using Sequenase (modified T 7 DNA polymerase; United States Biochemical) in place of the Klenow enzyme, or by the dideoxy method using a fluorescent dye primer 20 ) and Sequenase, on an automated DNA sequencer (Applied Biosystems). Template DNAs were prepared using M13mp18 or M13mp19 phage vector, as described by Messing. 14 ) Ordered deletion clones of M13 as the template DNA for sequencing were prepared with a Kilo-sequencing Deletion Kit (Takara Shuzo). Nucleotide sequence data were analyzed on the basis of data from the GENETYX Gene Information Treatment System (Software Development).
Bacillus sp. KSM -64 that had been blotted on a membrane. Thus, the 4.4-kb insert of pCL64 appeared to have come from the genomic DNA of Bacillus sp. KSM-64. Nucleotide sequence analysis The nucleotides of the 4.I-kb XhoI-HindIII fragment from pCL64 were sequenced. The 4126-bp nucleotide sequence is shown in Fig. 4. Two open reading frames (ORFs), designated ORF-I and ORF-2, encoding proteins of 244 and 822 amino acids (with Mr of 27,865 and 91,040, respectively) were identified in the sequence. ORF-2 begins at nucleotide I (AT G) and ends at nucleotide 2467 (TAA), which encodes the alkaline EG. Upstream from the initiation codon of ORF-2, there is a putative Shine-Dalgarno (SD) sequence, SD-2, which is complementary to a high degree to the 3' end of 16S rRNA from B. subtilis. 2 1) SD-2 starts at nucleotide -10 (AGGAGGT) and would have a free energy of binding (LtG) of -18.8 kcaljmol (- 78.7 kJ jmol), as calculated by the method of Tinoco et al.,22) if it bound to the 3' end of the 16S rRNA from B. subtilis. Upstream from this ORF, there are three sequences, P-I, P-2, and P-3, which are similar to the consensus sequence of the sigma A-type promoter of B. subtilis. 23) The P-I region starts at nucleotide -404 (TTTAAT) as the -35 region and at -381 (TATAAT) as the -10 region. The P-2 region starts at nucleotide -245 (GTAAGA) as the -35 region and at -219 (TATGAT) as
Sc Ec Hi Sp
Sc Kp
Assay of EG activity. E. coli cells carrying the recombinant plasmid were grown on LB broth supplemented with ampicillin (SO /lg/ml) at 37°C for 24 hr. Cells were harvested from a 3S-ml portion of the culture by centrifugation (9,800 x g, 10 min) at 4°C and were suspended in 7 m1 of 20mM NaH2P04/Na2HP04 buffer (pH 7.0). The cells in the suspension were disrupted by sonication at temperatures not exceeding 4°C. The cell debris was removed by centrifugation at 12,000 x 9 for IS min at 4°C and the resulting cell-free extract was used for assays of enzymatic activity. B. subtilis ISW1214 carrying the recombinant plasmid was grown at 30°C for 3 days on PY broth supplemented with lS/lg/ml tetracycline. Two ml of the culture were centrifuged at 12,000 x 9 for 10 min and the supernatant obtained was used as the solution of enzyme. EG activity was then assayed as described previously.7) One unit (U) of enzymatic activity was defined as the amount of enzyme that produced 1.0/lmol of reducing sugar as glucose per min under the conditions of the reaction.
Sc
Hp
pHY300PLK 4.9 kb
Hi Xh
1~_H_in_d_rn________~________~1 Hindrn
11 igation Sp
Results and Discussion Cloning of the EG gene The EG-expressing plasmid, pCL64, contained one 4.4-kb HindIII insert, and its restriction map is shown in Fig. I. E. coli HBIOI harboring pCL64 produced active intracellular EG, to a level that corresponded to approximately 1000 U jliter broth, when cells were grown at 37°C for 24 hr in LB broth containing ampicillin. The expressed EG was active from pH 5 to 13, with maximum activity at pH 9.5. The profile of the pH-activity curve of the expressed enzyme coincided with that of the alkaline EG produced by Bacillus sp. KSM-64, as shown in Figs. 2A and 2B. To clarify the origin of the 4.4-kb insert in pCL64, hybridization analysis was done using the digoxigeninlabelled 4.4-kb insert of pCL64 as a probe. Figure 3 shows that the labelled insert of pCL64 hybridized with a 4.4-kb fragment in HindUI digests of the genomic DNA from
Sc Kp
Hp
Ec Srn Hi Xh Fig. 1.
Construction of pHCL64 from pCL64 and pHY300PLK.
Thick and thin lines show the cloned genes (ORF-l and ORF-2) and the vectors (pBR322 and pHY300PLK), respectively. Arrows around circles indicate the positions and the orientations of the sequences that encode ORF-I and ORF-2 (the alkaline EG gene) from Bacillus sp. KSM-64, and the ampicillin (Ap)- and tetracycline (Tc)-resistance genes in the vector plasmids. Abbreviations for restriction sites are: Ec, EcoRI; Hi, HindIII; Hp, HpaI; Kp, Kpnl; Sc, Seal; Sp, Sphl; Sm, Smal; Xh, XhoI.
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pH Fig. 2. pH-Activity Profiles of the Alkaline Endoglucanases Produced by Bacillus sp. KSM-64 (A), by E. coli HB101 Carrying pCL64 (B), and by B. subtilis ISW1214 Carrying pHCL64 (C). McIlvaine buffer (e, pH 6.2-7.7), sodium phosphate buffer (., pH 6.6-7.6), glycine/NaOH buffer (0, pH 8.2-10.8), and Na zHP0 4 /NaOH buffer (... , 11.0-12.1)
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were used. Assays were done at 40°C with indicated buffers at 0.1 glycine/NaOH buffer was taken as 100% in each case.
A
M,
at various pH values, using 1.0% (w/v) CMC as substrate. The enzymatic activity at pH 9.5 in 0.1
B 1 234
1 234
kb --23.1-9.4-- - - 6.7--4.4--
---
-2.3-
M
of ORF-1, there is a putative SD sequence (AAAGGA), which starts at nucleotide -1269 (a L1G of - 11.8 kcal/mol (-49.4kJ/mol)). In addition, an IR is found upstream from the initiation codon of ORF-1 (IR-1, nucleotides -1320 to -1291) and two IRs downstream from the stop codon of the sequence (IR-2 and IR-3 at nucleotides -510 to -478 and -402 to -375, respectively). The L1G values for these sequences, IR-1, IR-2, and IR-3, for stem-loop structures, were calculated to be -23.8 kcal/mol (-99.6 kJ Imol), - 16.8 kcal/mol (- 70.3 kJ Imol), and - 11.2 kcall mol ( - 46.9 kJ Imo!), respectively.
----- 2. 0 ----
_____
Fig. 3.
O.56~
Hybridization Analysis of the HindIII Insert of pCL64.
A-DNA digested with HindIII (lane I) or EcoRI (lane 2), or DNA from Bacillus sp. KSM-64 digested with HindIII (lane 3) or EcoRI (lane 4) was electrophoresed on a 0.8% (w/v) agarose gel and transferred onto a Zeta-Probe Blotting Membrane. The DNA digests on the membrane were hybridized with the digoxigenin-ll-dUTPlabelled 4.4-kb HindIII fragment of pCL64. A, results of agarose gel electrophoresis of digests of KSM-64 DNA; B, autoradiograms of digests of KSM-64 DNA hybridized with labelled HindIII probe.
