Molecular characterization of a dimeric intracellular maltogenic amylase of Bacillus subtilis SUH4-2

An additional amylase besides the typical alpha-amylase was detected in the cytoplasm of Bacillus subtilis SUH4-2, an isolate from Korean soil. The corresponding gene encoded a maltogenic amylase, which hydrolyzed cyclodextrin or starch to maltose and glucose; pullulan to panose; acarbose to glucose and acarviosine-glucose. Maltogenic amylase of B. subtilis SUH4-2 transferred sugar molecules to form various branched oligosaccharides upon the hydrolysis of substrates. The enzyme existed in a monomer-dimer equilibrium with a molar ratio of 3:2 in 50 mM KH(2)PO(4)-NaOH buffer (pH 7.0). The maltogenic amylase is most likely to be associated with carbohydrate metabolism in the cytoplasm, since the nucleotide sequence of the gene was highly homologous to the yvdF gene of B. subtilis 168, which is located in a gene cluster involved in maltose/maltodextrin utilization.     

Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli

A gene corresponding to a maltogenic amylase (MAase) in Lactobacillus gasseri ATCC 33323 (lgma) was cloned and expressed in Escherichia coli. The recombinant LGMA was efficiently purified 24.3-fold by one-step Ni-NTA affinity chromatography. The final yield and specific activity of the purified recombinant LGMA were 68% and 58.7 U/mg, respectively. The purified enzyme exhibited optimal activity for beta-CD hydrolysis at 55 degrees C and pH 5. The relative hydrolytic activities of LGMA to beta-CD, soluble starch or pullulan was 8:1:1.9. The activity of LGMA was strongly inhibited by most metal ions, especially Zn(2+), Fe(2+), Co(2+) and by EDTA. LGMA possessed some unusual properties distinguishable from typical MAases, such as being in a tetrameric form, having hydrolyzing activity towards the alpha-(1,6)-glycosidic linkage and being inhibited by acarbose.     

Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and β-cyclodextrin (β-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and β-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched β-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the α-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.Bacillus subtilis can utilize polysaccharides such as starch, glycogen, and amylose as carbon sources by hydrolyzing them into smaller maltodextrins via the action of extracellular α-amylase (AmyE) (14). In B. subtilis, α-glucosidase encoded by malL has been known to contribute to maltodextrin metabolism in the cell (40, 41). Schönert et al. (42) reported that maltose is transported by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in B. subtilis. They also reported that maltodextrins with degrees of polymerization (DP) of 3 to 7 (G3 to G7) are taken up via a maltodextrin-specific (Mdx) ATP-binding cassette (ABC) transport system (42). This system is made up of a maltodextrin-binding protein (MdxE) and two membrane proteins (MdxF and MdxG), as well as an ATPase (MsmX). The basic model proposed for the transport and metabolism of maltooligosaccharides includes a series of carbohydrate-hydrolyzing and -transferring enzymes. However, the enzymatic hydrolysis of maltodextrins and glycogen, providing a major energy reservoir in prokaryotes, was not reflected in the model, due probably to a lack of experimental analysis. Unlike those in Bacillus spp., the transport and metabolic systems for maltodextrins in Escherichia coli have been investigated extensively (7, 9, 10). A model for maltose metabolism involving an α-glucanotransferase (MalQ), a maltodextrin glucosidase (MalZ), and a maltodextrin phosphorylase (MalP) was proposed previously based on analyses of the breakdown of ¹⁴C-labeled maltodextrins in various knockout mutants (10).Ninety bacterial genomes were analyzed to identify the enzymes involved in sugar metabolism, and the results suggested that bacterial enzymes for the synthesis and degradation of glycogen belong to the glucosyltransferase and glycosidase/transglycosidase families, respectively. Free-living bacteria such as B. subtilis carry a minimal set of enzymes for glycogen metabolism, encoded by the glg operon of five genes. The four genes most proximal to the promoter encode enzymes for the synthesis of glycogen, including a branching enzyme (glgB), an ADP-glucose phyrophosphorylase (glgC and glcD), and a glycogen synthase (glgA). On the other hand, the most distal gene, glgP, encodes a glycogen phosphorylase (a member of glycosyltransferase family 35) (13, 18), which degrades glycogen branches by forming glucose-1-phosphate (glucose-1-P). B. subtilis carries two additional enzymes encoded at separate loci, a maltogenic amylase (MAase [YvdF, encoded at 304°]) and a pullulanase (AmyX, encoded at 262°), which have been known to degrade glycogen in vitro (15, 31). These two enzymes are ubiquitous among Bacillus spp. and may play an important role in glycogen and maltodextrin metabolism in the bacteria (see Table S1 in the supplemental material).The MAase YvdF in B. subtilis 168 and its homologue in B. subtilis SUH4-2 share 99% identity at both the nucleotide and amino acid sequence levels (4). MAase (EC 3.2.1.133) is a multisubstrate enzyme that acts on substrates such as cyclodextrin (CD), maltooligosaccharides, pullulan, starch, and glycogen (4). MAase belongs to a subfamily of glycoside hydrolase family 13, along with cyclodextrinase (EC 3.2.1.54), neopullulanase (EC 3.2.1.135), and Thermoactinomyces vulgaris R-47 α-amylase II (46). Although the catalytic properties and tertiary structure of MAase have been studied extensively (33), its physiological role in the bacterial cell is yet to be elucidated. The expression pattern of MAase in B. subtilis 168 has been investigated by monitoring the β-galactosidase activity expressed from the yvdF promoter in defined media containing various carbon sources (20). The yvdF promoter is induced in medium containing maltose, starch, or β-CD but is repressed in the presence of glucose, fructose, sucrose, or glycerol as the sole carbon source. In a previous study, Spo0A, a master regulator determining the life cycle of B. subtilis, was shown to be related to the expression of MAase in a positive manner (20). Kiel et al. (18) reported that the glycogen operon in B. subtilis is turned on during sporulation by RNA polymerase containing σ^E. This finding indicated that MAase, along with glycogen phosphorylase and pullulanase, might be involved in the metabolism of maltodextrin and glycogen in vivo.Pullulanases are capable of hydrolyzing the α-1,6-glycosidic linkages of pullulan to form maltotriose (2, 11, 15, 28, 31, 38). In particular, type I pullulanases have been reported to hydrolyze the α-1,6-glycosidic linkages in branched oligosaccharides such as starch, amylopectin, and glycogen, forming maltodextrins linked by α-1,4-glycosidic linkages (11). Pullulanase is also known as a debranching enzyme. The enzymatic properties and three-dimensional structure of AmyX from B. subtilis 168 were investigated by Malle et al. (28). However, to date, the physiological function of pullulanase encoded by amyX has not been investigated.The aim of this study was to elucidate the physiological functions of MAase and pullulanase, specifically concentrating on their roles in the degradation of maltodextrin and glycogen in B. subtilis. For this purpose, studies of the localization of the enzymes, the accumulation of glycogen, and the distribution of glycogen side chains were performed using the wild type and knockouts of MAase- and pullulanase-related genes.     

Isolation and expression of a constitutive variant of the chloramphenicol-inducible plasmid gene cat-86 under control of the Bacillus subtilis 168 amylase promoter

The amyR1 region controls the regulated expression of the Bacillus subtilis 168 amylase gene amyE. When cloned into the B. subtilis promoter-cloning plasmid pPL603, amyR1 has been shown to activate expression of the promoter-indicator gene cat-86. In this chimeric plasmid, p5' alpha B10, cat-86 expression was maximal in stationary phase B. subtilis cells and cat-86 expression was repressible by glucose. Both these properties are similar to the regulated expression of the B. subtilis amyE gene. In addition, cat-86 expression in p5' alpha B10 was inducible with chloramphenicol (Cm). The inducibility phenotype of cat-86 has been shown to be independent of the promoter that is used to activate the gene, and inducibility has been suggested to result from the presence of a pair of inverted-repeat sequences that span the ribosome-binding site (RBS) for cat-86. A spontaneous deletion mutant of p5' alpha B10 was isolated, p5' alpha B10 delta 1, in which cat-86 expression was constitutive with respect to Cm, but the basic pattern of amyR1-directed regulation of cat-86 was intact. The rightward deletion endpoint was within the upstream member of the pair of inverted repeats that immediately precede cat-86. This result is therefore consistent with the role proposed for the inverted repeats in Cm inducibility. The leftward endpoint of the deletion is within the amyR1 region and thus allows a more precise determination of the functional domain of amyR1.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis.

Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.     

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.     

枯草杆菌表达载体pUB23的构建及地衣杆菌α-淀粉酶基因的表达

将克隆的解淀粉芽胞杆菌强启动子经DNA序列分析后连接到能在枯草杆菌中复制的质粒pUB18上,构建枯草杆菌表达载体pUB23。为了测试构建的表达载体能否表达外源基因,将地衣杆菌抉失了启动子的α-淀粉酶基因接到pUB23上启动子的下游,组建重组质粒,转化枯草杆菌QB1130(amy~-),获得能分泌α-淀粉酶的转化株,证明缺失了启动子的结构基因在pUB23上克隆启动子的启动下获得表达。酶活力测定结果表明,表达水平是用原启动子时的2.5倍.     

Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis

A novel phytase gene ( phyL) was cloned from Bacillus licheniformis by multiple steps of degenerate and inverse PCR. The coding region of the phyL gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. This gene, together with a phytase gene ( 168phyA) identified in the B. subtilis strain 168 genome by a homology search, was cloned and over-expressed in B. subtilis using a phi105MU331 prophage vector system. Up to 35 units of phytase/ml were secreted into the culture media; and mature enzymes of around 44-47 kDa were purified for characterization. Both phytases exhibited broad temperature and pH optima and showed high thermostability. Of the two, the phytase encoded by phyL exhibited higher thermostability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95 degrees C for 10 min. With their neutral pH optima and good temperature stabilities, these Bacillus phytases are good candidates for animal feed applications and transgenic studies.     

Cloning and characterization of the beta-amylase gene from Bacillus polymyxa.

The gene for beta-amylase was isolated from Bacillus polymyxa by molecular cloning in B. subtilis. B. subtilis cells containing this gene express and secrete an amylase which resembles the B. polymyxa beta-amylase and barley beta-amylase in terms of the products it generates during carbohydrate hydrolysis. Starch hydrolysis with this beta-amylase produces maltose, not glucose, whereas maltotriose and cycloheptaose are resistant to the action of this beta-amylase. The enzyme has a molecular weight of approximately 68,000. Restriction endonuclease mapping demonstrated that the DNA inserted in pBD64 and containing the gene is approximately 3 kilobases in length.     

利用枯草芽孢杆菌ytkA和ywoF基因启动子与信号肽重组分泌表达mpd基因

用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。     

Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene.

The bolA gene, which is involved in the morphogenetic pathways of Escherichia coli, was sequenced and two potential promoters were identified. Expression from promoter P1, proximal to the bolA structural gene is specifically induced during the transition to the stationary phase of growth. This promoter contains an unusual--10 region (CGGCTAGTA), which defines a new class of E. coli promoters necessary for the dramatic increase in the rate of synthesis of a large set of proteins during the cessation of logarithmic growth. This conclusion was confirmed by identifying two additional E. coli promoters and one plasmid promoter, which also were induced during the transition to the stationary phase of growth. Analysis of proteins produced during the exponential and stationary phases of growth in a bolA null mutant suggest a possible role for the BolA protein in the induction of the expression of penicillin-binding protein 6 (PBP6) in the transition to the stationary phase. Supporting this hypothesis is the presence of a putative DNA-binding domain within the bolA coding sequence.     

Cloning, nucleotide sequence, and enzymatic characterization of an alpha-amylase from the ruminal bacterium Butyrivibrio fibrisolvens H17c.

A Butyrivibrio fibrisolvens amylase gene was cloned and expressed by using its own promoter on the recombinant plasmid pBAMY100 in Escherichia coli. The amylase gene consisted of an open reading frame of 2,931 bp encoding a protein of 976 amino acids with a calculated Mr of 106,964. In E. coli(pBAMY100), more than 86% of the active amylase was located in the periplasm, and TnphoA fusion experiments showed that the enzyme had a functional signal peptide. The B. fibrisolvens amylase is a calcium metalloenzyme, and three conserved putative calcium-binding residues were identified. The amylase showed high sequence homology with other alpha-amylases in the three highly conserved regions which constitute the active centers. These and other conserved regions were located in the N-terminal half, and no similarity with any other amylase was detected in the remainder of the protein. Deletion of approximately 40% of the C-terminal portion of the amylase did not result in loss of amylolytic activity. The B. fibrisolvens amylase was identified as an endo-alpha-amylase by hydrolysis of the Phadebas amylase substrate, hydrolysis of gamma-cyclodextrin to maltotriose, maltose, and glucose and the characteristic shape of the blue value and reducing sugar curves. Maltotriose was the major initial hydrolysis product from starch, although extended incubation resulted in its hydrolysis to maltose and glucose.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis.

We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic alpha-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacillus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known alpha-amylase sequences. We found 83% homology with the 48-kDa alpha-amylase part of the Bacillus polymyxa beta-alpha-amylase polyprotein and 50% homology with Taka amylase A of Aspergillus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacillus amyloliquefaciens alpha-amylase gene.