Characterization of a novel endo-levanase and its gene from Bacillus sp. L7

A novel endo-levanase producing bacterium belonging to the Bacillus family has been isolated from soil. The enzyme was characterized and found to have no exo-β-fructofuranosidase activity. The endo-levanase gene was cloned and sequenced. Homology searches have shown that the C-terminal domain of the enzyme is homologous to a number of known β-fructofuranosidases, including exo-levanase from Bacillus subtilis and yeast invertases. The N-terminal region of the endo-levanase which is homologous to the C-terminal sequence of the B. subtilis levanase appears to be a levan-binding domain.     

Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase

Summary The structural gene for the enzyme levanase of Bacillus subtilis (SacC) was cloned in Escherichia coli. The cloned gene was mapped by PBS1 transduction near the sacL locus on the B. subtilis chromosome, between leu4 and aroD. Expression of the enzyme was demonstrated both in B. subtilis and in E. coli. The presence of sacC allowed E. coli to grow on sucrose as the sole carbon source. The complete nucleotide sequence of sacC was determined. It includes an open reading frame of 2,031 bp, coding for a protein with calculated molecular weight of 75,866 Da, including a putative signal peptide similar to precursors of secreted proteins found in Bacilli. The apparent molecular weight of purified levanase is 73 kDa. The sacC gene product was characterized in an in vitro system and in a minicellproducing strain of E. coli, confirming the existence of a precursor form of levanase of about 75 kDa. Comparison of the predicted aminoacid sequence of levanase with those of the two other known -D-fructofuranosidases of B. subtilis indicated a homology with sucrase, but not with levansucrase. A stronger homology was detected with the N-terminal region of yeast invertase, suggesting the existence of a common ancestor.     

Cloning, sequencing, and disruption of a levanase gene of Bacillus polymyxa CF43.

The Bacillus polymyxa CF43 lelA gene, expressing both sucrose and fructan hydrolase activities, was isolated from a genomic library of B. polymyxa screened in Bacillus subtilis. The gene was detected as expressing sucrose hydrolase activity; B. subtilis transformants did not secrete the lelA gene product (LelA) into the extracellular medium. A 1.7-kb DNA fragment sufficient for lelA expression in Escherichia coli was sequenced. It contains a 548-codon open reading frame. The deduced amino acid sequence shows 54% identity with mature B. subtilis levanase and is similar to other fructanases and sucrases (beta-D-fructosyltransferases). Multiple-sequence alignment of 14 of these proteins revealed several previously unreported features. LelA appears to be a 512-amino-acid polypeptide containing no canonical signal peptide. The hydrolytic activities of LelA on sucrose, levan, and inulin were compared with those of B. subtilis levanase and sucrase, confirming that LelA is indeed a fructanase. The lelA gene in the chromosome of B. polymyxa was disrupted with a chloramphenicol resistance gene (cat) by "inter-gramic" conjugation: the lelA::cat insertion on a mobilizable plasmid was transferred from an E. coli transformant to B. polymyxa CF43, and B. polymyxa transconjugants containing the lelA::cat construct replacing the wild-type lelA gene in their chromosomes were selected directly. The growth of the mutant strain on levan, inulin, and sucrose was not affected.     

Molecular cloning of the gene for 2,6-beta-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2

The gene encoding a 2,6-beta-D-fructan 6-levanbiohydrolase (LF2ase) (EC 3.2.1.64) that converts levan into levanbiose was cloned from the genomic DNA of Streptomyces exfoliatus F3-2. The gene encoded a signal peptide of 37 amino acids and a mature protein of 482 amino acids with a total length of 1560 bp and was successfully expressed in Escherichia coli. The similarities of primary structure were observed with levanases from Clostridium acetobutylicum, Bacillus subtilis, B. stearothermophilus (51.0-54.3%) and with LF2ase from Microbacterium levaniformans (53.9%). The enzyme from S. exfoliatus F3-2 shared the conserved six domains and the completely conserved five amino acid residues with family 32 glycosyl hydrolases, which include levanase, inulinase, and invertase. These observations led to the conclusion that the enzyme belongs to family 32 glycosyl hydrolases.     