the -10 region. The P-3 region starts at nucleotide -123 (TTGAGA) as the -35 region and at -100 (GTTACT) as the -10 region. In addition, an inverted repeat sequence (IR), which resembles a prokaryotic p-independent transcription terminator,24) is 22 nucleotides downstream from the stop codon of ORF-2 (IR-4, nucleotides 2488 to 2526). The L1G value for IR-4 for a stem-loop structure was calculated to be - 18.4 kcal/mol (-77.0 kJ/mol). Therefore, a combination of these potential promoter (P-1, P-2, or P-3) and terminator (IR-4) sequences may be required for the expression of the alkaline EG gene. ORF-1 (nucleotides -1256 (ATG) to -524 (TAA)) encodes an unidentified protein. Upstream from the initiation codon
Homology of the amino acid sequences encoded by ORF-J and 0 RF-2 to those of other enzymes The amino acid sequence of the alkaline EG, deduced from ORF-2, from Bacillus sp. KSM-64, was compared with those of an acid EG,25) neutral EGs, 3,26,27) and alkaline EGs 13 ,28,29) that have been reported to date for the genus Bacillus. The sequence of amino acids of this enzyme has 99.6% homology to that of the alkaline enzyme from alkalophilic Bacillus sp. no. 1139. 28 ) There are only 12 differences in nucleotides and 4 differences in amino acids between the two genes for EGs. The gene from Bacillus sp. KSM-64 has a insertion of one nucleotide, at nucleotide 2399 of this gene, as compared to the gene from Bacillus sp. no. 1139. As a result of the single insertion at this position, the deduced amino acid sequence of this EG is 22 amino acids longer than that of the EG from Bacillus sp. no. 1139. These results indicate that the additional 22 amino acid residues are not necessary for the activity of the EG. The deduced amino acid sequence of the EG from Bacillus sp. KSM -64 is also similar to those of other alkaline (38-72% homology)13,29) and neutral EGs (approximately 31 % homology),3,26,27) but it is not homologous to that of the acid EG. 25 ) The amino acid sequence deduced from ORF-1 has 34% homology to that of pseudouridine synthase from E. coli. 30) Pseudouridine is found in the tRNA of B. subtilis,31) but there are no reports of cloning of the gene for pseudouridine synthase from the genus Bacillus. We are
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Alkaline Endoglucanase Gene from Bacillus X hoi
CTC - 1400
GAGTGGAGCAAGAGGCTTCTTAACTCGTTCATGAATGTAGAATAGAAACA ATGGAAAAGCTAAATTAAAGAAAAAACTTTGGGGTTATTGCAICAATCl§ IR-1 -1300 ¢ ORF-1 CGATAACCCCTTAAATGCTAACTACATAGATAAAGGAAGATAAAATGAAC AATTATAAACTAATGATTCAATATGATGGTGGTCGATACAAAGGTTGGCA 50-1 •••••• M N N Y K L M I Q Y 0 G G R Y K G WQ (I)
(10)
-1200
GCGTCTTGGGAACGGTGAAAATACGATTCAAGGTAAAATTGAAACGGTTT TATCAGAGATGGTAGGTAGAAAAATAGAGATTATAGGGTCTGGTAGAACA R L G N G E N T I Q G K lET V L 5 E M V G R K I E I I G S G R T (53)
- I I 00
GATGCTGGTGTCCATGCTCTTGGACAAGTGGCTAATGTAAAATTAAGCGA AAATTTTACAGTAAAAGAGGTTAAAGAGTATTTGAATCGTTATTTGCCTC D A G V HAL G Q V A N V K L SEN F T V K E V KEY L N R Y L P H (86)
ATGATATCAGTGTGACTGAGGTGACGCTAGTTCCAGATCGTTTTCACTCA AGGTATAACGCAAAGGACAAAACCTATCTTTATAAAATTTGGAATGAGGA DIS V T E V T L V P D R F H 5 R Y N A K D K T Y L Y K I W NED (119)
TTATACTCATCCGTTTATGCGTAAGTACAGCTTGCACATCGAAAAGAAAT TACATATTGATAACATGGTAAAAGCAAGTCAACTTTTCGTAGGAGAACAT Y T H P F M R K Y 5 L HIE K K L HID N M V K A 5 Q L F V G E H (153)
GATTTTACAGCTTTTTCTAATGCTAAATCTAAAAAGAAGACAAATACGAG AACGATTCACTCTATAACTATTCAAGATAATCAAGGATTTATAGACATTA OFT A F 5 N A K 5 KKK TNT R T I H 5 I T I Q 0 N Q G FlO I R (186)
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GGGTTTGTGGAGATGGTTTTCTTTATAACATGGTTAGAAAAATGGTAGGG ACTTTGATTGAGGTTGGTCTAGGTGAAAAGGAACCTGAACAAGTACTTAC V C G D G FLY N M V R K M V G T LIE V G L G EKE P E Q V L T (219)
CATTTTAGAGTCAAAAGATAGAAGCCAAGCAGGATTTGCCGATGCAACCG GCTTATATTTAGAGGGAATTTCTTTTTAAATTGAATACGGAATAAAATCA I L E S K D R 5 Q A G FAD A T G L Y LEG I 5 F * ~TAAACAGGT~CTGATTTTATTTTTTTGAATTTTTTTGAGAACTAAAGA
TTGAAATAGAAGTAGAAGACAACGGACATAAGAAAATTGTATTAGTTTTA
IR-2 P-l
NapV
ATTATAGAA~ACGCT~TTCTATAATTATTTATACCTAGAACGAAAATACT
GTTTCGAAAGCGGTTTACTATAAAACCTTATATTCCGGCTCTTTTTTTAA
IR-3 P-2 ACAGGGGGTGAAAATTCACTCTAGTATTCTAATTTCAACATGCTATAATA AATTTGTAAGACGCAATATACATCTTTTTTTTATGATATTTGTAAGCGGT 11 i n , I I (H
p •
P-3
I)
TAACCTTGTGCTATATGCCGATTTAGGAAGGGGGTAGATTGAGTCAAGTA GTCATAATTTAGATAACTTATAAGTTGTTGAGAAGCAGGAGAGAATCTGG GTTACTCACAAGTTTTTTAAAACATTATCGAAAGCACTTTCGGTTATGCT TATGAATTTAGCTATTTGATTCAATTACTTTAATAATTTTAGGAGGTAAT SO-2 ••••• .1. ¢ ORF-2 (alkal ine EG) ATGATGTTAAGAAAGAAAACAAAGCAGTTGATTTCTTCCATTCTTATTTT AGTTTTACTTCTATCTTTATTTCCGACAGCTCTTGCAGCAGAAGGAAACA M M L R K K T K Q LIS S I L I L V L L L S L F PTA L A A E G N T (34)
CTCGTGAAGACAATTTTAAACATTTATTAGGTAATGACAATGTTAAACGC CCTTCTGAGGCTGGCGCATTACAATTACAAGAAGTCGATGGACAAATGAC RED N F K H L L G NON V K R P SEA GAL Q L Q E V 0 G Q M T (67)
ATTAGTAGATCAACATGGAGAAAAAATTCAATTACGTGGAATGAGTACAC ACGGATTACAATGGTTTCCTGAGATCTTGAATGATAACGCATACAAAGCT L V D Q H G E K I Q L R G M 5 T H G L Q W F PEl L N DNA Y K A (100)
CTTGCTAACGATTGGGAATCAAATATGATTCGTCTAGCTATGTATGTCGG TGAAAATGGCTATGCTTCAAATCCAGAGTTAATTAAAAGCAGAGTCATTA LAN 0 W E 5 N M I R LAM Y V G ENG Y A 5 N PEL I K 5 R V I K (134)
B 1'1 I I
AAGGAATAGATCTTGCTATTGAAAATGACATGTATGTCATCGTTGATTGG CATGTACATGCACCTGGTGATCCTAGAGATCCCGTTTACGCTGGAGCAGA G I D L A lEN D M Y V I V D W H V HAP G D P R D P V Y A G A E (167)
AGATTTCTTTAGAGATATTGCAGCATTATATCCTAACAATCCACACATTA TTTATGAGTTAGCGAATGAGCCAAGTAGTAACAATAATGGTGGAGCTGGG D F F R D I A A L Y P N N PHI lYE LAN E P 5 5 N N N G GAG (200)
601
ATTCCAAATAATGAAGAAGGTTGGAATGCGGTAAAAGAATACGCTGATCC AATTGTAGAAATGTTACGTGATAGCGGGAACGCAGATGACAATATTATCA I P NNE E G W N A V KEY A 0 P I V E M L R D 5 G N ADD N I I I (2311
701
TTGTGGGTAGTCCAAACTGGAGTCAGCGTCCTGACTTAGCAGCTGATAAT CCAATTGATGATCACCATACAATGTATACTGTTCACTTCTACACTGGTTC V G S P N W 5 Q R _ POL A A D N P I DOH H T M Y T VHF Y T G 5 (267)
ACATGCTGCTTCAACTGAAAGCTATCCGCCTGAAACTCCTAACTCTGAAA GAGGAAACGTAATGAGTAACACTCGTTATGCGTTAGAAAACGGAGTAGCA H A A S T E 5 Y P PET P N S ERG N VMS N TRY ALE N G V A (300)
90 I
GTATTTGCAACAGAGTGGGGAACTAGCCAAGCAAATGGAGATGGTGGTCC TTACTTTGATGAAGCAGATGTATGGATTGAGTTTTTAAATGAAAACAACA V FAT E W G T 5 Q A N G D G G P Y F D E A 0 V W I E F L N E N N I Fig. 4.
