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BACTERIOLOGY, Mar. 1990, p. 1576-1586
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Molecular Cloning, Expression, and Characterization of Endo-3-1,4-Glucanase Genes from Bacillus polymyxa and Bacillus circulanst STEPHEN D. BAIRD,1'2t DOUGLAS A. JOHNSON,' AND VERNER L. SELIGY2* Department of Biology, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5,1 and Molecular Genetics Section, Division of Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada KIA 0R62 Received 11 August 1989/Accepted 11 December 1989
Endo-0-1,4-glucanase genes from Bacillus circulans and from B. polymyxa were cloned by direct expression by using bacteriophage M13mp9 as the vector. The enzymatic activity of the gene products was detected by using either the Congo red assay or hydroxyethyl cellulose dyed with Ostazin Brilliant Red H-3B. The B. circulans and B. subilis PAP115 endo-0-1,4-glucanase genes were shown to be homologous by the use of restriction endonuclease site mapping, DNA-DNA hybridization, Si nuclease digestion after heteroduplex formation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein products. Analysis of the nucleotide sequence of 3.1 kilobase pairs of cloned B. polymyxa DNA revealed two convergently transcribed open reading frames (ORFs) consisting of 398 codons (endoglucanase) and 187 codons (ORF2) and separated by 374 nucleotides. The coding region of the B. polymyxa endoglucanase gene would theoretically produce a 44-kilodalton preprotein. Expression of the B. polymyxa endoglucanase in Escherichia coli was due to a fusion of the endoglucanase gene at codon 30 with codon 9 of the lacZ oa-peptide gene. The B. polymyxa endoglucanase has 34% amino acid similarity to the Clostridium thermocellum ceiB endoglucanase sequence but very little similarity to endoglucanases from other BaciUus species. ORF2 has 28% amino acid similarity to the NH2-terminal half of the E. coli lac repressor protein, which is responsible for DNA binding. Cellulose is degraded to varying extents by a variety of organisms, depending upon the properties of the cellulosic substrate and the types of glycosidases produced (8). Highly efficient cellulolytic bacteria and fungi generally produce one or more enzymes from each of the three classes of enzymes required to degrade microcrystalline cellulose to glucose. In contrast, members of the industrially important Bacillus bacteria are generally not considered to be cellulolytic organisms because none of them have so far revealed a complete cellulase enzyme complex needed to degrade native cellulose. Several related endo-,B-1,4-glucanase genes have been isolated and sequenced from different species of the Bacillus genus (14, 24, 25, 28, 30, 44, 48-50). To extend this database further, we screened 16 additional Bacillus species for endo-P-1,4-glucanase genes and found that, aside from B. subtilis, only B. circulans and B. polymyxa have such genes. Here we show that the cloned B. polymyxa endoglucanase gene is not similar to the B. subtilis gene or the B. circulans gene that we also cloned. Nucleotide sequence comparisons involving all other available cellulase genes revealed that the B. polymyxa gene is related to the celB gene of Clostridium thermocellum, therefore representing a new class of Bacillus endoglucanases.
screened for cellulolytic activity by growth on agar plates containing 0.01% (wt/vol) carboxymethyl cellulose (CMcellulose; degree of polymerization, 7; medium viscosity; Sigma Chemical Co.) and Luria Bertanini medium. Escherichia coli JM103 for bacteriophage M13 propagation and expression was grown by the method of Messing (32). E. coli HB101 was grown with Luria Bertanini medium unless otherwise stated. All media used for the maintenance of pUC plasmids in E. coli contained 35 ,ug of ampicillin per ml. Cloning methodology. The Bacillus genomic DNA was prepared as described by Saito and Miura (46). RNA was removed from the DNA preparations with RNase A treatment followed by ethanol (EtOH) precipitation. The plasmid pUC9 and the replicative form of bacteriophage M13 were prepared as described by Maniatis et al. (31). Partially digested genomic DNA was ligated to the linearized replicative form of M13mp9 and transformed into E. coli by the method of Hanahan (19), but without incubation of the transformed cells at 37°C before plating. To detect cellulaseproducing, Congo red-positive M13 (CR+) clones, 0.1% CM-cellulose was used in the top agar. Activity was determined by flooding the agar plate with 10 ml of Congo red (1 mglml) for 15 min and destaining it with 1 M NaCl, as described by Teather and Wood (53). Hydroxyethylcellulose dyed with Ostazin Brilliant Red H-3B (OBR-cellulose; see reference 5) was used in the top agar at a concentration of 0.1% for secondary and tertiary screening of the clones.
MATERIALS AND METHODS
Bacterial strains and cloning vectors. The Bacillus strains used in this study and the temperatures that they were grown at are listed in Table 1. All Bacillus species were initially *
Protein analysis. Intracellular and extracellular proteins from recombinant E. coli were electrophoresed in denaturing gels by the method of Laemmli (27), by using a Mini Protean II Dual Slab cell electrophoresis apparatus (Bio-Rad Laboratories). To obtain intracellular proteins, cells were pelleted and washed twice in 1/10 the original cell volume and then suspended in the same volume of Tris hydrochloride-EDTA
Corresponding author.
t National Research Council of Canada publication 31,100. t Present address: 20 Julian Ave., Ottawa, Ontario, Canada K1Y OSS.
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TABLE 1. Bacillus species and strains screened for endoglucanase activity Species and Temp Other designation Activity NRCC strain' (OC) 30 B. alvei 9062 37 B. amyloliquifaciens 2147 USDA 616 37 B. brevisb 9084 30 B. cereusb 9003 + USDA 729 30 B. circulansb 9024 37 B. coagulans 2851 37 B. licheniformisb 9012 37 B. macerans 9074 30 B. megaterium 2852 30 B. pantothenticus 9026 + 30 B. polymyxab 2822 37 B. pumilusb 9066 30 B. sphaericus 9027 B. stearothermophilus 9001 55 + ATCC 6051 B. subtilisb 3052 37 + USDA 231 37 9041 37 9044 + 37 9045 + 9048 37 + 37 9049 + USDA 703 9051 37 + USDA 1003 9052 37 + 37 9053 + 37 9057 + 37 Univ. of Alberta 9058 strain 231 + 37 PAP115 5990 B. thuringiensis subsp. 37 kurstaki 2278 a NRCC, National Research Council of Canada. b Species reported to have strains which produce cellulase.
