Modification of length, hydrophobic properties and electric charge of Bacillus subtilis alpha-amylase signal peptide and their different effects on the production of secretory proteins in B. subtilis and Escherichia coli cells

Summary Bacillus subtilis -amylase signal peptide, which consists of 33 amino acids, is functional in Escherichia coli cells.Lysine, glutamic acid, leucine, leucyl-leucine, or leucyl-leucyl-leucine was inserted between positions 28 and 29 of the -amylase signal peptide using site directed mutagenesis. DNAs encoding the wild-type and modified signal peptides were then fused in-frame to DNAs encoding the mature regions of the -lactamase of pBR322 and a thermostable -amylase. The secretion of -lactamase in E. coli cells was more inhibited by the modified signal peptides than that in B. subtilis cells, although the degree of inhibition varied and the inhibitory effect of each signal peptide was found to be similar in the two strains. In contrast, the difference in the inhibitory effect of each modified signal peptide was no longer detected in the case of the production of thermostable -amylase, except for the insertion of glutamic acid. Nearly 50% of thermostable -amylase in the precursor form was accumulated in the intracellular fraction of E. coli cells containing the DNAs for the modified signal peptides. The insertion of glutamic acid inhibited the secretion of the two enzymes in both B. subtilis and E. coli cells.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

Colony switching in an alpha-amylase-producing strain ofBacillus subtilis

Four colony variants (two stable and two unstable phenotypes) were observed inBacillus subtilis -10, an -amylase-hyperproducing strain. The stable variants lost the ability to produce -amylase, while the unstable ones reverted to the typical morphology after restreaking. Unstable expanding sectors appeared in typical colonies, and their appearance was influenced by the culture origin and age.     

Amplification of the amyE-tmrB region on the chromosome in tunicamycin-resistant cells of Bacillus subtilis

Summary In a class of tunicamycin-resistant mutants (tmrA7) of Bacillus subtilis, the production of extracellular -amylase is increase by about five fold. The tmrA7 characteristics (tunicamycin resistance and hyperproduction of extracellular -amylase) can be transferred to recipient cells by transformation. In the transformants and the original tmrA7 mutant, typical amplification of the region from 4 kb upstream of the amyE gene to the tmrB gene on the chromosome was detected. The repeating unit, 16 kb in size, repeats tandemly about five and ten times in the mutant and transformants, respectively, and the -amylase production is proportional to the copy number of the amyE gene. Simultaneous amplification of the tmrB gene, which is responsible for tunicamycin resistance in the multicopy state, and the -amylase structural gene (amyE) seems to be the cause of the pleiotropy of the tmrA7 mutation.     

Effect of polyethylene glycol on Bacillus subtilis α-amylase purification with raw and modified starch

Polyethylene glycol was found to enhance adsorption of Bacillus subtilis -amylase on starch in optimum concentration 10 % (w/w). Degree of adsorption at 12°C was increased from 83 to 98 % and from 30 to 81 % for cross-linked and raw starch, resp. Higher sorption capacity and easy desorption of -amylase without temperature or pH change was reached at 22 °C. Yield of -amylase 95 % and purification factor 8.3 were achieved on the cross-linked starch column. The method is suitable for -amylase isolation from PEG phase after its microbial production in aqueous two-phase systems.     

The effect of glucose on the expression of a clonedBacillus amyloliquefaciens α-amylase gene in strains ofBacillus subtilis

Bacillus subtilis strain 1A297 was shown to relieve the glucose repression of a clonedB. amyloliquefaciens -amylase gene carried on the hybrid plasmid pVC102 without affecting its temporal activation. However, glucose repression of -amylase occurred when pVC102, was introduced intoB. subtilis strain 1A289. Glucose repression was relieved by -methyl-d-glucoside, an analog of glucose that blocks its uptake. The relief of glucose repression in 1A297 did not act at the level of plasmid copy number. As 1A297 was capable of exerting glucose repression on a homologous chromosomally encoded gene, it is postulated that the putativetrans-acting product involved in glucose repression inB. subtilis (Nicholson and Chambliss, 1986, J. Bacteriol. 165:663–670) is altered in strain 1A297 and does not recognize theB. amyloliquefaciens -amylase gene.     