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100~TAGCTGGGCTAACTGGTCTTTAACGAATAAAAATGAAGTATCTGGTGCA TTTACACCATTCGAGTTAGGTAAGTCTAACGCAACAAGTCTTGACCCAGG S WAN W S L T N K N E V S G AFT P F E L G K S NAT S LOP G (367) 1101 n 1 GCCAGACCAAGTATGGGTACCAGAAGAGTTAAGTCTTTCTGGAGAATATG TACGTGCTCGTATTAAAGGTGTGAACTATGAGCCAATCGACCGTACAAAA PDQ V W V PEE L S L S G E Y V R A R I K G V N YEP lOR T K 00) 120 1 I TACACGAAAGTACTTTGGGACTTTAATGATGGAACGAAGCAAGGATTTGG AGTGAATGGAGATTCTCCAGTTGAAGATGTAGTTATTGAGAATGAAGCGG Y T K V L W 0 F N 0 G T K Q G F G V N G 0 S P V E 0 V V lEN E A G K p
(4
SCI
(434l
I 30 l O r
I
I
GCGCTTTAAAACTTTCAGGATTAGATGCAAGTAATGATGTTTCTGAAGGT AATTACTGGGCTAATGCTCGTCTTTCTGCCGACGGTTGGGGAAAAAGTGT A L K L S G LOA S NOV S E G N Y WAN A R L SAD G W G K S V
(4 67)
I 4 0 I
TGATATTTTAGGTGCTGAAAAACTTACTATGGATGTGATTGTTGATGAGC CGACCACGGTATCAATTGCTGCAATTCCACAAGGGCCATCAGCCAATTGG OIL G A E K L T M 0 V I V 0 E P T T V S I A A I P Q G P SAN W(500)
I
50 I GTTAATCCAAATCGTGCAATTAAGGTTGAGCCAACTAATTTCGTACCGTT AGGAGATAAGTTTAAAGCGGAATTAACTATAACTTCAGCTGACTCTCCAT V N P N R A I K V E P T N F V P L G 0 K F K A E L TIT SAD S P S
(534)
I B0 I
S phi
CGTTAGAAGCTATTGCGATGCATGCTGAAAATAACAACATCAACAACATC ATTCTTTTTGTAGGAACTGAAGGTGCTGATGTTATCTATTTAGATAACAT LEA I A M H A E N N N INN I I L F V GTE GAD V I Y LON I (567)
170 I TAAAGTAATTGGAACAGAAGTTGAAATTCCAGTTGTTCATGATCCAAAAG GAGAAGCTGTTCTTCCTTCTGTTTTTGAAGACGGTACACGTCAAGGTTGG K V I GTE V E I P V V HOP K G E A V L P S V FED G T R Q G W
(600) I 80 I
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GACTGGGCTGGAGAGTCTGGTGTGAAAACAGCTTTAACAATTGAAGAAGC AAACGGTTCTAACGCGTTATCATGGGAATTTGGATACCCAGAAGTAAAAC o WAG E S G V K TAL TIE E A N G S N A L S W E F GYP E V K P (634)
I gO I
CTAGTGATAACTGGGCAACAGCTCCACGTTTAGATTTCTGGAAATCTGAC TTGGTTCGCGGTGAAAATGATTATGTAACTTTTGATTTCTATCTAGATCC SON W A TAP R L 0 F W K SOL V R G END Y V T F 0 F Y LOP (667) Ace I 200 I AGTTCGTGCAACAGAAGGCGCAATGAATATCAATTTAGTATTCCAGCCAC CTACTAACGGGTATTGGGTACAAGCACCAAAAACGTATACGATTAACTTT V RAT EGA M N I N L V F Q P P T N G Y W V Q A P K T Y TIN F (700) 2 I 0 I GATGAATTAGAGGAAGCGAATCAAGTAAATGGTTTATATCACTATGAAGT GAAAATTAACGTAAGAGATATTACAAACATTCAAGATGACACGTTACTAC DEL E E A N Q V N G L Y H Y E V KIN V R 0 I T N I Q DDT L L R (73 4 ) 220 ) GTAACATGATGATCATTTTTGCAGATGTAGAAAGTGACTTTGCAGGGAGA GTCTTTGTAGATAATGTTCGTTTTGAGGGGGCTGCTACTACTGAGCCGGT N M M I I FAD V E S 0 FAG R V F VON V R F EGA A T T E P V
( 7 67)
2 3 0 I
TGAACCAGAGCCAGTTGATCCTGGCGAAGAGACGCCGCCTGTCGATGAGA AGGAAGCGAAAAAAGAACAAAAAGAAGCAGAGAAAGAAGAGAAAGAAGCA E PEP V 0 P GEE T P P V 0 EKE A K K E Q K E A EKE EKE A (800)
2 40 I
GTAAAAGAAGAAAAGAAAGAAGCTAAAGAAGAAAAGAAAGCAATCAAAAA TGAGGCTACGAAAAAATAATCTAATAAACTAGTTATAGGGTTATCTAAAG V K E E K K E A K E E K K A I K N EAT K K * (822) 2 5 0 I B. I I I GTCT¥ATG~AGATCTTTTAGATAACCTTTTTTTGCATAACTGGACATAGA ATGGTTATTAAAGAAAGCAAGGTGTTTATACGATATTAAAAAGGTAGCGA
R-4
260 l O r
I
I
TTTTAAATTGAAACCTTTAATAATGTCTTGTGATAGAATGATGAAGTAAT TTAAGAGGGGGAAACGAAGTGAAAACGGAAATTTCTAGTAGAAGAAAAAC HI n d I I I 270 1 AGACCAAGAAATACTGCAAGCTT
Fig. 4.
Nucleotide Sequence of the XhoI-HindIII Fragment of pCL64.
The nucleotide sequence of the coding strand is given from the 5' to the 3'-end, and the deduced amino acid sequences, ORF-l and ORF-2, are shown under the corresponding ORFs. Numbering of the nucleotides starts from the initiation codon, ATG, of ORF-2 (the coding sequence of the alkaline EG). Three putative -35 and -10 regions upstream from ORF-2 (P-l, P-2, and P-3) are indicated by dashed lines above the sequence. The possible ribosome-binding sites found upstream both from ORF-l (SD-l) and from ORF-2 (SD-2) are indicated by arrow-heads. Converging horizontal arrows beneath the sequence indicate terminator-like, inverted-repeat sequences (IR-l, IR-2, IR-3, and IR-4). A possible catabolite repression operator-like sequence is indicated by a pair of dashed lines. Restriction sites present in the sequence are also shown above the sequence.
now attempting to identify ORF-l as the pseudouridine synthase gene. Comparison of the gene from KSM-64 with the gene from KSM-64C Interestingly, a catabolite repression operator-like sequence, TGTAAGCGGTTAACC (nucleotides -210 to -196), is found just downstream from the putative promoter-like sequence, P-2, which is similar to the sequence of the catabolite repression operator (TGTAAGCGTTAACA) of the a-amylase gene (amyE) of B. subtilis. 32 )
Therefore, it was thought to be important to compare the EG gene from KSM-64 with that from the cataboliterepression insensitive mutant KSM -64C. However, we could demonstrate no differences in nucleotide sequence between the respective 1.7-kb XhoI-BglII regions that contain sequences for potential promoter, putative SD, and putative catabolite repression operator, for the transcription and expression of the alkaline EG gene (data not shown). Given the absence of differences in nucleotide sequences between the parent and the mutant, it is possible that the phenotype of the mutant is not caused by any change in nucleotides
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Alkaline Endoglucanase Gene from Bacillus Table I. Endoglucanase Activities Produced by E. coli HBlOl and B. subtilis ISW12l4 Cells Carrying Various Plasmids
3) 4)
EG activity expresseda in Plasmid
pBR322 pCL64 pHY300PLK pHCL64 a
b
5)
E. coli (U /liter /24 hr)
B. subtilis (U/liter/3 days)
o (6.2)b 940 (6.2) 0(4.9) 2000 (5.1)
6) 7)
o (2.7) 12,400 (2.5)
EG activity was produced intracellularly in the case of E. coli and extracellularly in the case of B. subtilis (see Materials and Methods). Numbers in parentheses show the degree of bacterial growth, expressed in terms of the absorbance of culture at 600 nm.