(0.01 M Tris hydrochloride-0.001 M EDTA, pH 8). The cell mixture was added to an equal volume of 2X Laemmli loading buffer. Cell lysis was completed by heating the samples for 5 min in a bath of boiling water before loading them onto the gel. Extracellular proteins were prepared and concentrated by ethanol precipitation by the method of Petre et al. (39) or by lyophilization. In the latter case, the lyophilate was resuspended in Tris hydrochloride-EDTA at 1/50 the original cell culture volume. Samples (100 ,ul) were then passed through two spin columns of Bio-Gel P6 polyacrylamide beads (Bio-Rad) equilibrated with 0.01 M sodium acetate, pH 6.0. The solution was lyophilized again and then suspended in 15 ,ul of Tris hydrochloride-EDTA. DNA Analysis. All DNA electrophoresis was carried out by using 0.7% agarose and Tris borate buffer (31). DNA was transferred from gels by alkaline blotting (43) onto Biotrans (ICN Pharmaceuticals Inc.) and examined by Southern analysis (52). Radioactively labeled probes were prepared via nick translation (31). The DNA fragments to be used for probes were prepared by either the S1 nuclease protection assay (see below) or by gel electrophoresis and transferred to DEAE paper (11). Hybridization of the probes to the various membranes was performed in the presence of 200 ,ug of sheared salmon sperm DNA heated in 1% sodium dodecyl sulfate (SDS) and 1 M NaCl per ml for 20 h at 65°C. Washes followed the Biotrans protocol. Ordered deletion clones for sequencing were prepared by the method of Dale et al. (9). Clones were sequenced by using the dideoxynucleotide chain termination method and [ot-S35]dATP label (6, 35) with either the E. coli Klenow fragment or the modified T7 DNA polymerase sequenase
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(United States Biochemical). Manipulation and analysis of DNA sequence data as well as the protein comparisons were performed with the IBI sequence analysis programs (41, 42) and the National Research Council of Canada COMPMOLGEN package of programs. The statistical significance of the protein comparisons was calculated by using the SEQDP program of the IDEAS package (22). Si nuclease protection assay. Hybridization of M13 singlestranded phage DNA carrying complementary strands of cloned DNA was carried out at 65°C for 20 min in S1 nuclease buffer, as described by Maniatis et al. (31). Subsequent digestion of the single-stranded M13 phage DNA portions was done at 37°C for 20 min by using 0.5 to 1 U of S1 nuclease (Sigma) per ,ug of DNA. The reactions were stopped by the addition of EDTA to a final concentration of 20 mM. When necessary, the nucleotides were removed and the buffer was changed by passage through two spin columns of Bio-Rad G50 agarose beads (31). RESULTS Screening for endoglucanases produced by BaciUlus spp. Sixteen species of Bacillus and 10 strains ofB. subtilis (Table 1) were examined for endoglucanase activity by plating on media containing 0.1% CM-cellulose and grown to a minimum colony size of 5 mm. Detection of activity was visualized because of the lack of Congo red dye adhering to hydrolyzed CM-cellulose in the vicinity of each colony (53). Only B. circulans, B. polymyxa, and all the strains of B. subtilis, with the exception of strain NRCC 9044, displayed activity. The genomic DNAs of the 16 species were digested with PstI and analyzed by the Southern method by using a 3.1-kilobase-pair (kbp) PstI fragment which contains the complete gene for the B. subtilis endocellulase (30, 49). Positive hybridization to the PstI fragment was detected in DNAs from B. circulans and all B. subtilis strains except NRCC 9044. Isolation of endoglucanase genes by expression in M13 phage. A preliminary cloning of genomic DNA was performed by using PstI-cut B. subtilis (NRCC 3052) DNA and PstI-cleaved M13mp9. With equivalent weights of genomic DNA and vector added to the ligation mixture, 1 in 5 x 103 to 1 in 6 x 103 plaques containing inserts gave haloes after staining with Congo red. It was found that phage recovered from halo-producing plaques were still viable following staining with Congo red and destaining with 1 M NaCl. This method was then used to clone the B. polymyxa and B. circulans endoglucanase genes except that the genomic DNAs used for ligation to vector DNA were from partial PstI digestions in case PstI cleaved the genes. Although initial plates were confluent (-75 to 100 plaques per cm2), haloes were still detectable around active clones when the Congo red assay was used. Since staining and destaining of plates can mix phage from all the plaques on the plate, picks of positive clones were rarely pure. To avoid this problem in a secondary screening, we used OBR-cellulose (5) in the top agar for direct detection of activity. This method was not used for primary screening because the visual contrast of the background color to the clear halo is not very intense. Two CR+ clones (Bp-P1 and Bp-P2) were isolated from the B. polymyxa bank of approximately 104 clones. Clone Bp-P1 contained a 1.6-kbp single PstI DNA fragment, whereas clone Bp-P2 contained the same 1.6-kbp fragment and a 0.2-kbp PstI DNA fragment. The removal of the 0.2-kbp DNA from Bp-P2 had no effect on activity. The orientation of the 1.6-kbp PstI DNA fragments relative to the
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transcription of the P-galactosidase--cx-peptide coding gene in M13mp9 was the same in both clones. Three CR+ clones (Bc-P1, Bc-P2, and Bc-P3) were isolated from 104 clones
from the B. circulans bank. All three clones contained the same 3. 1-kbp PstI DNA fragment in the same orientation as the oa-peptide gene. Two CR+ clones (pCH.7 and pCH.9) were also recovered from a B. circulans bank constructed from genomic DNA partially digested with HindIll and from the fully digested pUC8 plasmid. Both of these plasmids contained a 3.4-kbp HindIII fragment with a single PstI site approximately 0.3 kbp from one end, indicating that the same genomic section of DNA was cloned in each case. DNA characterization of clones. The B. polymyxa 1.6-kbp PstI and the B. circulans 3.4-kbp HindIII DNA fragments were subcloned into M13mpl9 in the opposite orientation to that in which they were originally found (B. polymyxa Bp-opl9 and B. circulans Bc-H4). Since the B. circulans gene is expressed in both orientations, this gene most likely has its own E. coli-like promoter sequence(s), as was found previously for the B. subtilis endocellulase gene (30, 49). However, this is not the case for the B. polymyxa gene, since expression of this gene occurred only in the same orientation as the promoter of the P-galactosidase o-peptide gene. Further subcloning of the 1.6-kbp PstI fragment into the reading frame shift vectors (pUC9.0, pUC9.1, and pUC9.2 [20]) indicated that only pUC9.0 gave activity. This vector has exactly the same reading frame as that of M13mp9, which means that expression of the B. polymyxa endoglucanase gene depends on both the lac promoter as well as the ATG codon of the lacZ ot-peptide. The DNA hybridization analysis shown in Fig. 1A and B indicates that the B. polymyxa gene is not related to the genes of B. circulans and B. subtilis. The presence of a single fragment in both the lanes of HindIll- and PstI-digested B. polymyxa genomic DNA in Fig. 1B suggests that this is also a single-copy gene. In contrast, B. circulans and B. subtilis have homologous fragments that are distinguishable in Fig. 1A only by a HindIII site located immediately downstream of the TGA stop codon of the B. subtilis endocellulase gene (30). Since S1 nuclease digestion of the hybrid formed between M13 B. circulans clone Bc-H4 and M13 B. subtilis clone Bs-P3.2 (which contains the putative complementary sequence in the 3.1-kbp PstI fragment [30]) shown in Fig. 2 produces a single 3-kbp fragment, this suggests that there are no large sequence differences between the two clones. The restriction endonuclease maps of the endoglucanase genes from the three Bacillus species are presented in Fig. 1C. These maps were constructed from the Southern analysis (Fig. 1A and B) and the nucleotide sequence of the B. polymyxa (see Fig. 6 and 7) and B. subtilis (30) genes. Expression of the cloned genes. For comparative studies of the endocellulases, we found that the high-copy pUC plasmid vectors gave significantly better yields than the M13 vectors. The plasmids of each of the three Bacillus endocellulase clones were transformed into E. coli HB101 instead of the standard JM series used for pUC vectors. This was done to avoid possible experimental error due to an unexplained low-level activity observed with the CM-cellulose viscosity assay with E. coli JM103 (30) but not with HB101 (A. Lo,
personal communication). The pUC clones were grown overnight in 2x yeast tryp-
tone, and the intracellular and extracellular proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) as shown in Fig. 3. Two similarly sized protein bands measuring 52 and 35 kilodaltons (kDa), respectively, were produced by both the B. circulans endoglucanase clone
FIG. 1. Comparative mapping of endoglucanase genes. (A) Results of a Southern blot hybridized with the B. circulans 3.4-kbp HindIII fragment carrying the endoglucanase gene. (B) Results of a second blot hybridized with the 1.6-kbp Pstl fragment encoding the B. polymyxa endoglucanase gene. Genomic DNAs of B. subtilis PAP115 (lane Bs), B. circulans (lane Bc), and B. polymyxa (lane Bp) were digested with Hindlll (H), EcoRI (E), or Pstl (P). (C) Restriction site maps of the three genes were constructed from the data of the type shown in panels A and B and sequence data from Fig. 6 and 7. Arrows denote the putative region and direction of transcription of genes. The location and direction of transcription of the B. subtilis gene were described by MacKay et al. (29). ORF2 upstream of the B. polymyxa endoglucanase gene refers to a second ORF which was found in the sequence data shown in Fig. 6 and is possibly transcribed.
