Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing

A series of promoter hybrids has been constructed by exchanging the ? 35 and ? 10 regions of lacUV5, tet, and trp promoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the ? 35 and ? 10 regions of the promoter. Derivatives of the tet promoter with one or two extra base pairs in this spacer region and constructions of the lac:: tet hybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: ? 35, ? 10) were trp:: lac and trp:: tet suggesting a high efficiency for the ? 35 region of the trp promoter. The weakest promoters were tet:: trp, lac:: trp and lac::tet indicating a weak ? 10 region for the trp promoter and the importance of ? 35 to ? 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.     

Optimizing the promoter and ribosome binding sequence for expression of human single chain urokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response

Summary The expression of recombinant single-chain urokinase-like plasminogen activator (rscuPA) in Escherichia coli was optimized by fusing the puk gene to different promoters and ribosome binding sequences. Comparison of the tac, trp and P _L promoters showed that expression was maximal under tac control. Variation in the ribosome binding sequence and its distance to the AUG start codon yielded a further slight improvement of expression. The largest increase in rscuPA expression was achieved by variations in the host strain and growth conditions. In E. coli DG75 grown at 37°C maximal expression was achieved 30 min after induction and decreased gradually until 240 min after induction. Growth at 30°C yielded maximal expression 60 min after induction and resulted in reduced activity at longer times. Western blot analysis of the products showed that degradation of rscuPA was much larger at 37°C than at 30°C. Using E. coli CAG630 carrying the htpR mutation, which avoids heat shock response, for expression of rscuPA eliminated the instability of the product at both temperatures. Expression in this strain was even more efficient than in E. coli JM101 carrying the lon mutation. It is concluded that induction of the general heat-shock response in E. coli must be avoided to obtain stabilization of rscuPA. This drastically improves the overall yield of rscuPA from recombinant E. coli strains.     

The construction of lambda transducing phages containing deletions defining regulatory elements of the lac and trp operons in E. coli

Summary Two sets of plaque-forming transducing phages have been isolated which carry parts of the tryptophan (trp) and lactose (lac) operons. The ptrp-lac set of phages carry the trp and lac operons in the same orientation connected by deletions which enter the lac regulatory region from the i side. These deletions start at various sites in or near the trp operon and end either late in the lac i gene, within the promoter, between the promoter and the operator, within the operator, between the operator and the z gene, or very early in the z gene. Starting with one particular trp-lac fusion strain, a series of transducing phages were isolated which contain varying portions of the trp operon extending from the trp A gene towards the trp operator. The other set of phages, which are designated ptrp/lac, carry trp and lac in opposing orientations. These ptrp/lac phages contain deletions which remove all of the lac structural genes and end between the operator and the z gene.A preliminary report of a portion of this work was presented at the Thirteenth International Congress of Genetics, Berkeley, 1973.     

Phage lambda pL promoter controlled α-amylase expression inEscherichia coli during fermentation

Summary Phage lambda p_L promoter controlled expression of theBacillus stearothermophilus gene coding for a thermostable -amylase inE. coli was studied in shake flask cultures and in a laboratory fermenter. At an inducible temperature (40 °C) the final cell density was lower, but the total enzyme activity produced ca. 80% higher than at a non-inducible temperature (30 °C). Moreover, 17% of the total enzyme activity was found in the culture medium. The -amylase yield, production rate and proportion secreted were further increased by shifting the fermentation temperature after certain period of bacterial growth rather than at the beginning of fermentation.     

Kinetics of β-glucosidase production by Escherichia coli recombinants harboring heterologous bgl genes

Maximum productivities of -glucosidase by E. coli recombinants, under the control of either lacZ or GALI promoters, were 33 ± 10 and 100 ± 5 IU l^–1 h^–1, respectively.GAL1 promoter of pYES2 enabled the E. colirecombinants to produce 3.1- and 15.1-fold more -glucosidase than that supported by lacZ promoter of pUC18 in E. coli recombinants and donor, respectively.     

