Cloning and expression in Escherichia coli of Clostridium thermocellum DNA encoding β-glucosidase activity

Sau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in a Lac⁻mutant of Escherichia coli HB101. Five clones expressing β-glucosidase activity were shown by restriction enzyme analysis to contain a common 4.4 kbp fragment of inserted DNA. Hybridization of recombinant plasmids with chromosomal DNA ratified the physical maps of the inserted DNA and was further used to confirm that the 4.4 kbp fragment was common to all five clones. Enzyme activity, comprising cellobiase and aryl-β-glucosidase, was similar with respect to substrate specificity for each of the five clones, and was expressed independently of the orientation of the cloned DNA. A differential effect of temperature on activity of the cellobiase and aryl-β-glucosidase activities was observed but in other respects, the properties of the cloned β-glucosidase corresponded to those of the single β-glucosidase previously described for C. thermocellum.     

Isolation of an Arabidopsis thaliana casein kinase II β subunit by complementation in Saccharomyces cerevisiae

Casein kinase II is thought to play an essential role in the control of cell division and differentiation in all eukaryotes. Through complementation of a defective casein kinase II catalytic subunit gene from Saccharomyces cerevisiae, we isolated an Arabidopsis thaliana casein kinase II regulatory subunit homologue, CKB1. A second regulatory subunit was identified by low-stringency hybridization with CKB1.Casein kinase II from S. cerevisiae is composed of two catalytic () and two regulatory () subunits. Simultaneous disruption of the genes for the and subunits, CKA1 and CKA2, respectively, is lethal. Strain YDH8 has disruptions of CKA1 and CKA2; its viability depends on a temperature-sensitive allele of CKA2, cka2–8, carried on a centromeric plasmid. We screened an A. thaliana cDNA library, whose inserts are under the control of the galactose-inducible GAL10 promoter, for cDNAs which enabled YDH8 cells to grow at the restrictive temperature. One cDNA, CKB1, was isolated by this screen which had homology to cDNAs of casein kinase II subunits. A second cDNA, CKB2, was isolated by hybridization and was also able to suppress the YDH8 mutant phenotype.The proteins encoded by CKB1 and CKB2 are 80% identical. The carboxy-terminal two thirds of both proteins is ca. 54% identical to the regulatory subunits of casein kinase II from other species. The amino termini are unrelated to any other known proteins. CKB1 and CKB2 lack the conserved autophosphorylation site characteristic of animal subunits, but have potential casein kinase II phosphorylation sites in the same region. Suppression of the cka1 cka2–8 mutant phenotype occurs by interaction of CKB1 with the defective, cka2–8-encoded, catalytic subunit. Cells with disruptions in CKA1 and CKA2 are not rescued by expression of CKB1.     

Characterisation of an Arabidopsis thaliana cDNA encoding a novel thylakoid lumen protein imported by the ΔpH-dependent pathway

An Arabidopsis thaliana (L.) Heynh. cDNA encoding a novel 16-kDa protein (P16) of the chloroplast thylakoid lumen has been characterised. The function of the protein is unknown but it shares some sequence similarity with alpha allophycocyanins. P16 is synthesised with a bipartite, lumen-targeting presequence, and import experiments demonstrated that this protein follows the ΔpH-dependent pathway. Analysis of the thylakoid transfer peptide revealed two unusual features. Firstly, the key targeting determinant is predicted to be a twin-arginine followed by a highly hydrophobic residue two residues later, rather than at the third position as in most transfer peptides. Secondly, the C-terminal domain of the transfer peptide contains multiple charged residues which may help to prevent mistargeting by the Sec-type protein translocase. Received: 16 October 1998 / Accepted: 29 October 1998     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.     

Cloning and expression of an Oerskovia xanthineolytica β-1,3-glucanase in Escherichia coli

An Escherichia coli recombinant system produced a soluble -1,3-glucanase (BglII) cloned from Oerskovia xanthineolytica. The protein was obtained in a truncated form derived from the complete polypeptide. Cell fractionation studies show that 80% of the glucanase was retained in the periplasmic space after 20 h of induction. If cells were grown with glycine, 60% of the glucanase was released from the periplasm     

Crystal structure of the single cystathionine β-synthase domain-containing protein CBSX1 from Arabidopsis thaliana