Production of heterologous proteins in Bacillus subtilis: the effect of the joint between signal sequence and mature protein on yield

Summary We have previously made a set of DNA constructs by fusing the mature part of Bacillus licheniformis -amylase with the signal sequence of B. amyloliquefaciens -amylase at various distances from the signal sequence cleavage site. We observed that the level of -amylase production in B. subtilis depended strongly on the distance of the junction from the signal sequence cleavage site, with quite a sharp optimum distance. To test whether the effect is limited to the pair of -amylase signal sequence and mature protein, we analysed the protein production in a set of constructs in which an Escherichia coli \-lactamase was similarly joined at different distances from the -amylase signal sequence. Also in this case the distance seemed to be an important factor in affecting the level of production in B. subtilis. The observed effect might depend on the modulation of pre-protein folding, which in turn could affect the secretion level. Offsprint requests to: M. Sibakov     

The relation of proton motive force,adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

The magnitude of the proton motive force (p) and its constituents, the electrical () and chemical potential (-ZpH), were established for chemostat cultures of a protease-producing, relaxed (rel ^–) variant and a not protease-producing, stringent (rel ⁺) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of p with the specific growth rate was found. The calculated intracellular pH (pH_in) was not constant but inversely related to . This change in pH_in might be related to regulatory functions of metabolism but a regulatory role for pH_in itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low and an increased value at higher . For both limitations, the ATP/ADP ratio was directly related to .The phosphorylation potential (G'p) was invariant with . From the values for G'p and p, a variable H⁺/ATP-stoichiometry was inferred: H⁺/ATP=1.83+0.52µ, so that at a given H⁺/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for =0 to _max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.Abbreviations CCCP carbonylcyanide-p-trichloromethoxyphenylhydrazone - DW dry weight of biomass - F Faraday's constant, 96.6 J/(mV × mol) - Fo chemostat outflow-rate (ml/h) - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - G'p phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and P_i - G'⁰p standard Gibbs energy change at specified conditions - H⁺/ATP number of protons translocated through - ATP synthase in synthesis of one ATP - H⁺/O protons translocated during transfer of 2 electrons from substrate to oxygen - specific growth rate (1/h) - _H+ transmembrane electrochemical proton potential, J/mol - M_b molar weight (147.6 g/mol) of bacteria with general cell formula C_6.0H_10.8O_3.0N_1.2 - pH_out,in extracellular, intracellular pH - P_i (intracellular) inorganic phosphate - p proton motive force, mV - pH transmembrane pH-difference - transmembrane electrical potential, mV - P/O number of ADP phosphorylated to ATP upon reduction of one O^2– to H₂O by two electrons transferred through the electron transfer chain - P/O (H⁺/O) × (H⁺/ATP)^–1 - P/O_F, P/O_N P/O with the two electrons donated by resp. (NADH + H⁺) and FADH - q specific rate of consumption or production (mol/g DW × h) - rel ⁺,rel ^– stringent, relaxed genotype - R universal gas constant, 8.36 J/(mol × degree) - T absolute temperature - TPMP⁺ triphenylmethylphosphonium ion - TPP⁺ tetraphenyl phosphonium ion - Y growth yield, g DW/mol - Z conversion constant=61.8 mV for 310 K (37 °C) - ZpH transmembrane proton potential or chemical potential, mV     

Evolution of the response patterns to dietary carbohydrates and the developmental differentiation of gene expression of α-amylase in Drosophila

Intraspecific variation of -amylase activity in D. melanogaster and D. immigrans, which is distantly related to D. melanogaster, and interspecific variation of -amylase activity in 18 Drosophila species were examined. The amount of intraspecific variation of -amylase activities measured in terms of coefficient of variation in D. melanogaster and D. immigrans was one-half and one-tenth or less, respectively, of the interspecific variation in 18 Drosophila species. We also surveyed the response patterns of -amylase activity to dietary carbohydrates at the larval and adult stages. The levels of -amylase activity depended on both repression by dietary glucose (glucose repression) and induction by dietary starch (starch induction). In general, our data suggest that glucose repression was conserved among species at both stages while starch induction was mainly observed in larvae, although the degree of the response depended on species. In D. lebanonensis lebanonensis and D. serrata, larvae expressed electrophoretically different -amylase variants (isozymes) from those of adult flies. These results may suggest that the regulatory systems responsible both for the response to environment and developmental expression are different among species in Drosophila. Correspondence to: T. Yamazaki     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