pCH.9 and the B. subtilis clone pC6.5,
as indicated in Fig. 3A and C. The sizes of these bands are in agreement with published results for pC6.5 (29). Figures 3A and C also show that the plasmid pC2.2 encoding the B. polymyxa endoglucanase gene produced a single major 37-kDa protein, which is mainly cell associated. Figure 3B shows the proteins in the supernatant that have been precipitated with EtOH, whereas Fig. 3C shows the extracellular proteins concentrated by lyophilization. The B. circulans endoglucanase was selectively precipitated by EtOH, but the B. polymyxa endoglucanase was not. The endoglucanase of B. circulans shown in Fig. 3B was also selectively precipitated relative to E. coli proteins, since both the 52- and 35-kDa bands appear relatively much darker by Coomassie staining than in Fig. 3C. The B. subtilis endoglucanase was precipitated by EtOH, just as was the B. circulans endoglucanase (data not shown). This result suggests that the B. circulans and B. subtilis endocellulases may be more hydrophilic than most of the E. coli proteins and the B. polymyxa endocellulase. It was not possible to recover activity of any of the endoglucanases from E. coli or from the original Bacillus hosts after electro-
VOL. 172, 1990
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Intracellular bp
A 'JC 3 pC2.2
pC0-zQ pCH.7
200-
97
68
'7250
'3584
52
43-
'2686
'1809
37 la
25 7-
FIG. 2. Homology between B. circulans and B. subtilis endoglucanase genes as determined by Si nuclease protection. Singlestranded DNAs of M13 clones were annealed together, digested with S1 nuclease, and electrophoresed as described in Materials and Methods. Lanes: Bc, B. circulans M13 clone Bc-H4 annealed to the oppositely oriented B. circulans clone Bc-HI; Bc/Bs, B. circulans clone Bc-HI hybridized to the B. subtilis clone SBO (29); M, marker DNA composed of linearized M13mp7, pBR325, pBR328, pUC8, and pBR322 digested with either PstI and HindIll, BglI, or TaqI. bp, Base pairs.
phoresis of the SDS-denatured proteins by the method of Beguin (2). Activity was retained if the samples were not heated before loading, but the resulting zymograms did not correlate with the sizes of the fully denatured proteins (data not shown). Cloning the 5' end of the B. polymyxa endoglucanase gene. It was evident from the first nucleotide sequencing results of the B. polymyxa 1.6-kbp PstI DNA fragment that part of the 5' end of the endoglucanase gene was missing. A large open reading frame (ORF) of 369 codons was found to be in frame with the first 9 codons of the lacZ a-peptide ORF of the vectors mp9 and pUC9 (55). The exact fusion sequence is given in Fig. 4A along with an example of increased endoglucanase production by inducing the lac operon with isopropyl-p-D-thiogalactopyranoside (IPTG) (Fig. 4B and C). A 2.35-kbp EcoRI fragment, which was expected to contain the 5' end of the endoglucanase gene (Fig. 1), was cloned in order to fuse it with the already cloned portion of the gene. Genomic DNA digested with EcoRI in the size range of 2 to 2.8 kbp was ligated with the dephosphorylated EcoRI M13mpl9 replicative form. After transfection in E. coli JM103, the plaques were screened with a radioactive DNA probe made from the 800-base-pair PstI-EcoRI fragment located at the 5' end of the previously cloned PstI fragment. Two of the six clones (Bp-E1.2 and Bp-E2.1) recovered from this screening were shown to have the 2.35-kbp EcoRI fragment. When cut with PstI, this EcoRI fragment gave an 820base-pair fragment, which contained most of the coding region of the endoglucanase gene, and a 1.53-kbp piece of DNA, which coded for 87 nucleotides of the 5' region of the protein coding sequence plus the putative promoter region of the gene (see Fig. 6 and 7). The 1.53-kbp PstI-EcoRI fragment was ligated to the original 1.6-kbp PstI fragment and fractionated by electrophoresis on a preparative gel in order to purify the desired 3.1-kbp product. This 3.1-kbp EcoRI-PstI fragment was cloned by using both mpl8 and mpl9 vectors. Single-stranded DNAs of these clones were analyzed by the Si nuclease protection assay using the
18.7-
_,
Extracellular
EtOH ppt.
B M
pUIC8 pC22 pC-H7
C
Lyophilized pUC 8 pC2.2 pCH ,' ;co 5
FIG. 3. Analysis of cell-associated and extracellular proteins from E. coli cells expressing the endoglucanase genes of B. subtilis, B. circulans, or B. polymyxa. Cell-associated and extracellular protein samples were prepared as described in Materials and Methods. (A) Intracellular endoglucanase gene products. Sizes of the gene products (marked with dots in all panels) are indicated on the right, and sizes of the protein size markers are indicated on the left. (B) Proteins precipitated from culture supernatants with EtOH. (C) Proteins recovered from lyophilized supematants. Twenty times more cell culture was required for samples loaded in gels of panels B and C than panel A. Lanes: M, high-molecular-mass BRL protein size markers; pC2.2, pUC9 plus the B. polymyxa 1.6-kbp Pstl fragment; pC6.5, pUC8 plus the B. subtilis 3.1-kbp PstI fragment; pCH7, pUC8 plus the B. circulans 3.4-kbp Hindlll fragment.