8) 9) 10) 11)
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in the regulatory region of the EO gene but it is caused by a change in the repressor protein or some other regulatory mechanism( s). Expression of EG in E. coli and B. subtilis To compare the production of EO in E. coli HBIOI with that in B. subtilis ISW1214, we subcloned the 4.4-kb fragment of pCL64 into pHY300PLK and the recombinant plasmid obtained was designated pHCL64, as is also shown in Fig. 1. The pH-activity profile of the pHCL64-encoded EO expressed in B. subtilis (Fig. 2C) coincided with that of the native enzyme produced by Bacillus sp. KSM-64 (Fig. 2A) and that of the pCL64-encoded enzyme expressed in E. coli (Fig. 2B). E. coli carrying pHCL64 expressed high intracellular EO activity, 2000 U/liter, in LB broth (growth at 37°C for 24 hr), as shown in Table I. B. subtilis carrying the same plasmid expressed very high EO activity extracellularly at 12,400 U/liter when PY culture medium was used (growth at 30°C for 3 days). This rate of extracellular production of the pHCL64-encoded enzyme by B. subtilis was much greater than those reported to date for Bacillus cells transformed with plasmids that contain the EO genes of various origins. 33 - 35) It appears that one of the potential promoter-like sequences, P-l, P-2, or P-3, functions in the high expression of the alkaline EO gene. We have no idea why such promoter-like sequences are repeated in the alkaline EO gene or how these sequences might function in vivo if they actually have individual physiological significance. Tandemly overlapping promoters have been found in several genes of Bacillus species and are considered to participate in quantitative or growth stage-specific regulation of gene expression. 36 - 38) However, different types of promoters overlap in these cases. We are now attempting to identify the actual promoter for the alkaline EO gene by the deletion method and S I nuclease mapping.
12) 13) 14) 15) 16) 17) 18) 19) 20)
21)
22)
23)
24) 25) 26) 27) 28) 29) 30) 31) 32)
33) 34)
Acknowledgments. We wish to thank Dr. K. Horikoshi, the Institute of Physical and Chemical Research, for supplying Bacillus sp. no. 1139 and for discussing this work with us.
References 1) 2)
W. M. Fogarty, P. J. Griffin, and A. M. Joyce, Process Biochem., 9, 11-24 (1974). F. G. Priest, Bacterial. Rev., 41, 711-753 (1977).
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L. M. Robson and G. H. Chambliss, J. Bacterial., 169,2017-2025 (1987). T.-M. Enari and P. H. Markkanen, Proc. Am. Soc. Brew. Chem., 33, 13-17 (1975). B. Cantwell, G. Brazil, J. Hurley, and D. McConnell, Proceedings of the 20th European Brewing Conversion Congress, IRL Press Oxford, U.K., 1985, pp. 259-266. S. Kawai, H. Okoshi, K. Ozaki, S. Shikata, K. Ara, and S. Ito, Agric. Bioi. Chem., 52, 1425-1431 (1988). S. Ito, S. Shikata, K. Ozaki, S. Kawai, K. Okamoto, S. Inoue, A. Takei, Y. Ohta, and T. Satoh, Agric. Bioi. Chem., 53, 1275-1281 (1989). S. Shikata, K. Saeki, H. Okoshi, T. Y oshimatsu, K. Ozaki, S. Kawai, and S. Ito, Agric. Bioi. Chem., 54, 91-96 (1990). H. Okoshi, K. Ozaki, S. Shikata, K. Oshino, S. Kawai, and S. Ito, Agric. Bioi. Chem., 54, 83-89 (1990). T. Yoshimatsu, K. Ozaki, S. Shikata, Y. Ohta, K. Koike, S. Kawai, and S. Ito, J. Gen. Microbiol., 136, 1973-1979 (1990). S. Ito, Y. Ohta, M. Shimooka, M. Takaiwa, K. Ozaki, S. Adachi, and K. Okamoto, Agric. Bioi. Chem., 55, 2387-2391 (1991). M. Murata, E. Hoshino, M. Yokosuka, and A. Suzuki, J. Am. Oil Chem. Soc., 68, 553-558 (1991). K. Ozaki, S. Shikata, S. Kawai, S. Ito, and K. Okamoto, J. Gen. Microbial., 136, 1327-1334 (1990). J. Messing, Methods Enzymol., 101, 20-78 (1983). S. Chang and S. N. Cohen, Mol. Gen. Genet., 168,111-115 (1979). H. Saito and K. Miura, Biochim. Biophys. Acta, 72, 619-629 (1963). H. C. BirnboimandJ. Doly, Nucleic Acids Res., 7,1513-1523 (1979). R. M. Teather and P. J. Wood, Appl. Environ. Microbial., 43, 777-780 (1982). F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. A cad. Sci. U.S.A., 74, 5463-5467 (1977). L. M. Smith, J. Z. Sanders, R. J. Kaiser, P. Hughes, C. Dodd, C. R. Connell, C. Heiner, S. B. H. Kent, and L. E. Hood, Nature, 321, 674-679 (1986). P. W. Hager and J. C. Rabinowitz, in "The Molecular Biology of the Bacilli," Vol II, ed. by D. A. Dubnau, Academic Press, Orlando, U.S.A., 1985, pp. 1-32. 1. Tinoco, Jr., P. N. Borer, B. Dengler, M. D. Levine, O. C. Uhlenbeck, D. M. Crothers, and J. Gralla, Nature New Bioi., 246, 40---41 (1973). C. P. Moran, Jr., N. Lang, S. F. J. LeGrice, G. Lee, M. Stephens, A. L. Sonenshein, J. Pero, and R. Losick, Mol. Gen. Genet., 186, 339-346 (1982). S. Adhya and M. Gottesman, Ann. Rev. Biochem., 47, 967-996 (1978). K. Ozaki, N. Sumitomo, and S. Ito, J. Gen. Microbial., 137, 2299-2305 (1991). R. M. MacKay, A. Lo, G. Willick, M. Zuker, S. Baird, M. Dove, F. Moranelli, and V. Se1igy, Nucleic Acids Res., 14, 9159-9170 (1986). A. Nakamura, T. Uozumi, and T. Beppu, Eur. J. Biochem., 164, 317-320 (1987). F. Fukumori, T. Kudo, Y. Narahashi, and K. Horikoshi, J. Gen. Microbiol., 132, 2329-2335 (1986). F. Fukumori, N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacterial., 168, 479---485 (1986). P. J. Arps, C. C. Marvel, B. C. Rubin, D. A. Tolan, E. E. Penhoet, and M. E. Winkler, Nucleic Acids Res., 13, 5297-5315 (1985). B. VoId, J. Bacterial., 127, 258-267 (1976). W. L. Nicholson, Y.-K. Park, T. M. Henkin, M. Won, M. J. Weickert, J. A. Gaskell, and G. H. Chambliss, J. Mol. Bioi., 198, 609-618 (1987). L. M. Robson and G. H. Chambliss, J. Bacterial., 165, 612-619 (1986). E. Soutschek-Bauer and W. L. Staudenbauer, Mol. Gen. Genet., 208, 537-541 (1987). G. Joliff, A. Edelman, A. Klier, and G. Rapoport, Appl. Environ. Microbiol., 55, 2739-2744 (1989). W. C. Johnson, C. P. Moran, Jr., and R. Losick, Nature, 302, 800-804 (1983). H. C. Wong, H. E. Schnepf, and H. R. Whiteley, J. Bioi. Chem., 258, 1960-1967 (1983). P.-Z. Wang and R. H. Doi,J. Bioi. Chem., 259,8619-8625 (1984).
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Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis Nobuyuki Sumitomo, Katsuya Ozaki, Shuji Kawai & Susumu Ito To cite this article: Nobuyuki Sumitomo, Katsuya Ozaki, Shuji Kawai & Susumu Ito (1992) Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis, Bioscience, Biotechnology, and Biochemistry, 56:6, 872-877, DOI: 10.1271/bbb.56.872 To link to this article: http://dx.doi.org/10.1271/bbb.56.872
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Biosci. Biotech. Biochem., 56 (6), 872-877, 1992
Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus suhtilis Nobuyuki SUMITOMO, Katsuya OZAKI, Shuji KAWAI, and Susumu ITot Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-34, Japan Received October 21, 1991
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The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HBIOI. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-l and ORF-2. The protein deduced from ORF-l was composed of 244 amino acids with an Mr of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an Mr of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGT AAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.