麦芽糖诱导软化芽孢杆菌α-环糊精葡萄糖基转移酶在枯草杆菌中的表达

通过PCR扩增软化芽孢杆菌α-环糊精葡萄糖基转移酶基因,将基因片段克隆到大肠杆菌-枯草杆菌穿梭载体pGJ103中,转化枯草杆菌WB600得基因工程菌进行外源表达。在1.5%的麦芽糖初始发酵培养基上摇瓶培养,48 h后重组枯草杆菌产酶活性为6.1U/ml。通过单因素分析和响应面分析对重组枯草杆菌产CGT酶摇瓶发酵条件进行优化。分析得到培养基关键组分麦芽糖,玉米淀粉和酵母粉三者最佳浓度分别为:15.5g/L,13g/L和20g/L。在此条件下,摇瓶培养36h后α-CGT酶活性为17.6U/ml,5L罐分批发酵30h后酶活达到20U/ml (水解活性为1.4×10⁴ IU/ml)。     

蛋白激发子PeaT1在枯草芽胞杆菌中的分泌表达及重组菌株提高小麦抗旱和促生的作用

PeaT1是从极细链格孢菌Alternaria tenuissima中分离的一种蛋白激发子,具有促进植物生长和诱导植物产生系统获得抗性的功能,为了实现peaT1基因在枯草芽胞杆菌Bacillus subtilis中的分泌表达,增加其应用途径,从枯草芽胞杆菌基因组DNA中分别扩增获得P43启动子和nprB基因的信号肽序列,并用SOE (Splicing by over lapping extension) 方法与peaT1基因连接,将连接产物克隆到大肠杆菌-枯草芽胞杆菌穿梭表达载体pHY300-PLK上,构建了重组表达载体pHY43N-peaT1。将重组载体转化枯草芽胞杆菌WB800菌株,SDS-PAGE和Western blotting分析证实,在NprB信号肽的引导下,枯草芽胞杆菌成功分泌表达了PeaT1蛋白。构建的重组菌株能够显著增强幼苗抗旱性,提高小麦株高。     

嗜碱性芽孢杆菌PB92碱性蛋白酶分别在大肠杆菌和枯草芽孢杆菌中表达

从Bacillus alcalophillus PB92中扩增出碱性蛋白酶基因Mapr,Mapr分别插入到大肠杆菌载体pET-22b( )和枯草芽孢杆菌载体pWB980中构建成重组分泌型表达载体pET22b( )-Mapr、pWB980-Mapr。碱性蛋白酶基因分别在大肠杆菌宿主BL21和枯草芽孢杆菌DB104中得到表达。SDS-PAGE分析,重组蛋白酶的分子量为28kD。在大肠杆菌,所得酶活为231U/ml,而在枯草芽孢杆菌,其酶活为1563U/ml。大概是由于碱性蛋白酶在枯草芽孢杆菌折叠成熟机制与大肠杆菌的不同造成的。     

Gene cloning,expression, and crystallization of a thermostable exo-inulinase from Geobacillus stearothermophilus KP1289

The gene ( inuA) encoding exo-inulinase (EC 3.2.1.80) was cloned from the thermophilic Geobacillus stearothermophilus ( Bacillus stearothermophilus) KP 1289 growing at between 41 degrees C and 69 degrees C. The inuA gene consisted of 1,482 bp encoding a protein of 493 amino acids. The deduced polypeptide of molecular mass ( M) 56,744 Da showed strong sequence similarity to Pseudomonas mucidolens exo-inulinase, Bacillus subtilis levanase, Paenibacillus polymyxa ( Bacillus polymyxa) fructosyltransferase, and so on, indicating that the enzyme belonged to glycosyl hydrolase family 32. The M of the purified exo-inulinase, expressed in Escherichia coli HB101, was estimated as approximately 54,000 Da by both SDS-PAGE and gel filtration. These results suggested that the active form of the enzyme is a monomer. The enzyme was active between 30 and 75 degrees C with an optimum at 60 degrees C. The properties were identical to those of the native enzyme. Additionally, for the first time for a prokaryotic GH32 protein, crystals of the recombinant enzyme were obtained.     

Saccharomyces cerevisiae homoserine kinase is homologous to prokaryotic homoserine kinases

The Saccharomyces cerevisiae gene (THR1) encoding homoserine kinase (HK; EC 2.7.1.39) was cloned by complementation in yeast. Disruption of the THR1 gene results in threonine auxotrophy in yeast. Comparison of the amino acid sequences of yeast and bacterial HKs reveals substantial similarity.     

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.     

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.     