2.35-kbp EcoRI cloned fragment (i.e., Bp-E1.2 and the oppositely oriented Bp-E4.1) to show that the 5' and 3' fragments were indeed in the proper orientation to each other. Correct orientation gave only one 2.35-kbp DNA fragment (Fig. 5). If the orientation of either the 3' or 5' fragment respective to the other was incorrect, no fragment or either the 1.53-kbp or 0.8-kbp fragment would be recovered after S1 nuclease treatment. As a control, the 1.6-kbp PstI fragment used for this experiment was also ligated into pUC9 to show that it could still confer activity. Although the 1.6-kbp PstI-pUC9 clones exhibited activity, none of the approximately 103 resulting clones from the 5'-3' gene fusion expressed endoglucanase activity either in the presence or absence of IPTG. DNA sequencing of the complete B. polymyxa endoglucanase gene. Both the 1.6-kbp PstI fragment and the nonoverlapping
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FIG. 4. B. polymyxa endoglucanase 5'-end fusion to the lacZ a-peptide. (A) Fusion sequences the endoglucanase and az-peptide genes. The 5' end of the a-peptide gene ends at the PstI site, with the amino acids for this region in bold type. The B. polymyxa DNA, which starts at the PstI site, codes for a 369-amino-acid ORF that is in frame with the 5' end of the lacZ a-peptide gene. (B) Bp-P1 and E. coli JM101 plated with B media and CM-cellulose with or without the gratuitous inducer of the lac operon, IPTG.
section of the 2.35-EcoRI fragment were sequenced completely in both orientations. Most of the sequences were obtained from 3' deletion clones constructed by the method of Dale et al. (9). (i) 5' Region of DNA fragment. The DNA sequence 5' of the putative B. polymyxa endoglucanase gene is shown in Fig. 6 in its reverse complement. Upstream of the endoglucanase gene, there is a second ORF; this putative gene, ORF2, would be transcribed in the opposite direction to the endoglucanase gene. The translation of this putative 187amino-acid protein would begin at an ATG start codon at nucleotide 1064 and end at nucleotide 511. Figure 6 gives the DNA sequence, the putative translation product, and the regulatory sequences of ORF2. The amino acid sequence has also been used to search the NBRF and SWIS/PROT protein data bases. A 28% similarity was found with the first 186 amino acids of the E. coli lac repressor protein. This suggests that this protein may be a DNA-binding protein with some regulatory function of the endoglucanase gene or some other distal gene. The ORF2 sequence has a typical bacterial ribosomebinding site 5' of the start codon and -10 and -35 sequences that are similar to the major vegetative promoter sequences of E. coli and B. subtilis. There are also -10 and -35 sequences similar to the consensus recognition sequences for B. subtilis cr29 (21). In the 3' region, there is an imperfect
inverted repeat that could possibly function as a rho-independent transcription site (40). The DNA sequence of the endoglucanase gene, including some of the 5' sequence that overlaps with that in Fig. 6, is shown in Fig. 7. A typical Shine-Delgarno ribosome-binding site, GGAGG, was found 11 bases upstream from the putative ATG start codon. The 300 bases extending from the start codon were searched for consensus and known variants of the -10 and -35 sequences recognized by E. coli and B. subtilis RNA polymerases (3, 7, 16, 21, 38, 51, 54). The most E. coli-like sequences for the -10 and -35 positions marked in Fig. 7 are probably too far apart to be used by an E. coli promoter. The lack of typical -10 and -35 E. coli promoter sequences is in agreement with the lack of expression of the reconstituted B. polymyxa endoglucanase gene. A sequence resembling the -10 and -35 regions of the B. subtilis ur32 recognition sites is also marked. It is not known whether the variation of the recognition sites that exist in the B. polymyxa sequence would allow a B. subtilis polymerase with the u32 factor to transcribe the gene. (ii) Protein coding region and DNA sequence 3' to the gene. The PstI site which defines the 5' end of the original expression clone (M13 Bp-P1 or pC2.2) is marked at amino acid positions 29 to 31 in Fig. 7. The 28 amino acid codons 5' to the PstI site appear to be part of a putative secretion signal sequence. Comparison of this sequence to the B. polymyxa
B. POLYMYXA AND B. CIRCULANS ENDOGLUCANASE GENES
VOL. 172, 1990
A c
d
e
f
kbp
2.31.5-
0.8-
B 1
2 3 a b c d e
f
2.3-
0.8-
S1
FIG. 5. nuclease protection of B. polymyxa endoglucanase 5'-to-3' gene fusion clones. Two mpl8 (lanes a and b) and four mpl9 (lanes c to f) 5'-to-3' fusion clones were hybridized to either the B. polymyxa 2.35-kbp EcoRI clone E1.2 (A) or the oppositely oriented clone E4.1 (B) and digested with Si nuclease. The possible resulting sizes are as follows: 1.5-kbp fragment used to construct 5' regions of the gene (lane 1); 2.35 kbp for the Si nuclease protection assay of E2.1 and E4.1, the expected result of a proper fusion (lane 2); and S1 nuclease protection assay of the 2.35-kbp EcoRI clone E2.1 and the oppositely oriented 1.6-kbp PstI clone Bp-opl9, giving an 0.8-kbp overlap (lane 3). The clone in lane f is the only one that was not in the proper orientation.
,-amylase secretion signal sequence (23) indicates that both exhibit a similar distribution of charged and hydrophobic amino acids comparable to signal sequences of B. subtilis (30). The highest similarity of the putative B. polymyxa endoglucanase amino acid sequence with another protein was to the C. thermocellum CelB endoglucanase. An alignment was made by the MATCH program of these two sequences (Fig. 8). Direct matches are boxed. The overall similarity to the B. subtilis endoglucanase was very low, which agrees with the DNA results. No simple transcription termination sequence of the inverted repeat type followed by six to eight thymines (40) was found. Three inverted repeats were found in the 3' untranslated region and have been marked in Fig. 7. There is a possibility that transcription is not terminated properly by sequences
any sequence
of this DNA fragment when in E. coli. DISCUSSION
Derivatives of the phage M13 have been widely used in secondary cloning because of many useful DNA manipulation techniques, which include site-specific mutagenesis, single-stranded probe preparation, C-test determination of fragment orientation without DNA purification, isolation of heterologous duplex DNA by Si nuclease protection, and
1581
the dideoxy-chain termination method of sequencing. To our knowledge, this is the first report of the use of an M13 vector for cloning genes such as the endo-4-1,4-glucanase genes from B. circulans and B. polymyxa from genomic banks by direct expression of their enzyme products. The two substrates employed for direct screening of endoglucanase expression on plates were easy to use and circumvented the need for replica plating. The use of OBR-cellulose for screening of endoglucanase clones is also a new application. However, other researchers in the past have used the Congo red assay to clone and express endoglucanase genes by plasmid vectors in E. coli (1, 12, 15, 24, 25, 34, 37, 45, 49, 50, 56) and once by the bacteriophage lambda (26). The lambda vector L47-1 was used to isolate cellulase genes from Erwinia chrysanthemi in E. coli because it would lyse the cells in the lytic cycle, releasing enzymes that might not have been secreted by E. coli (26). However, lysis of the E. coli cells is not necessary for detecting expression of the E. chrysanthemi cellulase genes (15) or any other of the cellulase genes already mentioned. From the DNA-DNA hybridization results shown in Fig. 1A it appears