Numerous strains of the genus Bacillus produce extracellular endo-l,4-f3-glucanases (EG; Ee 3.2.l.4).1,2) The EGs of Bacillus have been the focus of some attention because of their potential use in the bioconversion of agricultural wastes into useful products 3) and in the brewing industry.4) For the latter purpose, an EG gene from Bacillus has been cloned and expressed in Saccharomyces cerevisiae. 5 ) Recently, we have isolated some alkalophilic and neutrophilic strains of Bacillus that produce alkaline EGs, the properties of which fulfill the essential requirements for enzymes to be used in laundry detergents. 6 - 8 ) We have purified and characterized some of these alkaline EGS. 9 ,lO) A mutant of one of our isolates, Bacillus sp. KSM-635,7,lO) is currently exploited for the large-scale industrial production of an alkaline EG for use in laundry detergents. ll ,12) To clarify the mechanism of action of EGs, with a special focus on differences in pH optima, we cloned and sequenced the gene for alkaline EG from Bacillus sp. KSM-635. The deduced amino acid sequence of the alkaline EG protein had significant homology to the amino acid sequences of other alkaline and neutral EGs produced by various strains of Bacillus, with some amino acid residues conserved in either the alkaline enzymes or the neutral enzymes, or in both types of enzyme. 13) Since the major factor limiting the inclusion of alkaline EG in detergents is the cost of the enzyme, cloning the gene for a suitable EG may also make possible a more detailed study of some aspects of enhanced production of the enzyme. In this paper, we describe the nucleotide sequence of the gene for an alkaline EG, suitable for use in laundry detergents, from Bacillus sp. KSM-64. 8 ) We also describe the construction of a recombinant plasmid from a shuttle vector and the alkaline EG-coding fragment and demonstrate that the EG gene in the plasmid can be expressed at high levels both in Escherichia coli and in
Bacillus subtilis.
Materials and Methods Bacterial strains, plasmids, and bacteriophages. Bacillus sp. KSM-64 and Bacillus sp. KSM-64C were used as sources of the EG gene. Bacillus sp. KSM-64C was derived from Bacillus sp. KSM-64 as a mutant that was insensitive to catabolite repression (S. Ito and K. Saeki, unpublished data). Escherichia coli HB 10 1 (F - hsdS20 recA 13 ara-14 proA2 lac Y 1 galK2 rpsL20 xyl-S mtl-l supE44 leuB6 thi-I), E .. coli JMI09 (recAl LJ(lac-pro) endAl gyrA96 thi-l hsdR17 supE44 relAl F'traD36 proAB lacJQZ LJMlS), and Bacillus subtilis ISW1214 (leuA8 metES hsdMl) were used as hosts for cloning and nucleotide sequencing. Plasmids pBR322 and pHY300PLK, and bacteriophages Ml3mp18 and M13mpl9 were used as the vectors. Culture media. E. coli strains carrying plasmids were grown on LB broth. 2 x YT broth was used for cultivation of E. coli JM 109 infected with M 13 bacteriophage. 14) B. subtilis ISWl2l4 was grown on PY broth that contained (w/v %) 2.0% polypepton (Nihon Pharmaceutical), 0.1 % Bacto yeast extract (Difco), 0.1 % KH 2 P0 4 , and O.S% NaC!. PYM medium that contained (w /v %) O.S% polypepton, 0.1 % Bacto yeast extract, 0.1 % KH 2 P0 4 , 0.02% MgS0 4 ' 7H 2 0, and 1.0% Na 2 C0 3 was used for the propagation of Bacillus sp. KSM-64 and Bacillus sp. KSM-64C. Solidified media contained 1.S% (w/v) Bacto agar (Difco). DM-3 agar plates, composed of O.S M disodium succinate (pH 7.0), O.S% casamino acid (Difco), O.S% yeast extract, 0.35% KH 2 P0 4 , O.lS% K 2 HP0 4 , O.S% glucose, 20mM MgCI 2 , 0.01 % bovine serum albumin (Sigma), and 0.8% Bacto agar, were used for the regeneration of protoplasts of B. subtilis ISWI214. 15 ) Preparation of DNA. The test bacilli were grown in PYM medium at 30°C overnight, with shaking, and pelleted by centrifugation (12,000 x g 20 min). Genomic DNA was prepared as described by Saito and Miura. 16 ) Plasmid DNA and the replicative form of M 13 bacteriophage DNA were isolated by the alkaline extraction procedure described by Birnboim and Doly. 1 7) Covalently closed circular DNA was purified by CsCl/ethidium bromide equilibrium density gradient centrifugation. Cloning of the EG gene. Genomic DNA from Bacillus and plasmid pBR322 were both digested with HindIII (Boehringer Mannheim) and ligated with T 4 DNA ligase (Boehringer Mannheim). The ligation mixture was used for transformation of competent E. coli HBI01 cells (purchased from Takara Shuzo). LB plates supplemented with SO Jlg/mi ampicillin
t To whom all correspondence should be addressed. The nucleotide sequence data reported in this paper have been submitted to the GenBank/EMBL Data Bank with the accession number M84963.
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Alkaline Endoglucanase Gene from Bacillus (sodium salt; Sigma), on which the transformants appeared, were overlaid with 1.0% (w/v) agar that contained O.S% (w/v) carboxymethy1cellulose (CMC; degree of substitution 0.68; Sanyo Kokusaku Pulp), 1.0% (w/v) NaCl, 0.1 mg/mllysozyme and SO mM glycine/NaOH buffer (pH 9.0). After incubation at 37°C for 6 hr, the activity of the EG produced by the transformants was detected by staining the overlaid CMC with Congo red dye, essentially as described by Teather and Wood. 18 ) After screening for ampicillin-resistant and EG-positive transformants of E. coli HB101, the plasmid, named pCL64, was obtained. B. subtilis ISW1214 was transformed by plasmid pHY300PLK or its derivatives by the protoplast method of Chang and Cohen. 1S ) Transformed cells were selected on DM-3 agar plates that contained tetracycline at lS/lg/ml (Sigma).
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Hybridization analysis of DNA digests. HindIII digests of the genomic DNA (S/lg) of Bacillus sp. KSM-64 were electrophoresed on a 0.8% (w/v) agarose gel and then fragments were electrophoretically transferred to a nylon membrane (Zeta-Probe Blotting Membrane, Bio-Rad) after denaturation by soaking the gel in 0.2 M NaOH/O.S M NaCI for 30 min at room temperature. Approximately 3/lg of the HindIII insert, isolated from pCL64, was labelled with digoxigenin-ll-dUTP and used as a probe for hybridization analysis of the HindIII digests of the genomic DNA on the membrane, by use of a DNA-Labelling and Detection Kit (Boehringer Mannheim). Nucleotide sequencing. The nucleotides of the 4.1-kb XhoI-HindIII region of pCL64 (see Fig. I) were sequenced by the dideoxy method of Sanger et al.,19) using an M13 Sequencing Kit (Takara Shuzo) and [1X- 32 PJdCTP (Amersham). Parts of the nucleotide sequence were identified by the dideoxy method using Sequenase (modified T 7 DNA polymerase; United States Biochemical) in place of the Klenow enzyme, or by the dideoxy method using a fluorescent dye primer 20 ) and Sequenase, on an automated DNA sequencer (Applied Biosystems). Template DNAs were prepared using M13mp18 or M13mp19 phage vector, as described by Messing. 14 ) Ordered deletion clones of M13 as the template DNA for sequencing were prepared with a Kilo-sequencing Deletion Kit (Takara Shuzo). Nucleotide sequence data were analyzed on the basis of data from the GENETYX Gene Information Treatment System (Software Development).
