Effects of Proline Analogues on the Formation of Alkaline Phosphatase in Escherichia coli

Two of the four proline analogues tested for their effect on the formation and activity of Escherichia coli alkaline phosphatase were able to substitute for proline in protein synthesis in a proline auxotroph. One of these, 3,4-dehydroproline, effectively replaced proline and led to formation of an active enzyme under conditions where no proline was present in the polypeptides. Substitution of azetidine-2-carboxylate for proline prevented active enzyme formation, producing instead altered monomeric forms of the alkaline phosphatase. These were detected with antibodies specific to denatured forms of the enzyme, and they were also characterized, together with cellular proteins, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Alkaline phosphatase, as well as several other proteins, is localized exterior to the bacterial cell cytoplasm in the periplasmic space. In the presence of azetidine-2-carboxylate, a substantial number of these periplasmic proteins retain their specific site of localization, and the denatured subunits of alkaline phosphatase were only detected in the periplasmic fraction of the cell. Thus, secretion of these proteins does not appear to require a high degree of specificity in the native structure of the polypeptide chain. The analogues 4-allohydroxyproline and 4-thiazolidine carboxylate were unable to substitute for proline in protein synthesis but they inhibited growth of E. coli.     

Expression and Localization of Escherichia coli Alkaline Phosphatase Synthesized in Salmonella typhimurium Cytoplasm

The Escherichia coli structural gene for alkaline phosphatase was inserted into Salmonella typhimurium by episomal transfer in order to determine whether this enzyme would continue to be localized to the periplasmic space of the bacterium even though it was formed in a cell that does not synthesize alkaline phosphatase. The S. typhimurium heterogenote synthesized alkaline phosphatase under conditions identical to that observed with E. coli. This enzyme appeared to be identical to that synthesized by E. coli, and was quantitatively released from the bacterial cell by spheroplast formation with lysozyme. These results showed that localization is not a property unique to the E. coli cell and suggested that, in E. coli, enzyme location is related to the structure of the protein. Formation of alkaline phosphatase in the S. typhimurium heterogenote was repressed in cells growing in a medium with excess inorganic phosphate, even though only one of the three regulatory genes for this enzyme is on the episome. Thus, S. typhimurium can supply the products of the other two regulatory genes essential for repression even though this bacterium seems to lack the structural gene for alkaline phosphatase.     

Induction of alkaline phosphatase in Escherichia coli: effect of procaine hydrochloride.

The effect of procaine hydrochloride, an anesthetic known to alter membrane structure, on the induced formation of alkaline phosphatase, a periplasmic enzyme, in Escherichia coli was investigated. Procaine hydrochloride specifically arrested the appearance of active alkaline phosphatase while permitting the induction of another enzyme, beta-galactosidase, which is internally localized. Evidence has been obtained to show that procaine hydrochloride does not arrest synthesis of inactive monomer subunits of the enzyme, indicating that the drug interferes in the conversion of monomer subunits to an active dimer enzyme.     

Cell surface-localized alkaline phosphatase of Escherichia coli as visualized by reaction product deposition and ferritin-labeled antibodies.

When cells of a wild-type Eschericia coli O8 strain bearing a complete lipopolysaccharide were incubated for alkaline phosphatase reaction product and examined by electron microscopy, the depostion of lead salts was to be observed primarily within the periplasmic space. A similar treatment of cells derived from this strain, which bears a highly abbreviated lipopolysaccharide, showed a mixed cell surface and periplasmic localization of reaction product, suggesting a surface association of a portion of the enzyme. To further explore this possibility, ferritin-antibody conjugates against the active enzyme and its irreversibly dissociated subunits were prepared and allowed to react with cells of both strains. The results obtained from these experiments revealed the presence of both the active enzyme and inactive subunits of the enzyme at the cell surface of the mutant strain. The evidence obtained offers further proof of the validity of the reaction product deposition technique and indicates that alkaline phosphatase may be associated with some component of the outer membrane in this organism. The observation of enzyme subunits at the cell surface further suggests that an association of these subunits with structural components of the cell envelope may provide a locus at which they may dimerize to form active enzyme.     