that both B. circulans and B. subtilis have very similar genomic organizations, at least in the vicinities of their respective endoglucanase genes. The endoglucanaserelated DNA fragments generated by digesting the genomic DNAs with PstI and EcoRI appear to be identical in size. The only difference is the extra HindIII site in the PAP115 genome located three nucleotides 3' from the stop codon of the endoglucanase gene and eight nucleotides from the inverted repeat, which may serve as the transcription termination site of the gene. Although the various assays we used to demonstrate similarity are not as precise as DNA sequencing, the B. circulans gene is most likely as close in homology to B. subtilis PAP115 as the two other B. subtilis P-1,4-endoglucanases genes reported so far. Compared with the 499 amino acids of the B. subtilis PAP115 endocellulase, the gene sequenced by Robson and Chambliss (44) differs by 42 amino acids and the gene sequenced by Nakamura et al. (33) differs by 31 amino acids. A xylanase gene from the same strain of B. circulans used in this work has recently been isolated and sequenced (57). We have compared the predicted amino acid sequence of this xylanase to one isolated from B. subtilis (35) and found very little difference between them (
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Molecular Cloning, Expression, and Characterization of Endo-3-1,4-Glucanase Genes from Bacillus polymyxa and Bacillus circulanst STEPHEN D. BAIRD,1'2t DOUGLAS A. JOHNSON,' AND VERNER L. SELIGY2* Department of Biology, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5,1 and Molecular Genetics Section, Division of Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada KIA 0R62 Received 11 August 1989/Accepted 11 December 1989
Endo-0-1,4-glucanase genes from Bacillus circulans and from B. polymyxa were cloned by direct expression by using bacteriophage M13mp9 as the vector. The enzymatic activity of the gene products was detected by using either the Congo red assay or hydroxyethyl cellulose dyed with Ostazin Brilliant Red H-3B. The B. circulans and B. subilis PAP115 endo-0-1,4-glucanase genes were shown to be homologous by the use of restriction endonuclease site mapping, DNA-DNA hybridization, Si nuclease digestion after heteroduplex formation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein products. Analysis of the nucleotide sequence of 3.1 kilobase pairs of cloned B. polymyxa DNA revealed two convergently transcribed open reading frames (ORFs) consisting of 398 codons (endoglucanase) and 187 codons (ORF2) and separated by 374 nucleotides. The coding region of the B. polymyxa endoglucanase gene would theoretically produce a 44-kilodalton preprotein. Expression of the B. polymyxa endoglucanase in Escherichia coli was due to a fusion of the endoglucanase gene at codon 30 with codon 9 of the lacZ oa-peptide gene. The B. polymyxa endoglucanase has 34% amino acid similarity to the Clostridium thermocellum ceiB endoglucanase sequence but very little similarity to endoglucanases from other BaciUus species. ORF2 has 28% amino acid similarity to the NH2-terminal half of the E. coli lac repressor protein, which is responsible for DNA binding. Cellulose is degraded to varying extents by a variety of organisms, depending upon the properties of the cellulosic substrate and the types of glycosidases produced (8). Highly efficient cellulolytic bacteria and fungi generally produce one or more enzymes from each of the three classes of enzymes required to degrade microcrystalline cellulose to glucose. In contrast, members of the industrially important Bacillus bacteria are generally not considered to be cellulolytic organisms because none of them have so far revealed a complete cellulase enzyme complex needed to degrade native cellulose. Several related endo-,B-1,4-glucanase genes have been isolated and sequenced from different species of the Bacillus genus (14, 24, 25, 28, 30, 44, 48-50). To extend this database further, we screened 16 additional Bacillus species for endo-P-1,4-glucanase genes and found that, aside from B. subtilis, only B. circulans and B. polymyxa have such genes. Here we show that the cloned B. polymyxa endoglucanase gene is not similar to the B. subtilis gene or the B. circulans gene that we also cloned. Nucleotide sequence comparisons involving all other available cellulase genes revealed that the B. polymyxa gene is related to the celB gene of Clostridium thermocellum, therefore representing a new class of Bacillus endoglucanases.
screened for cellulolytic activity by growth on agar plates containing 0.01% (wt/vol) carboxymethyl cellulose (CMcellulose; degree of polymerization, 7; medium viscosity; Sigma Chemical Co.) and Luria Bertanini medium. Escherichia coli JM103 for bacteriophage M13 propagation and expression was grown by the method of Messing (32). E. coli HB101 was grown with Luria Bertanini medium unless otherwise stated. All media used for the maintenance of pUC plasmids in E. coli contained 35 ,ug of ampicillin per ml. Cloning methodology. The Bacillus genomic DNA was prepared as described by Saito and Miura (46). RNA was removed from the DNA preparations with RNase A treatment followed by ethanol (EtOH) precipitation. The plasmid pUC9 and the replicative form of bacteriophage M13 were prepared as described by Maniatis et al. (31). Partially digested genomic DNA was ligated to the linearized replicative form of M13mp9 and transformed into E. coli by the method of Hanahan (19), but without incubation of the transformed cells at 37°C before plating. To detect cellulaseproducing, Congo red-positive M13 (CR+) clones, 0.1% CM-cellulose was used in the top agar. Activity was determined by flooding the agar plate with 10 ml of Congo red (1 mglml) for 15 min and destaining it with 1 M NaCl, as described by Teather and Wood (53). Hydroxyethylcellulose dyed with Ostazin Brilliant Red H-3B (OBR-cellulose; see reference 5) was used in the top agar at a concentration of 0.1% for secondary and tertiary screening of the clones.
MATERIALS AND METHODS
Bacterial strains and cloning vectors. The Bacillus strains used in this study and the temperatures that they were grown at are listed in Table 1. All Bacillus species were initially *
Protein analysis. Intracellular and extracellular proteins from recombinant E. coli were electrophoresed in denaturing gels by the method of Laemmli (27), by using a Mini Protean II Dual Slab cell electrophoresis apparatus (Bio-Rad Laboratories). To obtain intracellular proteins, cells were pelleted and washed twice in 1/10 the original cell volume and then suspended in the same volume of Tris hydrochloride-EDTA
Corresponding author.
t National Research Council of Canada publication 31,100. t Present address: 20 Julian Ave., Ottawa, Ontario, Canada K1Y OSS.
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TABLE 1. Bacillus species and strains screened for endoglucanase activity Species and Temp Other designation Activity NRCC strain' (OC) 30 B. alvei 9062 37 B. amyloliquifaciens 2147 USDA 616 37 B. brevisb 9084 30 B. cereusb 9003 + USDA 729 30 B. circulansb 9024 37 B. coagulans 2851 37 B. licheniformisb 9012 37 B. macerans 9074 30 B. megaterium 2852 30 B. pantothenticus 9026 + 30 B. polymyxab 2822 37 B. pumilusb 9066 30 B. sphaericus 9027 B. stearothermophilus 9001 55 + ATCC 6051 B. subtilisb 3052 37 + USDA 231 37 9041 37 9044 + 37 9045 + 9048 37 + 37 9049 + USDA 703 9051 37 + USDA 1003 9052 37 + 37 9053 + 37 9057 + 37 Univ. of Alberta 9058 strain 231 + 37 PAP115 5990 B. thuringiensis subsp. 37 kurstaki 2278 a NRCC, National Research Council of Canada. b Species reported to have strains which produce cellulase.