Bacillus sp. KSM -64 that had been blotted on a membrane. Thus, the 4.4-kb insert of pCL64 appeared to have come from the genomic DNA of Bacillus sp. KSM-64. Nucleotide sequence analysis The nucleotides of the 4.I-kb XhoI-HindIII fragment from pCL64 were sequenced. The 4126-bp nucleotide sequence is shown in Fig. 4. Two open reading frames (ORFs), designated ORF-I and ORF-2, encoding proteins of 244 and 822 amino acids (with Mr of 27,865 and 91,040, respectively) were identified in the sequence. ORF-2 begins at nucleotide I (AT G) and ends at nucleotide 2467 (TAA), which encodes the alkaline EG. Upstream from the initiation codon of ORF-2, there is a putative Shine-Dalgarno (SD) sequence, SD-2, which is complementary to a high degree to the 3' end of 16S rRNA from B. subtilis. 2 1) SD-2 starts at nucleotide -10 (AGGAGGT) and would have a free energy of binding (LtG) of -18.8 kcaljmol (- 78.7 kJ jmol), as calculated by the method of Tinoco et al.,22) if it bound to the 3' end of the 16S rRNA from B. subtilis. Upstream from this ORF, there are three sequences, P-I, P-2, and P-3, which are similar to the consensus sequence of the sigma A-type promoter of B. subtilis. 23) The P-I region starts at nucleotide -404 (TTTAAT) as the -35 region and at -381 (TATAAT) as the -10 region. The P-2 region starts at nucleotide -245 (GTAAGA) as the -35 region and at -219 (TATGAT) as
Sc Ec Hi Sp
Sc Kp
Assay of EG activity. E. coli cells carrying the recombinant plasmid were grown on LB broth supplemented with ampicillin (SO /lg/ml) at 37°C for 24 hr. Cells were harvested from a 3S-ml portion of the culture by centrifugation (9,800 x g, 10 min) at 4°C and were suspended in 7 m1 of 20mM NaH2P04/Na2HP04 buffer (pH 7.0). The cells in the suspension were disrupted by sonication at temperatures not exceeding 4°C. The cell debris was removed by centrifugation at 12,000 x 9 for IS min at 4°C and the resulting cell-free extract was used for assays of enzymatic activity. B. subtilis ISW1214 carrying the recombinant plasmid was grown at 30°C for 3 days on PY broth supplemented with lS/lg/ml tetracycline. Two ml of the culture were centrifuged at 12,000 x 9 for 10 min and the supernatant obtained was used as the solution of enzyme. EG activity was then assayed as described previously.7) One unit (U) of enzymatic activity was defined as the amount of enzyme that produced 1.0/lmol of reducing sugar as glucose per min under the conditions of the reaction.
Sc
Hp
pHY300PLK 4.9 kb
Hi Xh
1~_H_in_d_rn________~________~1 Hindrn
11 igation Sp
Results and Discussion Cloning of the EG gene The EG-expressing plasmid, pCL64, contained one 4.4-kb HindIII insert, and its restriction map is shown in Fig. I. E. coli HBIOI harboring pCL64 produced active intracellular EG, to a level that corresponded to approximately 1000 U jliter broth, when cells were grown at 37°C for 24 hr in LB broth containing ampicillin. The expressed EG was active from pH 5 to 13, with maximum activity at pH 9.5. The profile of the pH-activity curve of the expressed enzyme coincided with that of the alkaline EG produced by Bacillus sp. KSM-64, as shown in Figs. 2A and 2B. To clarify the origin of the 4.4-kb insert in pCL64, hybridization analysis was done using the digoxigeninlabelled 4.4-kb insert of pCL64 as a probe. Figure 3 shows that the labelled insert of pCL64 hybridized with a 4.4-kb fragment in HindUI digests of the genomic DNA from
Sc Kp
Hp
Ec Srn Hi Xh Fig. 1.
Construction of pHCL64 from pCL64 and pHY300PLK.
Thick and thin lines show the cloned genes (ORF-l and ORF-2) and the vectors (pBR322 and pHY300PLK), respectively. Arrows around circles indicate the positions and the orientations of the sequences that encode ORF-I and ORF-2 (the alkaline EG gene) from Bacillus sp. KSM-64, and the ampicillin (Ap)- and tetracycline (Tc)-resistance genes in the vector plasmids. Abbreviations for restriction sites are: Ec, EcoRI; Hi, HindIII; Hp, HpaI; Kp, Kpnl; Sc, Seal; Sp, Sphl; Sm, Smal; Xh, XhoI.
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...... u
ro
50
7
9
7
11
9
11
7
9
11
pH Fig. 2. pH-Activity Profiles of the Alkaline Endoglucanases Produced by Bacillus sp. KSM-64 (A), by E. coli HB101 Carrying pCL64 (B), and by B. subtilis ISW1214 Carrying pHCL64 (C). McIlvaine buffer (e, pH 6.2-7.7), sodium phosphate buffer (., pH 6.6-7.6), glycine/NaOH buffer (0, pH 8.2-10.8), and Na zHP0 4 /NaOH buffer (... , 11.0-12.1)
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were used. Assays were done at 40°C with indicated buffers at 0.1 glycine/NaOH buffer was taken as 100% in each case.
A
M,
at various pH values, using 1.0% (w/v) CMC as substrate. The enzymatic activity at pH 9.5 in 0.1
B 1 234
1 234
kb --23.1-9.4-- - - 6.7--4.4--
---
-2.3-
M
of ORF-1, there is a putative SD sequence (AAAGGA), which starts at nucleotide -1269 (a L1G of - 11.8 kcal/mol (-49.4kJ/mol)). In addition, an IR is found upstream from the initiation codon of ORF-1 (IR-1, nucleotides -1320 to -1291) and two IRs downstream from the stop codon of the sequence (IR-2 and IR-3 at nucleotides -510 to -478 and -402 to -375, respectively). The L1G values for these sequences, IR-1, IR-2, and IR-3, for stem-loop structures, were calculated to be -23.8 kcal/mol (-99.6 kJ Imol), - 16.8 kcal/mol (- 70.3 kJ Imol), and - 11.2 kcall mol ( - 46.9 kJ Imo!), respectively.
----- 2. 0 ----
_____
Fig. 3.
O.56~
Hybridization Analysis of the HindIII Insert of pCL64.
A-DNA digested with HindIII (lane I) or EcoRI (lane 2), or DNA from Bacillus sp. KSM-64 digested with HindIII (lane 3) or EcoRI (lane 4) was electrophoresed on a 0.8% (w/v) agarose gel and transferred onto a Zeta-Probe Blotting Membrane. The DNA digests on the membrane were hybridized with the digoxigenin-ll-dUTPlabelled 4.4-kb HindIII fragment of pCL64. A, results of agarose gel electrophoresis of digests of KSM-64 DNA; B, autoradiograms of digests of KSM-64 DNA hybridized with labelled HindIII probe.