The single cystathionine β-synthase (CBS) pair proteins from Arabidopsis thaliana have been identified as being a redox regulator of the thioredoxin (Trx) system. CBSX1 and CBSX2, which are two of the six Arabidopsis cystathione β-synthase domain-containing proteins that contain only a single CBS pair, have close sequence similarity. Recently, the crystal structure of CBSX2 was determined and a significant portion of the internal region was disordered. In this study, crystal structures of full-length CBSX1 and the internal loop deleted (Δloop) form are reported at resolutions of 2.4 and 2.2 Å, respectively. The structures of CBSX1 show that they form anti-parallel dimers along their central twofold axis and have a unique ～155° bend along the side. This is different from the angle of CBSX2, which is suggestive of the flexible nature of the relative angle between the monomers. The biochemical data that were obtained using the deletion as well as point mutants of CBSX1 confirmed the importance of AMP-ligand binding in terms of enhancing Trx activity.     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Transient β-glucuronidase activity after infiltration of Arabidopsis thaliana by Agrobacterium tumefaciens

Transient expression of the β-glucuronidase (GUS) gene introduced into Arabidopsis thaliana intact plants by T-DNA after vacuum infiltration of Agrobacterium tumefaciens was followed. The first incidence of GUS activity was found 2 - 3 d after treatment and a peak of activity one week after treatment in both A. thaliana races, Columbia and C24. GUS activity was sharply increased by cultivation of Arabidopsis plants at elevated temperature (29 °C) compared to cultivation at 25 °C. The density of inocula also influenced the GUS activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.     

Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli

Bifidobacterium infantis HL96 produces three -galactosidases (-gal I, II and III). A genomic bank of B. infantis was constructed in E. coli by using pBR322 as a cloning vector. Two E. coli transformants, BIG1 and BIG4, possessing -galactosidase activity, were selected from X-gal plates. They contained two different recombinant plasmids with insert DNA fragments of approx. 4.6 and 4.4 kb, respectively. The restriction maps of pBIG1 and pBIG4 were constructed. -Galactosidases from crude cell-free extracts of B. infantis and of two E. coli recombinants were analyzed by native PAGE and characterized by activity staining. pBIG1 and pBIG4 were shown to carry the genes for -gal I and -gal III, respectively. Optimal pH and temperature for hydrolytic activity of the native enzyme were 7.5 and 40°C, while those for recombinant BIG1 and BIG4 were 7.5, 50°C and 8.0, 40°C, respectively. © Rapid Science Ltd. 1998     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Cloning and expression in Escherichia coli of a Thermoanaerobacter cellulolyticus gene coding for heat-stable β-glucanase

Summary A 4.8 kb HindIII fragment of Thermoanaerobacter cellulolyticus DNA cloned in Escherichia coli was shown to direct the synthesis of -glucanase. The enzyme produced by the transformant was extremely heat-stable and the optimum temperature for the enzyme reaction was 80°C. The cloned enzyme could hydrolyse carboxymethyl cellulose and lichenan, but could not digest laminarin, xylan and cellobiose. Although T. cellulolyticus secreted cellulase(s) into the medium, most of the cloned enzyme activity was detected only in cytoplasm in the recombinant clone.     

Over-expression of cystathionine γ-synthase in Arabidopsis thaliana leads to increased levels of methionine and S-methylmethionine

Cystathionine γ-synthase (CGS, EC 4.2.99.9), the first committed enzyme in methionine biosynthesis, was over-expressed in Arabidopsis thaliana by introducing in the genome of this plant the coding sequence of the Arabidopsis enzyme under the control of the cauliflower mosaic virus 35S promoter. In order to target the recombinant protein to the chloroplast, the transgene included the sequence encoding the N-terminal transit peptide of Arabidopsis CGS. CGS activity and polypeptide were increased several fold in these plants. There was a markedly increased level of soluble methionine in the leaves of the transformed plants, up to 15-fold, indicating that CGS is a rate-limiting enzyme in this metabolic pathway. In addition, the transformed plants strongly over-accumulated S-methylmethionine, but not S-adenosylmethionine, in agreement with the view that S-methylmethionine corresponds to a storage form of labile methyl groups in plants and/or plays a role in preventing S-adenosylmethionine accumulation. The same strategy was used to increase the level of cystathionine β-lyase (CBL, EC 4.4.1.8), the second committed enzyme in methionine biosynthesis, in transformed A. thaliana. Despite an increase in both CBL activity and polypeptide in transformed Arabidopsis plants over-expressing Arabidopsis CBL, there was very little change in the contents of soluble methionine and S-methylmethionine, suggesting strongly that CBL is not rate limiting in the methionine biosynthetic pathway.     

Selective Inhibition of Escherichia coli in the Presence of Salmonella typhimurium by Phenethyl β-D-Galactopyranoside

Phenethyl β-d-galactopyranoside (PEG) was hydrolyzed by the β-galactosidase of Escherichia coli to form the toxic product phenethyl alcohol. Salmonella typhimurium did not hydrolyze PEG. In mixed culture, the ratio of S. typhimurium to E. coli was increased by growing the organisms in lactose broth containing 2.5% PEG. The high concentration of PEG required for inhibition of E. coli can be attributed to inadequate cell permeability rather than to prevention of β-galactosidase induction.