(0.01 M Tris hydrochloride-0.001 M EDTA, pH 8). The cell mixture was added to an equal volume of 2X Laemmli loading buffer. Cell lysis was completed by heating the samples for 5 min in a bath of boiling water before loading them onto the gel. Extracellular proteins were prepared and concentrated by ethanol precipitation by the method of Petre et al. (39) or by lyophilization. In the latter case, the lyophilate was resuspended in Tris hydrochloride-EDTA at 1/50 the original cell culture volume. Samples (100 ,ul) were then passed through two spin columns of Bio-Gel P6 polyacrylamide beads (Bio-Rad) equilibrated with 0.01 M sodium acetate, pH 6.0. The solution was lyophilized again and then suspended in 15 ,ul of Tris hydrochloride-EDTA. DNA Analysis. All DNA electrophoresis was carried out by using 0.7% agarose and Tris borate buffer (31). DNA was transferred from gels by alkaline blotting (43) onto Biotrans (ICN Pharmaceuticals Inc.) and examined by Southern analysis (52). Radioactively labeled probes were prepared via nick translation (31). The DNA fragments to be used for probes were prepared by either the S1 nuclease protection assay (see below) or by gel electrophoresis and transferred to DEAE paper (11). Hybridization of the probes to the various membranes was performed in the presence of 200 ,ug of sheared salmon sperm DNA heated in 1% sodium dodecyl sulfate (SDS) and 1 M NaCl per ml for 20 h at 65°C. Washes followed the Biotrans protocol. Ordered deletion clones for sequencing were prepared by the method of Dale et al. (9). Clones were sequenced by using the dideoxynucleotide chain termination method and [ot-S35]dATP label (6, 35) with either the E. coli Klenow fragment or the modified T7 DNA polymerase sequenase
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(United States Biochemical). Manipulation and analysis of DNA sequence data as well as the protein comparisons were performed with the IBI sequence analysis programs (41, 42) and the National Research Council of Canada COMPMOLGEN package of programs. The statistical significance of the protein comparisons was calculated by using the SEQDP program of the IDEAS package (22). Si nuclease protection assay. Hybridization of M13 singlestranded phage DNA carrying complementary strands of cloned DNA was carried out at 65°C for 20 min in S1 nuclease buffer, as described by Maniatis et al. (31). Subsequent digestion of the single-stranded M13 phage DNA portions was done at 37°C for 20 min by using 0.5 to 1 U of S1 nuclease (Sigma) per ,ug of DNA. The reactions were stopped by the addition of EDTA to a final concentration of 20 mM. When necessary, the nucleotides were removed and the buffer was changed by passage through two spin columns of Bio-Rad G50 agarose beads (31). RESULTS Screening for endoglucanases produced by BaciUlus spp. Sixteen species of Bacillus and 10 strains ofB. subtilis (Table 1) were examined for endoglucanase activity by plating on media containing 0.1% CM-cellulose and grown to a minimum colony size of 5 mm. Detection of activity was visualized because of the lack of Congo red dye adhering to hydrolyzed CM-cellulose in the vicinity of each colony (53). Only B. circulans, B. polymyxa, and all the strains of B. subtilis, with the exception of strain NRCC 9044, displayed activity. The genomic DNAs of the 16 species were digested with PstI and analyzed by the Southern method by using a 3.1-kilobase-pair (kbp) PstI fragment which contains the complete gene for the B. subtilis endocellulase (30, 49). Positive hybridization to the PstI fragment was detected in DNAs from B. circulans and all B. subtilis strains except NRCC 9044. Isolation of endoglucanase genes by expression in M13 phage. A preliminary cloning of genomic DNA was performed by using PstI-cut B. subtilis (NRCC 3052) DNA and PstI-cleaved M13mp9. With equivalent weights of genomic DNA and vector added to the ligation mixture, 1 in 5 x 103 to 1 in 6 x 103 plaques containing inserts gave haloes after staining with Congo red. It was found that phage recovered from halo-producing plaques were still viable following staining with Congo red and destaining with 1 M NaCl. This method was then used to clone the B. polymyxa and B. circulans endoglucanase genes except that the genomic DNAs used for ligation to vector DNA were from partial PstI digestions in case PstI cleaved the genes. Although initial plates were confluent (-75 to 100 plaques per cm2), haloes were still detectable around active clones when the Congo red assay was used. Since staining and destaining of plates can mix phage from all the plaques on the plate, picks of positive clones were rarely pure. To avoid this problem in a secondary screening, we used OBR-cellulose (5) in the top agar for direct detection of activity. This method was not used for primary screening because the visual contrast of the background color to the clear halo is not very intense. Two CR+ clones (Bp-P1 and Bp-P2) were isolated from the B. polymyxa bank of approximately 104 clones. Clone Bp-P1 contained a 1.6-kbp single PstI DNA fragment, whereas clone Bp-P2 contained the same 1.6-kbp fragment and a 0.2-kbp PstI DNA fragment. The removal of the 0.2-kbp DNA from Bp-P2 had no effect on activity. The orientation of the 1.6-kbp PstI DNA fragments relative to the
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transcription of the P-galactosidase--cx-peptide coding gene in M13mp9 was the same in both clones. Three CR+ clones (Bc-P1, Bc-P2, and Bc-P3) were isolated from 104 clones
from the B. circulans bank. All three clones contained the same 3. 1-kbp PstI DNA fragment in the same orientation as the oa-peptide gene. Two CR+ clones (pCH.7 and pCH.9) were also recovered from a B. circulans bank constructed from genomic DNA partially digested with HindIll and from the fully digested pUC8 plasmid. Both of these plasmids contained a 3.4-kbp HindIII fragment with a single PstI site approximately 0.3 kbp from one end, indicating that the same genomic section of DNA was cloned in each case. DNA characterization of clones. The B. polymyxa 1.6-kbp PstI and the B. circulans 3.4-kbp HindIII DNA fragments were subcloned into M13mpl9 in the opposite orientation to that in which they were originally found (B. polymyxa Bp-opl9 and B. circulans Bc-H4). Since the B. circulans gene is expressed in both orientations, this gene most likely has its own E. coli-like promoter sequence(s), as was found previously for the B. subtilis endocellulase gene (30, 49). However, this is not the case for the B. polymyxa gene, since expression of this gene occurred only in the same orientation as the promoter of the P-galactosidase o-peptide gene. Further subcloning of the 1.6-kbp PstI fragment into the reading frame shift vectors (pUC9.0, pUC9.1, and pUC9.2 [20]) indicated that only pUC9.0 gave activity. This vector has exactly the same reading frame as that of M13mp9, which means that expression of the B. polymyxa endoglucanase gene depends on both the lac promoter as well as the ATG codon of the lacZ ot-peptide. The DNA hybridization analysis shown in Fig. 1A and B indicates that the B. polymyxa gene is not related to the genes of B. circulans and B. subtilis. The presence of a single fragment in both the lanes of HindIll- and PstI-digested B. polymyxa genomic DNA in Fig. 1B suggests that this is also a single-copy gene. In contrast, B. circulans and B. subtilis have homologous fragments that are distinguishable in Fig. 1A only by a HindIII site located immediately downstream of the TGA stop codon of the B. subtilis endocellulase gene (30). Since S1 nuclease digestion of the hybrid formed between M13 B. circulans clone Bc-H4 and M13 B. subtilis clone Bs-P3.2 (which contains the putative complementary sequence in the 3.1-kbp PstI fragment [30]) shown in Fig. 2 produces a single 3-kbp fragment, this suggests that there are no large sequence differences between the two clones. The restriction endonuclease maps of the endoglucanase genes from the three Bacillus species are presented in Fig. 1C. These maps were constructed from the Southern analysis (Fig. 1A and B) and the nucleotide sequence of the B. polymyxa (see Fig. 6 and 7) and B. subtilis (30) genes. Expression of the cloned genes. For comparative studies of the endocellulases, we found that the high-copy pUC plasmid vectors gave significantly better yields than the M13 vectors. The plasmids of each of the three Bacillus endocellulase clones were transformed into E. coli HB101 instead of the standard JM series used for pUC vectors. This was done to avoid possible experimental error due to an unexplained low-level activity observed with the CM-cellulose viscosity assay with E. coli JM103 (30) but not with HB101 (A. Lo,
personal communication). The pUC clones were grown overnight in 2x yeast tryp-
tone, and the intracellular and extracellular proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) as shown in Fig. 3. Two similarly sized protein bands measuring 52 and 35 kilodaltons (kDa), respectively, were produced by both the B. circulans endoglucanase clone
FIG. 1. Comparative mapping of endoglucanase genes. (A) Results of a Southern blot hybridized with the B. circulans 3.4-kbp HindIII fragment carrying the endoglucanase gene. (B) Results of a second blot hybridized with the 1.6-kbp Pstl fragment encoding the B. polymyxa endoglucanase gene. Genomic DNAs of B. subtilis PAP115 (lane Bs), B. circulans (lane Bc), and B. polymyxa (lane Bp) were digested with Hindlll (H), EcoRI (E), or Pstl (P). (C) Restriction site maps of the three genes were constructed from the data of the type shown in panels A and B and sequence data from Fig. 6 and 7. Arrows denote the putative region and direction of transcription of genes. The location and direction of transcription of the B. subtilis gene were described by MacKay et al. (29). ORF2 upstream of the B. polymyxa endoglucanase gene refers to a second ORF which was found in the sequence data shown in Fig. 6 and is possibly transcribed.