Decay rates of Escherichia coli trp messenger RNA molecules lacking the normal 5''-terminal sequences.

Summary We have examined the stability in vivo of three mutant species of trp mRNA which differ from wild type in the nature of their 5-termini. These novel mRNA molecules originate from three mutationally generated promoters which lie within trp structural genes: trpE1423 lies near the carboxy-terminal end of trpE, trpD11 lies near the carboxy-terminal end of trpD (McPartland and Somerville, 1976) and trpC2121 lies near the center of trpC. When mRNA synthesis from the wild-type promoter is repressed by tryptophan, these strains still synthesize trp mRNA from their internal promoters at a relatively high efficiency in a constitutive fashion. The trp mRNA thus produced by the mutants lacks various lengths of the wild-type 5-proximal RNA sequence. These shortened trp mRNA molecules decayed exponentially, at about the same rate as that of normal trp mRNA whose synthesis originates at the authentic trp promoter. Chloramphenicol inhibited the degradation of 5-truncated trp mRNA fragments in a manner similar to that observed for wildtype trp mRNA, suggesting that the usual mechanism of mRNA decay is operative. We postulate that the initiation of mRNA decay at or near the 5-end does not require some special nucleotide sequence; rather the 5-proximal protion in general constitutes the target for nucleolytic attack.     

Metabolic burden in recombinant CHO cells: effect ofdhfr gene amplification andlacZ expression

Foreign protein production levels in two recombinant Chinese hamster ovary (CHO) cell lines were compared in cells transfected with different expression vectors. One vector pNL1 contained the gene for neomycin resistance (neo ^r ) and thelacZ gene which codes for intracellular -galactosidase, with both genes controlled by the constitutive simian virus (SV40) promoter. The other vector CDG contained the amplifiabledhfr gene andlacZ gene, controlled by the constitutive SV40 and cytomegalovirus (CMV) promoters, respectively. Cell growth and -galactosidase expression were compared quantitatively after cells were selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred in recombinant CHO cells in which thelacZ anddhfr genes were highly amplified and expressed. In contrast, the combined effects of the unamplifiedneo ^r gene andlacZ gene expression on the growth kinetics were small. Any metabolic burden caused bylacZ gene expression, which was evaluated separately from the effect ofneo ^r gene expression, must be negligible, as higher expression of -galactosidase (1.5×10^–6 units/cell) occurred in unamplified cells compared to the cells in whichlacZ was amplified by thedhfr-containing vector (3×10^–7 units/cell). Thus, the main factor causing severe growth reduction (metabolic burden) in cells containing the amplifieddhfr gene system was not overexpression of -galactosidase butdhfr andlacZ gene co-amplification anddhfr gene expression.     

A mutant rho ATPase from Escherichia coli that is temperature-sensitive in the presence of RNA

Summary The Escherichia coli mutant rho-115 suppresses lac operon polarity conferred by the lacZ::IS1 insertion MS319. The ATPase activity of purified rho-115 protein was maximal at 40°C, in contrast to 45°C for rho ⁺. At higher temperatures (50°C, 55°C), the fractions of activities at maximal temperature were consistently lower for rho-115 compared to rho ⁺. The 30-minute time course of rho-115 ATP hydrolysis was linear at 37°C but at 45°C the linear kinetics of hydrolysis reached a plateau between 10 and 15 minutes. The 30-minute time courses for rho ⁺ were linear at both 37°C and 45°C. The rho-115 and rho ⁺ ATPase activities were equally heat-stable during preincubation at 45°C in buffer. Inclusion of ATP during preincubation protected these rho proteins from inactivation to the same extent. The presence of polyC during preincubation protected rho ^- activity but produced substantial inactivation of rho-115 ATPase. The presence of polyU during preincubation gave similar results. Concentrations of polyC between 625 ng/ml and 100 g/ml yielded the same extent of rho-115 ATPase inactivation during preincubation at 45°C. Thermal inactivation of rho-115 ATPase by polyC was halted by shifting preincubation temperature from 45°C to 35°C, indicating that polyC-induced destabilization of rho-115 was irreversible.     