the -10 region. The P-3 region starts at nucleotide -123 (TTGAGA) as the -35 region and at -100 (GTTACT) as the -10 region. In addition, an inverted repeat sequence (IR), which resembles a prokaryotic p-independent transcription terminator,24) is 22 nucleotides downstream from the stop codon of ORF-2 (IR-4, nucleotides 2488 to 2526). The L1G value for IR-4 for a stem-loop structure was calculated to be - 18.4 kcal/mol (-77.0 kJ/mol). Therefore, a combination of these potential promoter (P-1, P-2, or P-3) and terminator (IR-4) sequences may be required for the expression of the alkaline EG gene. ORF-1 (nucleotides -1256 (ATG) to -524 (TAA)) encodes an unidentified protein. Upstream from the initiation codon
Homology of the amino acid sequences encoded by ORF-J and 0 RF-2 to those of other enzymes The amino acid sequence of the alkaline EG, deduced from ORF-2, from Bacillus sp. KSM-64, was compared with those of an acid EG,25) neutral EGs, 3,26,27) and alkaline EGs 13 ,28,29) that have been reported to date for the genus Bacillus. The sequence of amino acids of this enzyme has 99.6% homology to that of the alkaline enzyme from alkalophilic Bacillus sp. no. 1139. 28 ) There are only 12 differences in nucleotides and 4 differences in amino acids between the two genes for EGs. The gene from Bacillus sp. KSM-64 has a insertion of one nucleotide, at nucleotide 2399 of this gene, as compared to the gene from Bacillus sp. no. 1139. As a result of the single insertion at this position, the deduced amino acid sequence of this EG is 22 amino acids longer than that of the EG from Bacillus sp. no. 1139. These results indicate that the additional 22 amino acid residues are not necessary for the activity of the EG. The deduced amino acid sequence of the EG from Bacillus sp. KSM -64 is also similar to those of other alkaline (38-72% homology)13,29) and neutral EGs (approximately 31 % homology),3,26,27) but it is not homologous to that of the acid EG. 25 ) The amino acid sequence deduced from ORF-1 has 34% homology to that of pseudouridine synthase from E. coli. 30) Pseudouridine is found in the tRNA of B. subtilis,31) but there are no reports of cloning of the gene for pseudouridine synthase from the genus Bacillus. We are
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875
Alkaline Endoglucanase Gene from Bacillus X hoi
CTC - 1400
GAGTGGAGCAAGAGGCTTCTTAACTCGTTCATGAATGTAGAATAGAAACA ATGGAAAAGCTAAATTAAAGAAAAAACTTTGGGGTTATTGCAICAATCl§ IR-1 -1300 ¢ ORF-1 CGATAACCCCTTAAATGCTAACTACATAGATAAAGGAAGATAAAATGAAC AATTATAAACTAATGATTCAATATGATGGTGGTCGATACAAAGGTTGGCA 50-1 •••••• M N N Y K L M I Q Y 0 G G R Y K G WQ (I)
(10)
-1200
GCGTCTTGGGAACGGTGAAAATACGATTCAAGGTAAAATTGAAACGGTTT TATCAGAGATGGTAGGTAGAAAAATAGAGATTATAGGGTCTGGTAGAACA R L G N G E N T I Q G K lET V L 5 E M V G R K I E I I G S G R T (53)
- I I 00
GATGCTGGTGTCCATGCTCTTGGACAAGTGGCTAATGTAAAATTAAGCGA AAATTTTACAGTAAAAGAGGTTAAAGAGTATTTGAATCGTTATTTGCCTC D A G V HAL G Q V A N V K L SEN F T V K E V KEY L N R Y L P H (86)
ATGATATCAGTGTGACTGAGGTGACGCTAGTTCCAGATCGTTTTCACTCA AGGTATAACGCAAAGGACAAAACCTATCTTTATAAAATTTGGAATGAGGA DIS V T E V T L V P D R F H 5 R Y N A K D K T Y L Y K I W NED (119)
TTATACTCATCCGTTTATGCGTAAGTACAGCTTGCACATCGAAAAGAAAT TACATATTGATAACATGGTAAAAGCAAGTCAACTTTTCGTAGGAGAACAT Y T H P F M R K Y 5 L HIE K K L HID N M V K A 5 Q L F V G E H (153)
GATTTTACAGCTTTTTCTAATGCTAAATCTAAAAAGAAGACAAATACGAG AACGATTCACTCTATAACTATTCAAGATAATCAAGGATTTATAGACATTA OFT A F 5 N A K 5 KKK TNT R T I H 5 I T I Q 0 N Q G FlO I R (186)
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GGGTTTGTGGAGATGGTTTTCTTTATAACATGGTTAGAAAAATGGTAGGG ACTTTGATTGAGGTTGGTCTAGGTGAAAAGGAACCTGAACAAGTACTTAC V C G D G FLY N M V R K M V G T LIE V G L G EKE P E Q V L T (219)
CATTTTAGAGTCAAAAGATAGAAGCCAAGCAGGATTTGCCGATGCAACCG GCTTATATTTAGAGGGAATTTCTTTTTAAATTGAATACGGAATAAAATCA I L E S K D R 5 Q A G FAD A T G L Y LEG I 5 F * ~TAAACAGGT~CTGATTTTATTTTTTTGAATTTTTTTGAGAACTAAAGA
TTGAAATAGAAGTAGAAGACAACGGACATAAGAAAATTGTATTAGTTTTA
IR-2 P-l
NapV
ATTATAGAA~ACGCT~TTCTATAATTATTTATACCTAGAACGAAAATACT
GTTTCGAAAGCGGTTTACTATAAAACCTTATATTCCGGCTCTTTTTTTAA
IR-3 P-2 ACAGGGGGTGAAAATTCACTCTAGTATTCTAATTTCAACATGCTATAATA AATTTGTAAGACGCAATATACATCTTTTTTTTATGATATTTGTAAGCGGT 11 i n , I I (H
p •
P-3
I)
TAACCTTGTGCTATATGCCGATTTAGGAAGGGGGTAGATTGAGTCAAGTA GTCATAATTTAGATAACTTATAAGTTGTTGAGAAGCAGGAGAGAATCTGG GTTACTCACAAGTTTTTTAAAACATTATCGAAAGCACTTTCGGTTATGCT TATGAATTTAGCTATTTGATTCAATTACTTTAATAATTTTAGGAGGTAAT SO-2 ••••• .1. ¢ ORF-2 (alkal ine EG) ATGATGTTAAGAAAGAAAACAAAGCAGTTGATTTCTTCCATTCTTATTTT AGTTTTACTTCTATCTTTATTTCCGACAGCTCTTGCAGCAGAAGGAAACA M M L R K K T K Q LIS S I L I L V L L L S L F PTA L A A E G N T (34)
CTCGTGAAGACAATTTTAAACATTTATTAGGTAATGACAATGTTAAACGC CCTTCTGAGGCTGGCGCATTACAATTACAAGAAGTCGATGGACAAATGAC RED N F K H L L G NON V K R P SEA GAL Q L Q E V 0 G Q M T (67)
ATTAGTAGATCAACATGGAGAAAAAATTCAATTACGTGGAATGAGTACAC ACGGATTACAATGGTTTCCTGAGATCTTGAATGATAACGCATACAAAGCT L V D Q H G E K I Q L R G M 5 T H G L Q W F PEl L N DNA Y K A (100)
CTTGCTAACGATTGGGAATCAAATATGATTCGTCTAGCTATGTATGTCGG TGAAAATGGCTATGCTTCAAATCCAGAGTTAATTAAAAGCAGAGTCATTA LAN 0 W E 5 N M I R LAM Y V G ENG Y A 5 N PEL I K 5 R V I K (134)
B 1'1 I I
AAGGAATAGATCTTGCTATTGAAAATGACATGTATGTCATCGTTGATTGG CATGTACATGCACCTGGTGATCCTAGAGATCCCGTTTACGCTGGAGCAGA G I D L A lEN D M Y V I V D W H V HAP G D P R D P V Y A G A E (167)
AGATTTCTTTAGAGATATTGCAGCATTATATCCTAACAATCCACACATTA TTTATGAGTTAGCGAATGAGCCAAGTAGTAACAATAATGGTGGAGCTGGG D F F R D I A A L Y P N N PHI lYE LAN E P 5 5 N N N G GAG (200)
601
ATTCCAAATAATGAAGAAGGTTGGAATGCGGTAAAAGAATACGCTGATCC AATTGTAGAAATGTTACGTGATAGCGGGAACGCAGATGACAATATTATCA I P NNE E G W N A V KEY A 0 P I V E M L R D 5 G N ADD N I I I (2311
701
TTGTGGGTAGTCCAAACTGGAGTCAGCGTCCTGACTTAGCAGCTGATAAT CCAATTGATGATCACCATACAATGTATACTGTTCACTTCTACACTGGTTC V G S P N W 5 Q R _ POL A A D N P I DOH H T M Y T VHF Y T G 5 (267)
ACATGCTGCTTCAACTGAAAGCTATCCGCCTGAAACTCCTAACTCTGAAA GAGGAAACGTAATGAGTAACACTCGTTATGCGTTAGAAAACGGAGTAGCA H A A S T E 5 Y P PET P N S ERG N VMS N TRY ALE N G V A (300)
90 I
GTATTTGCAACAGAGTGGGGAACTAGCCAAGCAAATGGAGATGGTGGTCC TTACTTTGATGAAGCAGATGTATGGATTGAGTTTTTAAATGAAAACAACA V FAT E W G T 5 Q A N G D G G P Y F D E A 0 V W I E F L N E N N I Fig. 4.
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N.