pCH.9 and the B. subtilis clone pC6.5,
as indicated in Fig. 3A and C. The sizes of these bands are in agreement with published results for pC6.5 (29). Figures 3A and C also show that the plasmid pC2.2 encoding the B. polymyxa endoglucanase gene produced a single major 37-kDa protein, which is mainly cell associated. Figure 3B shows the proteins in the supernatant that have been precipitated with EtOH, whereas Fig. 3C shows the extracellular proteins concentrated by lyophilization. The B. circulans endoglucanase was selectively precipitated by EtOH, but the B. polymyxa endoglucanase was not. The endoglucanase of B. circulans shown in Fig. 3B was also selectively precipitated relative to E. coli proteins, since both the 52- and 35-kDa bands appear relatively much darker by Coomassie staining than in Fig. 3C. The B. subtilis endoglucanase was precipitated by EtOH, just as was the B. circulans endoglucanase (data not shown). This result suggests that the B. circulans and B. subtilis endocellulases may be more hydrophilic than most of the E. coli proteins and the B. polymyxa endocellulase. It was not possible to recover activity of any of the endoglucanases from E. coli or from the original Bacillus hosts after electro-
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Intracellular bp
A 'JC 3 pC2.2
pC0-zQ pCH.7
200-
97
68
'7250
'3584
52
43-
'2686
'1809
37 la
25 7-
FIG. 2. Homology between B. circulans and B. subtilis endoglucanase genes as determined by Si nuclease protection. Singlestranded DNAs of M13 clones were annealed together, digested with S1 nuclease, and electrophoresed as described in Materials and Methods. Lanes: Bc, B. circulans M13 clone Bc-H4 annealed to the oppositely oriented B. circulans clone Bc-HI; Bc/Bs, B. circulans clone Bc-HI hybridized to the B. subtilis clone SBO (29); M, marker DNA composed of linearized M13mp7, pBR325, pBR328, pUC8, and pBR322 digested with either PstI and HindIll, BglI, or TaqI. bp, Base pairs.
phoresis of the SDS-denatured proteins by the method of Beguin (2). Activity was retained if the samples were not heated before loading, but the resulting zymograms did not correlate with the sizes of the fully denatured proteins (data not shown). Cloning the 5' end of the B. polymyxa endoglucanase gene. It was evident from the first nucleotide sequencing results of the B. polymyxa 1.6-kbp PstI DNA fragment that part of the 5' end of the endoglucanase gene was missing. A large open reading frame (ORF) of 369 codons was found to be in frame with the first 9 codons of the lacZ a-peptide ORF of the vectors mp9 and pUC9 (55). The exact fusion sequence is given in Fig. 4A along with an example of increased endoglucanase production by inducing the lac operon with isopropyl-p-D-thiogalactopyranoside (IPTG) (Fig. 4B and C). A 2.35-kbp EcoRI fragment, which was expected to contain the 5' end of the endoglucanase gene (Fig. 1), was cloned in order to fuse it with the already cloned portion of the gene. Genomic DNA digested with EcoRI in the size range of 2 to 2.8 kbp was ligated with the dephosphorylated EcoRI M13mpl9 replicative form. After transfection in E. coli JM103, the plaques were screened with a radioactive DNA probe made from the 800-base-pair PstI-EcoRI fragment located at the 5' end of the previously cloned PstI fragment. Two of the six clones (Bp-E1.2 and Bp-E2.1) recovered from this screening were shown to have the 2.35-kbp EcoRI fragment. When cut with PstI, this EcoRI fragment gave an 820base-pair fragment, which contained most of the coding region of the endoglucanase gene, and a 1.53-kbp piece of DNA, which coded for 87 nucleotides of the 5' region of the protein coding sequence plus the putative promoter region of the gene (see Fig. 6 and 7). The 1.53-kbp PstI-EcoRI fragment was ligated to the original 1.6-kbp PstI fragment and fractionated by electrophoresis on a preparative gel in order to purify the desired 3.1-kbp product. This 3.1-kbp EcoRI-PstI fragment was cloned by using both mpl8 and mpl9 vectors. Single-stranded DNAs of these clones were analyzed by the Si nuclease protection assay using the
18.7-
_,
Extracellular
EtOH ppt.
B M
pUIC8 pC22 pC-H7
C
Lyophilized pUC 8 pC2.2 pCH ,' ;co 5
FIG. 3. Analysis of cell-associated and extracellular proteins from E. coli cells expressing the endoglucanase genes of B. subtilis, B. circulans, or B. polymyxa. Cell-associated and extracellular protein samples were prepared as described in Materials and Methods. (A) Intracellular endoglucanase gene products. Sizes of the gene products (marked with dots in all panels) are indicated on the right, and sizes of the protein size markers are indicated on the left. (B) Proteins precipitated from culture supernatants with EtOH. (C) Proteins recovered from lyophilized supematants. Twenty times more cell culture was required for samples loaded in gels of panels B and C than panel A. Lanes: M, high-molecular-mass BRL protein size markers; pC2.2, pUC9 plus the B. polymyxa 1.6-kbp Pstl fragment; pC6.5, pUC8 plus the B. subtilis 3.1-kbp PstI fragment; pCH7, pUC8 plus the B. circulans 3.4-kbp Hindlll fragment.