Comparison of promoter activities of heterologous and homologous promoters in Bacillus subtilis

Summary We have constructed promoter probe vectors with the Escherichia coli galactokinase monitoring system that can be used in Bacillus subtilis. In vivo studies with these vectors demonstrated that the E. coli trp and tac(trp::lac) promoter regions could be utilized in B. subtilis. These promoter regions and the promoter region for the erythromycin resistance gene originating from Staphylococcus aureus were preferentially utilized during the stationary growth phase of B. subtilis, whereas the B. subtilis P21K and P29K promoters were utilized during the exponential growth phase and decreased rapidly during the stationary phase. The apparent strength of these promoters of E. coli in B. subtilis, in terms of galactokinase units, was comparable with those of the B. subtilis promoters.     

Establishment,counts, and identification of the fibrolytic microflora in the digestive tract of rabbit. Influence of feed cellulose content

Dam-mediated adenine methylation at GATC sites can interfere with gene expression. By use oflacZ fusion technology, the efficiency oftrpR andtrpS promoters (which contain a GATC site) and oftrp (the target of TrpR repressor) was analyzed indam ⁺ anddam ^– backgrounds. In exponentially growing cells, thedam mutation leads to an increased activity oftrpR promoter but does not affecttrpS ortrp promoters. The Dam-mediated induction oftrpR was, however, found to be repressed bytrpR-mediated autoregulation. In contrast,trp-lacZ directed-galactosidase activity was increased at least sixfold indam ^– cells in late logarithmic growth phase. Indam ⁺ cells, expression oftrp-lacZ was similarly late-growth-phase induced, albeit to a reduced extent. Hence, we propose that enhancement of growth phase-dependent gene induction constitutes a previously unidentified trait ofdam mutation. This finding is discussed in the context of the pleiotropic phenotype ofdam mutation.     

The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a -transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.     

Operon fusions of Mu d1(Ap,lac) to thel-proline biosynthetic genes ofescherichia coli K-12

Operon fusions to the promoter of either theproA,proB, orproC genes of the proline biosynthetic pathway were obtained by the use of the Mu d1(Ap,lac) bacteriophage. These fusions were further stabilized by transformation with plasmid pGW600 containing the wildtype Mu repressor gene or by transduction with phage pSG1. The level of -galactosidase in the fusion strains was not affected by the presence of exogenously addedl-proline or high concentrations of NaCl in the growth medium. A Tn5 insertion nearproBA increased -galactosidase expression 140- to 200-fold in strains carrying theproA-lac andproB-lac fusions, but the level of this enzyme was unaltered in strains carrying theproC-lac fusion. The Tn5 insertion increased intracellular proline concentrations 8- to 10-fold, suggesting that mechanisms other than allosteric inhibition may regulate proline biosynthesis, but did not confer osmotolerance to cells growing in a medium with a high concentration of salt.     

Identification of a developmentally regulated sialidase inEimeria tenella that is immunologically related to theTrypanosoma cruzi enzyme