The glycolipid specificity ofErythrina cristagalli agglutinin

The interaction of¹²⁵I-labeledErythrina cristagalli agglutinin (ECA) with neutral glycosphingolipids on thin layer chromatograms was examined by the overlay technique followed by radioautography. The lectin bound topara-globoside with a sensitivity about 10 times higher than to lactosylceramide or globoside, in agreement with the specificity of the lectin forN-acetyllactosamine. The lower limit of detection ofpara-globoside was about 0.66 nmol. The specific binding of ECA to this glycolipid was confirmed by a highly sensitive enzyme-linked lectin assay (ELLA), utilizing the horseradish peroxidase-avidin-biotin system for detection of bound lectin. Overlays of neutral glycosphingolipid extracts from human erythrocyte membranes and from human granulocytes with ECA demonstrated that the lectin can be employed for the detection of small amounts ofpara-globoside in biological materials also in the presence of excess globoside. No staining was obtained when thin layer chromatograms of neutral glycosphingolipid extracts from rabbit erythrocyte membranes were overlayed with¹²⁵I-ECA. Afterin situ treatment of the chromatograms with -galactosidase, the lectin bound to several components, one of which had a mobility corresponding to that of the pentahexosylceramide Gal3Gal4GlcNAc3Gal4Glc1Cer, the major neutral glycosphingolipid of rabbit erythrocytes, thus providing further evidence for the specificity of ECA forpara-globoside.Abbreviations GSL glycosphingolipid(s) - CDH lactosylceramide, Gal4Glc1Cer - CTH trihexosylceramide, Gal4Gal4Glc1Cer - GLOB globoside, GalNac3Gal4Gal4Glc1Cer - PG para-globoside, Gal4GlcNAc3Gal4Glc1Cer - AsG_M1 asialo-G_M1, Gal3GalNAc4Gal4Glc1Cer - FORS Forsmann antigen, GalNAc3GalNAc3Gal4Gal4Glc1Cer - CPH pentahexosylceramide, Gal3Gal4GlcNAc3Gal4Glc1Cer - ECA Erythrina cristagalli agglutinin - SBA soybean agglutinin - PBS phosphate-buffered saline - PVP-40 polyvinylpyrrolidone M.W. 40000 - BSA bovine serum albumin - HRP-avidin horseradish peroxidase conjugated to avidin - ELLA enzyme-linked lectin assay - ELISA enzyme-linked immunosorbent assay - PMNL polymorphonuclear leukocytes - HPTLC high performance thin layer chromatography     

Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of Delgold tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a Delgold background, and PB19, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between Delgold and PB19, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between Delgold and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between Delgold and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Nucleotide sequence of an endo-β-1,4-glucanase gene fromBacillus subtilis CK-2

The gene encoding endo--1,4-glucanase inBacillus subtilis CK-2 was cloned intoEscherichia coli DH5, and the nucleotide sequence determined. The 1500 bp gene encodes a protein of 499 amino-acid residues with a calculated molecular mass of 55 261, and is equipped with a typicalB. subtilis signal peptide. Nucleotide sequence comparison revealed only 2 basepairs deviation between this gene and the endo--1,4-glucanase gene ofB. subtilis PAP115, and 93% to 95% homology was found between the amino acid sequences of these enzymes and otherB. subtilis endo--1,4-glucanases. Regions of similarity were also observed between the carboxy-terminal part of these enzymes and the part of theB. lautus PL236celA enzyme constituting the cellulose-binding domain.     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Location of β-amylase sequences in wheat and its relatives

Summary A -amylase cDNA clone isolated from barley has been used to locate -amylase encoding sequences on wheat, rye, and Aegilops umbellulata chromosomes by hybridisation to restriction endonuclease digested DNA obtained from wheat aneuploid and wheat-alien addition lines. Structural genes were identified on homoeologous group 4 and 5 chromosomes, confirming the results of isozyme studies. In addition, a further set of structural genes was found on homoeologous group 2 chromosomes. It is proposed that there are two homoeoallelic series, -Amy-1 on group 4 or 5 chromosomes, and -Amy-2 on group 2 chromosomes. Evidence is presented that each locus contains one or two -amylase structural genes, and it is suggested that the large number of isozymes seen upon IEF are due to post-translational modifications.     

Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express α-amylase and glucoamylase separately or as bifunctional fusion proteins

Eight constructions involving the Bacillus subtilis -amylase gene (amyE), a mouse pancreatic -amylase cDNA (AMY2) and an Aspergillus awamori glucoamylase cDNA (glaA) were prepared: three fusion genes, involving one -amylase and the glucoamylase, two double-cassette plasmids (expressing one or other -amylase and the glucoamylase) and three single-cassette plasmids, expressing the individual coding sequences. Following transformation of each plasmid into Saccharomyces cerevisiae, a plate test revealed that the largest starch hydrolysis halo was produced by the strain bearing the B. subtilis -amylase/glucoamylase fusion (BsAAase/GAase), and the smallest halo by the one expressing the mouse pancreatic -amylase/glucoamylase fusion (MAAase/GAase). When assayed for enzymatic activity in liquid medium, the strains bearing the fusion and the double-cassette plasmids involving B. subtilis -amylase and the glucoamylase exhibited both enzymic activities. Moreover, the BsAAase/GAase hybrid was able to adsorb and digest raw starch. The MAAse/GAase fusion protein was found to exhibit only -amylase activity. Finally, the capacity to grow on soluble and corn starch was tested in liquid medium for the strains bearing plasmids coding for the fusion proteins and the separate enzymes. The strain carrying the double-cassette BsAAase + GAase, which produced one of the smallest hydrolysis haloes in the place test, showed the best performance, not only in digesting soluble and corn starch but also in using all of the hydrolysis products for growth. The transformant bearing the BsAAase/GAase fusion was able to grow on soluble starch, but not on corn starch.