2.35-kbp EcoRI cloned fragment (i.e., Bp-E1.2 and the oppositely oriented Bp-E4.1) to show that the 5' and 3' fragments were indeed in the proper orientation to each other. Correct orientation gave only one 2.35-kbp DNA fragment (Fig. 5). If the orientation of either the 3' or 5' fragment respective to the other was incorrect, no fragment or either the 1.53-kbp or 0.8-kbp fragment would be recovered after S1 nuclease treatment. As a control, the 1.6-kbp PstI fragment used for this experiment was also ligated into pUC9 to show that it could still confer activity. Although the 1.6-kbp PstI-pUC9 clones exhibited activity, none of the approximately 103 resulting clones from the 5'-3' gene fusion expressed endoglucanase activity either in the presence or absence of IPTG. DNA sequencing of the complete B. polymyxa endoglucanase gene. Both the 1.6-kbp PstI fragment and the nonoverlapping
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FIG. 4. B. polymyxa endoglucanase 5'-end fusion to the lacZ a-peptide. (A) Fusion sequences the endoglucanase and az-peptide genes. The 5' end of the a-peptide gene ends at the PstI site, with the amino acids for this region in bold type. The B. polymyxa DNA, which starts at the PstI site, codes for a 369-amino-acid ORF that is in frame with the 5' end of the lacZ a-peptide gene. (B) Bp-P1 and E. coli JM101 plated with B media and CM-cellulose with or without the gratuitous inducer of the lac operon, IPTG.
section of the 2.35-EcoRI fragment were sequenced completely in both orientations. Most of the sequences were obtained from 3' deletion clones constructed by the method of Dale et al. (9). (i) 5' Region of DNA fragment. The DNA sequence 5' of the putative B. polymyxa endoglucanase gene is shown in Fig. 6 in its reverse complement. Upstream of the endoglucanase gene, there is a second ORF; this putative gene, ORF2, would be transcribed in the opposite direction to the endoglucanase gene. The translation of this putative 187amino-acid protein would begin at an ATG start codon at nucleotide 1064 and end at nucleotide 511. Figure 6 gives the DNA sequence, the putative translation product, and the regulatory sequences of ORF2. The amino acid sequence has also been used to search the NBRF and SWIS/PROT protein data bases. A 28% similarity was found with the first 186 amino acids of the E. coli lac repressor protein. This suggests that this protein may be a DNA-binding protein with some regulatory function of the endoglucanase gene or some other distal gene. The ORF2 sequence has a typical bacterial ribosomebinding site 5' of the start codon and -10 and -35 sequences that are similar to the major vegetative promoter sequences of E. coli and B. subtilis. There are also -10 and -35 sequences similar to the consensus recognition sequences for B. subtilis cr29 (21). In the 3' region, there is an imperfect
inverted repeat that could possibly function as a rho-independent transcription site (40). The DNA sequence of the endoglucanase gene, including some of the 5' sequence that overlaps with that in Fig. 6, is shown in Fig. 7. A typical Shine-Delgarno ribosome-binding site, GGAGG, was found 11 bases upstream from the putative ATG start codon. The 300 bases extending from the start codon were searched for consensus and known variants of the -10 and -35 sequences recognized by E. coli and B. subtilis RNA polymerases (3, 7, 16, 21, 38, 51, 54). The most E. coli-like sequences for the -10 and -35 positions marked in Fig. 7 are probably too far apart to be used by an E. coli promoter. The lack of typical -10 and -35 E. coli promoter sequences is in agreement with the lack of expression of the reconstituted B. polymyxa endoglucanase gene. A sequence resembling the -10 and -35 regions of the B. subtilis ur32 recognition sites is also marked. It is not known whether the variation of the recognition sites that exist in the B. polymyxa sequence would allow a B. subtilis polymerase with the u32 factor to transcribe the gene. (ii) Protein coding region and DNA sequence 3' to the gene. The PstI site which defines the 5' end of the original expression clone (M13 Bp-P1 or pC2.2) is marked at amino acid positions 29 to 31 in Fig. 7. The 28 amino acid codons 5' to the PstI site appear to be part of a putative secretion signal sequence. Comparison of this sequence to the B. polymyxa
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A c
d
e
f
kbp
2.31.5-
0.8-
B 1
2 3 a b c d e
f
2.3-
0.8-
S1
FIG. 5. nuclease protection of B. polymyxa endoglucanase 5'-to-3' gene fusion clones. Two mpl8 (lanes a and b) and four mpl9 (lanes c to f) 5'-to-3' fusion clones were hybridized to either the B. polymyxa 2.35-kbp EcoRI clone E1.2 (A) or the oppositely oriented clone E4.1 (B) and digested with Si nuclease. The possible resulting sizes are as follows: 1.5-kbp fragment used to construct 5' regions of the gene (lane 1); 2.35 kbp for the Si nuclease protection assay of E2.1 and E4.1, the expected result of a proper fusion (lane 2); and S1 nuclease protection assay of the 2.35-kbp EcoRI clone E2.1 and the oppositely oriented 1.6-kbp PstI clone Bp-opl9, giving an 0.8-kbp overlap (lane 3). The clone in lane f is the only one that was not in the proper orientation.
,-amylase secretion signal sequence (23) indicates that both exhibit a similar distribution of charged and hydrophobic amino acids comparable to signal sequences of B. subtilis (30). The highest similarity of the putative B. polymyxa endoglucanase amino acid sequence with another protein was to the C. thermocellum CelB endoglucanase. An alignment was made by the MATCH program of these two sequences (Fig. 8). Direct matches are boxed. The overall similarity to the B. subtilis endoglucanase was very low, which agrees with the DNA results. No simple transcription termination sequence of the inverted repeat type followed by six to eight thymines (40) was found. Three inverted repeats were found in the 3' untranslated region and have been marked in Fig. 7. There is a possibility that transcription is not terminated properly by sequences
any sequence
of this DNA fragment when in E. coli. DISCUSSION
Derivatives of the phage M13 have been widely used in secondary cloning because of many useful DNA manipulation techniques, which include site-specific mutagenesis, single-stranded probe preparation, C-test determination of fragment orientation without DNA purification, isolation of heterologous duplex DNA by Si nuclease protection, and
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the dideoxy-chain termination method of sequencing. To our knowledge, this is the first report of the use of an M13 vector for cloning genes such as the endo-4-1,4-glucanase genes from B. circulans and B. polymyxa from genomic banks by direct expression of their enzyme products. The two substrates employed for direct screening of endoglucanase expression on plates were easy to use and circumvented the need for replica plating. The use of OBR-cellulose for screening of endoglucanase clones is also a new application. However, other researchers in the past have used the Congo red assay to clone and express endoglucanase genes by plasmid vectors in E. coli (1, 12, 15, 24, 25, 34, 37, 45, 49, 50, 56) and once by the bacteriophage lambda (26). The lambda vector L47-1 was used to isolate cellulase genes from Erwinia chrysanthemi in E. coli because it would lyse the cells in the lytic cycle, releasing enzymes that might not have been secreted by E. coli (26). However, lysis of the E. coli cells is not necessary for detecting expression of the E. chrysanthemi cellulase genes (15) or any other of the cellulase genes already mentioned. From the DNA-DNA hybridization results shown in Fig. 1A it appears that both B. circulans and B. subtilis have very similar genomic organizations, at least in the vicinities of their respective endoglucanase genes. The endoglucanaserelated DNA fragments generated by digesting the genomic DNAs with PstI and EcoRI appear to be identical in size. The only difference is the extra HindIII site in the PAP115 genome located three nucleotides 3' from the stop codon of the endoglucanase gene and eight nucleotides from the inverted repeat, which may serve as the transcription termination site of the gene. Although the various assays we used to demonstrate similarity are not as precise as DNA sequencing, the B. circulans gene is most likely as close in homology to B. subtilis PAP115 as the two other B. subtilis P-1,4-endoglucanases genes reported so far. Compared with the 499 amino acids of the B. subtilis PAP115 endocellulase, the gene sequenced by Robson and Chambliss (44) differs by 42 amino acids and the gene sequenced by Nakamura et al. (33) differs by 31 amino acids. A xylanase gene from the same strain of B. circulans used in this work has recently been isolated and sequenced (57). We have compared the predicted amino acid sequence of this xylanase to one isolated from B. subtilis (35) and found very little difference between them (