Sporozoites and merozoites of three species ofEimeria, E. tenella, E. maxima, andE. necatrix, that cause diarrhea in chickens worldwide, were examined for their expression of sialidase (SA) activity. The enzyme was found in three species, and the activity of merozoites was 10–20 times higher than that of sporozoites. The enzyme was resistant to degradation by proteases that are normally present in the intestine, a site inhabited by theEimeria parasites, and it was relatively resistant to heat, with optimum activity being at 40°C, which is within the range of temperature in the chicken intestine (40–43°C).E. tenella SA was immuniprecipitated by monoclonal and polyclonal antibodies raised against theTrypanosoma cruzi SA (TCSA), and enzyme activity was neutralized by these antibodies.E. tenella SA was identified by immunoblots as a doublet of molecular weight 190 000 and 180 000 using, as a probe, anti-TCSA antibodies and antibodies against a synthetic peptide (TR) derived from the long tandem repeat domain of TCSA. Binding of the monoclonal and polyclonal antibodies toE. tenella was completely blocked by TR, but not by an irrelevant peptide (BR). Therefore,E. tenella expresses a developmentally regulated SA that is structurally related to theT. cruzi counterpart. Because of the high SA activity in merozoites, and by analogy with other SA-producing microbes that inhabit mucin-rich epithelia, we suggest that theEimeria SA plays a role in desialylating intestinal mucins to reduce viscosity of the local environment and thereby facilitate parasite migration. The enzyme could also play a role in host cell-parasite interaction.Abbreviations SA sialidase (neuraminidase) - Neu5Ac N-acetylneuraminic acid - 4-MU-Neu5Ac 2-(4-methylumbelliferyl)--N-acetyl-d-neuraminic acid - BSA bovine serum albumin - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PNA peanut agglutinin - Ab antibody - TCN-2 monoclonal antibody toT. cruzi sialidase, anti-Ars, monoclonal antibody top-azophenylarsonate - TCSA Trypanosoma cruzi sialidase     

Enhanced hydantoinase and N-carbamoylase activity on immobilisation of Agrobacterium tumefaciens

Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C. 3.5.1) activity on reaction with N-carbamylglycine. The hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) and N-carbamoylase activities remained stable over 4 weeks storage at 4°C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase. The pH optima of the immobilised hydantoinase and N-carbamoylase enzymes decreased to pH 7 and pH 8, respectively. The temperature optimum remained at 40°C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50°C.     

Cloning and sequencing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its expression in Escherichia coli

Summary A cyclcodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at –108 to –67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (M _r) and enzymatic properties as follows: M _r, 80 000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55°C; and stability below 45°C. The yields of the products from starch as the substrate were 25% for -and 5% for -cyclodextrin.The nucleotide and deduced amino acid sequence data reported in this paper have been deposited in the DDBJ, EMBL and Genbank Nucleotide Sequence Databases under the accession number D90243 Offprint requests to: T. Uozumi     

Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis

Summary Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the -galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.     

Expression of the guanosine-inosine kinase gene from Exiguobacterium acetylicum in Corynebacterium ammoniagenes and phosphorylation of inosine to produce inosine 5′-monophosphate

The guanosine-inosine kinase gene (gsk) isolated from Exiguobacterium acetylicum was expressed in an ATP-regenerating strain, Corynebacterium ammoniagenes. In order to induce its expression, three promoters (those for the Escherichia coli tac, Escherichia coli trp, and Corynebacterium glutamicum odhA gene) with the corresponding ribosome-binding sequences were examined. The E. coli trp promoter was most efficient with regard to inducing the expression of gsk in C. ammoniagenes. Further, the resulting strain, which has both inosine kinase activity and ATP-regenerating activity, was used to induce the phosphorylation of inosine to produce inosine 5′-monophosphate (5′-IMP), which is widely used as a flavor enhancer; approximately 80 g of 5′-IMP/l was produced with a molar conversion ratio of 80%.     

Relief of polarity in E. coli depleted of 30S ribosomal subunits.

Summary Escherichia coli was depleted of ribosomes by a thermal shock at 47° C which quantitatively destroyed the 30S ribosomal subunits. During recovery in minimal medium at 30° C RNA is synthesized while protein synthesis resumes only after about 90 min. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Lac mRNA from a series of lac nonsense mutants was examined in both heated and untreated cells. It was found that the polar effect of nonsense mutation is relieved in the absence of ribosomes and that this relief is due to the synthesis of larger mRNA molecules. Since Rho remained active in thermally treated cells, premature termination at secondary signals within the lac operon must also depend on the presence of active ribosomes.Abbreviations used SSC 0.15 M Nacl, 0.015 M sodium citrate (pH 7.0) - mRNA messenger ribonucleic acid - IPTG isopropyl--D-thiogalactopyranoside - cAMP adenosine 3: 5-cyclic monophosphoric acid - LDS lithium dodecyl sulfate - TCA trichloroacetic acid The paper forms part of the first author's M.Sc. thesis