876
SUMITOMO
et al. (334)
100~TAGCTGGGCTAACTGGTCTTTAACGAATAAAAATGAAGTATCTGGTGCA TTTACACCATTCGAGTTAGGTAAGTCTAACGCAACAAGTCTTGACCCAGG S WAN W S L T N K N E V S G AFT P F E L G K S NAT S LOP G (367) 1101 n 1 GCCAGACCAAGTATGGGTACCAGAAGAGTTAAGTCTTTCTGGAGAATATG TACGTGCTCGTATTAAAGGTGTGAACTATGAGCCAATCGACCGTACAAAA PDQ V W V PEE L S L S G E Y V R A R I K G V N YEP lOR T K 00) 120 1 I TACACGAAAGTACTTTGGGACTTTAATGATGGAACGAAGCAAGGATTTGG AGTGAATGGAGATTCTCCAGTTGAAGATGTAGTTATTGAGAATGAAGCGG Y T K V L W 0 F N 0 G T K Q G F G V N G 0 S P V E 0 V V lEN E A G K p
(4
SCI
(434l
I 30 l O r
I
I
GCGCTTTAAAACTTTCAGGATTAGATGCAAGTAATGATGTTTCTGAAGGT AATTACTGGGCTAATGCTCGTCTTTCTGCCGACGGTTGGGGAAAAAGTGT A L K L S G LOA S NOV S E G N Y WAN A R L SAD G W G K S V
(4 67)
I 4 0 I
TGATATTTTAGGTGCTGAAAAACTTACTATGGATGTGATTGTTGATGAGC CGACCACGGTATCAATTGCTGCAATTCCACAAGGGCCATCAGCCAATTGG OIL G A E K L T M 0 V I V 0 E P T T V S I A A I P Q G P SAN W(500)
I
50 I GTTAATCCAAATCGTGCAATTAAGGTTGAGCCAACTAATTTCGTACCGTT AGGAGATAAGTTTAAAGCGGAATTAACTATAACTTCAGCTGACTCTCCAT V N P N R A I K V E P T N F V P L G 0 K F K A E L TIT SAD S P S
(534)
I B0 I
S phi
CGTTAGAAGCTATTGCGATGCATGCTGAAAATAACAACATCAACAACATC ATTCTTTTTGTAGGAACTGAAGGTGCTGATGTTATCTATTTAGATAACAT LEA I A M H A E N N N INN I I L F V GTE GAD V I Y LON I (567)
170 I TAAAGTAATTGGAACAGAAGTTGAAATTCCAGTTGTTCATGATCCAAAAG GAGAAGCTGTTCTTCCTTCTGTTTTTGAAGACGGTACACGTCAAGGTTGG K V I GTE V E I P V V HOP K G E A V L P S V FED G T R Q G W
(600) I 80 I
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GACTGGGCTGGAGAGTCTGGTGTGAAAACAGCTTTAACAATTGAAGAAGC AAACGGTTCTAACGCGTTATCATGGGAATTTGGATACCCAGAAGTAAAAC o WAG E S G V K TAL TIE E A N G S N A L S W E F GYP E V K P (634)
I gO I
CTAGTGATAACTGGGCAACAGCTCCACGTTTAGATTTCTGGAAATCTGAC TTGGTTCGCGGTGAAAATGATTATGTAACTTTTGATTTCTATCTAGATCC SON W A TAP R L 0 F W K SOL V R G END Y V T F 0 F Y LOP (667) Ace I 200 I AGTTCGTGCAACAGAAGGCGCAATGAATATCAATTTAGTATTCCAGCCAC CTACTAACGGGTATTGGGTACAAGCACCAAAAACGTATACGATTAACTTT V RAT EGA M N I N L V F Q P P T N G Y W V Q A P K T Y TIN F (700) 2 I 0 I GATGAATTAGAGGAAGCGAATCAAGTAAATGGTTTATATCACTATGAAGT GAAAATTAACGTAAGAGATATTACAAACATTCAAGATGACACGTTACTAC DEL E E A N Q V N G L Y H Y E V KIN V R 0 I T N I Q DDT L L R (73 4 ) 220 ) GTAACATGATGATCATTTTTGCAGATGTAGAAAGTGACTTTGCAGGGAGA GTCTTTGTAGATAATGTTCGTTTTGAGGGGGCTGCTACTACTGAGCCGGT N M M I I FAD V E S 0 FAG R V F VON V R F EGA A T T E P V
( 7 67)
2 3 0 I
TGAACCAGAGCCAGTTGATCCTGGCGAAGAGACGCCGCCTGTCGATGAGA AGGAAGCGAAAAAAGAACAAAAAGAAGCAGAGAAAGAAGAGAAAGAAGCA E PEP V 0 P GEE T P P V 0 EKE A K K E Q K E A EKE EKE A (800)
2 40 I
GTAAAAGAAGAAAAGAAAGAAGCTAAAGAAGAAAAGAAAGCAATCAAAAA TGAGGCTACGAAAAAATAATCTAATAAACTAGTTATAGGGTTATCTAAAG V K E E K K E A K E E K K A I K N EAT K K * (822) 2 5 0 I B. I I I GTCT¥ATG~AGATCTTTTAGATAACCTTTTTTTGCATAACTGGACATAGA ATGGTTATTAAAGAAAGCAAGGTGTTTATACGATATTAAAAAGGTAGCGA
R-4
260 l O r
I
I
TTTTAAATTGAAACCTTTAATAATGTCTTGTGATAGAATGATGAAGTAAT TTAAGAGGGGGAAACGAAGTGAAAACGGAAATTTCTAGTAGAAGAAAAAC HI n d I I I 270 1 AGACCAAGAAATACTGCAAGCTT
Fig. 4.
Nucleotide Sequence of the XhoI-HindIII Fragment of pCL64.
The nucleotide sequence of the coding strand is given from the 5' to the 3'-end, and the deduced amino acid sequences, ORF-l and ORF-2, are shown under the corresponding ORFs. Numbering of the nucleotides starts from the initiation codon, ATG, of ORF-2 (the coding sequence of the alkaline EG). Three putative -35 and -10 regions upstream from ORF-2 (P-l, P-2, and P-3) are indicated by dashed lines above the sequence. The possible ribosome-binding sites found upstream both from ORF-l (SD-l) and from ORF-2 (SD-2) are indicated by arrow-heads. Converging horizontal arrows beneath the sequence indicate terminator-like, inverted-repeat sequences (IR-l, IR-2, IR-3, and IR-4). A possible catabolite repression operator-like sequence is indicated by a pair of dashed lines. Restriction sites present in the sequence are also shown above the sequence.
now attempting to identify ORF-l as the pseudouridine synthase gene. Comparison of the gene from KSM-64 with the gene from KSM-64C Interestingly, a catabolite repression operator-like sequence, TGTAAGCGGTTAACC (nucleotides -210 to -196), is found just downstream from the putative promoter-like sequence, P-2, which is similar to the sequence of the catabolite repression operator (TGTAAGCGTTAACA) of the a-amylase gene (amyE) of B. subtilis. 32 )
Therefore, it was thought to be important to compare the EG gene from KSM-64 with that from the cataboliterepression insensitive mutant KSM -64C. However, we could demonstrate no differences in nucleotide sequence between the respective 1.7-kb XhoI-BglII regions that contain sequences for potential promoter, putative SD, and putative catabolite repression operator, for the transcription and expression of the alkaline EG gene (data not shown). Given the absence of differences in nucleotide sequences between the parent and the mutant, it is possible that the phenotype of the mutant is not caused by any change in nucleotides
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Alkaline Endoglucanase Gene from Bacillus Table I. Endoglucanase Activities Produced by E. coli HBlOl and B. subtilis ISW12l4 Cells Carrying Various Plasmids
3) 4)
EG activity expresseda in Plasmid
pBR322 pCL64 pHY300PLK pHCL64 a
b
5)
E. coli (U /liter /24 hr)
B. subtilis (U/liter/3 days)
o (6.2)b 940 (6.2) 0(4.9) 2000 (5.1)
6) 7)
o (2.7) 12,400 (2.5)
EG activity was produced intracellularly in the case of E. coli and extracellularly in the case of B. subtilis (see Materials and Methods). Numbers in parentheses show the degree of bacterial growth, expressed in terms of the absorbance of culture at 600 nm.
8) 9) 10) 11)
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in the regulatory region of the EO gene but it is caused by a change in the repressor protein or some other regulatory mechanism( s). Expression of EG in E. coli and B. subtilis To compare the production of EO in E. coli HBIOI with that in B. subtilis ISW1214, we subcloned the 4.4-kb fragment of pCL64 into pHY300PLK and the recombinant plasmid obtained was designated pHCL64, as is also shown in Fig. 1. The pH-activity profile of the pHCL64-encoded EO expressed in B. subtilis (Fig. 2C) coincided with that of the native enzyme produced by Bacillus sp. KSM-64 (Fig. 2A) and that of the pCL64-encoded enzyme expressed in E. coli (Fig. 2B). E. coli carrying pHCL64 expressed high intracellular EO activity, 2000 U/liter, in LB broth (growth at 37°C for 24 hr), as shown in Table I. B. subtilis carrying the same plasmid expressed very high EO activity extracellularly at 12,400 U/liter when PY culture medium was used (growth at 30°C for 3 days). This rate of extracellular production of the pHCL64-encoded enzyme by B. subtilis was much greater than those reported to date for Bacillus cells transformed with plasmids that contain the EO genes of various origins. 33 - 35) It appears that one of the potential promoter-like sequences, P-l, P-2, or P-3, functions in the high expression of the alkaline EO gene. We have no idea why such promoter-like sequences are repeated in the alkaline EO gene or how these sequences might function in vivo if they actually have individual physiological significance. Tandemly overlapping promoters have been found in several genes of Bacillus species and are considered to participate in quantitative or growth stage-specific regulation of gene expression. 36 - 38) However, different types of promoters overlap in these cases. We are now attempting to identify the actual promoter for the alkaline EO gene by the deletion method and S I nuclease mapping.
12) 13) 14) 15) 16) 17) 18) 19) 20)
21)
22)
23)
24) 25) 26) 27) 28) 29) 30) 31) 32)
33) 34)
Acknowledgments. We wish to thank Dr. K. Horikoshi, the Institute of Physical and Chemical Research, for supplying Bacillus sp. no. 1139 and for discussing this work with us.
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