短小芽孢杆菌碱性蛋白酶基因启动子的克隆、鉴定及其应用

利用TAIL-PCR(Thermal asymmetric interlaced PCR)从短小芽孢杆菌基因组中扩增到碱性蛋白酶基因编码区上游的启动子片段。对该片段的序列测定和分析表明, 此片段长797 bp, 但与基因表达有关的序列长约390 bp。对启动子片段进行不同长度的缺失突变, 以获得最小的基因启动子片段, 结果表明, 该基因起始密码子上游约160 bp的DNA片段就可以启动基因的表达。将含有该片段的碱性蛋白酶基因WApQ3插入大肠杆菌-芽孢杆菌穿梭质粒载体pSUGV4中, 构建了碱性蛋白酶基因表达质粒pSUBpWApQ3。将该质粒分别转入枯草芽孢杆菌和短小芽孢杆菌中表达, 可在胞外检测到碱性蛋白酶活性, 最高酶活分别为466.5 U/mL和3060 U/mL。     

Sequence of the indoleglycerol phosphate synthase (trpC) gene from Rhodobacter capsulatus.

We have isolated, cloned, and sequenced the indoleglycerol phosphate synthase gene (trpC) from Rhodobacter capsulatus. Normalized alignment scores comparing the trpC gene of R. capsulatus with the trpC genes of other bacterial species are reported. An unexpected degree of similarity to the trpC gene of Bacillus subtilis was found.     

外源木聚糖酶在枯草芽胞杆菌中信号肽筛选

[目的]本试验旨在筛选引导表达外源木聚糖酶基因高效分泌的信号肽,为枯草芽胞杆菌木聚糖酶高效分泌表达系统提供元件.[方法]构建信号肽筛选载体,载体是以含壮观霉素抗性基因的大肠-枯草穿梭载体为基本骨架,目标蛋白为耐碱性木聚糖酶,可在麦芽糖启动子Pglv诱导下表达.从枯草芽胞杆菌A1747基因组中扩增获得24个Sec途径信号肽,并将其全部链接到至筛选载体上,并在枯草芽胞杆菌WB700中实现表达分泌.重组菌在3%麦芽糖诱导下培养24h后用DNS法测定上清酶活.[结果]成功构建信号肽筛选载体pGPSX及24个表达载体,实现木聚糖酶表达分泌.且不同信号肽对于引导外源木聚糖酶分泌能力不同,其中YnfF信号肽引导分泌目标蛋白效率最高,上清酶活为37.2IU/mL.[结论]试验证明在枯草杆菌中对外源蛋白进行信号肽筛选是提高其分泌的有效途径,并获得了针对木聚糖酶高效分泌信号肽YnfF.     

Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168.

A beta-D-fructofuranosidase -- called levanase -- capable of the hydrolysis of sucrose, inulin and levans has been identified in Bacillus subtilis Marburg. This enzyme can not be detected in strain 168. However, sacL mutations -- mapped on the chromosome of strain 168 between the pheA and aroD reference markers -- lead to constitutive levanase synthesis. This synthesis is repressed by carbon sources such as glucose, glycerol or sucrose.     

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase

Respiratory-deficient mutants of Saccharomyces cerevisiae assigned to pet complementation group G72 are impaired in mitochondrial protein synthesis. The loss of this activity has been correlated with the inability of the mutants to acylate the two methionyl-tRNAs of yeast mitochondria. A nuclear gene (MSM1) capable of complementing the respiratory deficiency has been cloned by transformation of the G72 mutant C122/U3 with a yeast genomic library. In situ disruption of the MSM1 gene in a wild-type haploid strain of yeast induces a respiratory-deficient phenotype but does not affect the ability of the mutant to grow on fermentable substrates indicating that the product of MSM1 functions only in mitochondrial protein synthesis. Mitochondrial extracts prepared from the mutant with the disrupted copy of MSM1 were found to be defective in acylation of the two mitochondrial methionyl-tRNAs thereby confirming the identity of MSM1 as the structural gene for the mitochondrial methionyl-tRNA synthetase. The sequence of the protein encoded by MSM1 is similar to the Escherichia coli and yeast cytoplasmic methionyl-tRNA synthetases. Based on the primary-sequence similarities of the three proteins, the mitochondrial enzyme appears to be more related to the bacterial than to the yeast cytoplasmic methionyl-tRNA synthetase.