Induction of alkaline phosphatase in Escherichia coli. Effect of phenethyl alcohol.

Induction of alkaline phosphatase, an enzyme located in the periplasmic region of Escherichia coli, was inhibited by phenethyl alcohol, an agent believed to alter the cell membrane structure. Studies to elucidate mechanism of this inhibition showed that while phenethyl alcohol arrested the incorporation of [3H]leucine into active alkaline phosphatase, it did allow substantial incorporation of the label into inactive monomer subunits of the enzyme. These results suggest that phenethyl alcohol may not interfere with the de novo synthesis of monomer subunits of the enzyme but arrest conversion of these into active dimer enzyme presumably by its primary action on the cell membrane structure.     

Purification of Escherichia coli alkaline phosphatase on an ion-exchange high-performance liquid chromatographic column using carboxymethyl dextrans

Carboxymethyl dextrans (CM-Ds) were used on an HPLC ion-exchange column to obtain significantly enriched alkaline phosphatase (EC 3.1.3.1) from a sample of Escherichia coli periplasmic space proteins without significant loss of enzymatic activity. The ability of CM-Ds to separate alkaline phosphatase even when the column was 80-85% saturated with protein demonstrates the potential for high column capacity using CM-Ds. In addition, the fractions containing alkaline phosphatase and CM-Ds were reapplied to the same ion-exchange column under different buffer conditions and purified to homogeneity by salt gradient elution chromatography, thus demonstrating the compatibility of CM-Ds with the latter chromatographic method. The two-step chromatographic procedure yielded enzyme of purity comparable to that of electrophoretically purified E. coli alkaline phosphatase obtained commercially. These studies demonstrate that HPLC displacement chromatography is a mild procedure which allows rapid, quantitative purification of an enzyme. Scaling up with larger columns should allow purification of enzymes of a commercial basis.     

High-level overproduction of <Emphasis Type="Italic">Thermus</Emphasis> enzymes in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermophilus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes corresponding to an alkaline phosphatase (a periplasmic enzyme in T. thermophilus) and that corresponding to a beta-glycosidase (an intracellular enzyme) in Escherichia coli and in S. lividans. Comparison of the production in both hosts revealed that the expression of active protein achieved in S. lividans was much higher than in E. coli, especially in the case of the periplasmic enzyme. In fact, the native signal peptide of the T. thermophilus phosphatase was functional in S. lividans, being processed at the same peptide bond in both organisms, allowing the overproduction and secretion of this protein to the S. lividans culture supernatant. As in E. coli, the thermostability of the expressed proteins allowed a huge purification factor upon thermal denaturation and precipitation of the host proteins. We conclude that S. lividans is a very efficient and industry-friendly host for the expression of thermophilic proteins from Thermus spp.     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Topology of the Escherichia coli uhpT sugar-phosphate transporter analyzed by using TnphoA fusions.

The Escherichia coli uhpT protein catalyzes the active transport of sugar-phosphates by an obligatory exchange mechanism. To examine its transmembrane topology, we isolated a collection of uhpT-phoA fusions encoding hybrid proteins of different lengths from the N terminus of UhpT fused to alkaline phosphatase by using transposon TnphoA. These fusions displayed different levels of alkaline phosphatase activity, although comparable levels of full-length UhpT-PhoA proteins were produced in maxicells of both high- and low-activity fusions. The full-length protein species were unstable and were degraded to the size of the alkaline phosphatase moiety in the case of a high-activity fusion or to small fragments in the case of a low-activity fusion. The enzyme activity present in low-activity fusions appeared to result from export of a small proportion of the fusion proteins to the periplasmic space. Although fusions were not obtained in all predicted extramembranous loops, the deduced topology of UhpT was consistent with a model of 12 membrane-spanning regions oriented with the amino and carboxyl termini in the cytoplasm.     

Growth defects of Escherichia coli cells which contain the gene of an alpha-amylase from Bacillus coagulans on a multicopy plasmid

An alpha-amylase gene from Bacillus coagulans has previously been cloned in Escherichia coli and shown to direct the synthesis of an enzymically active protein of 60,000 Dal (Cornelis et al., 1982). In one particular E. coli host, strain HB101, amylase was found to accumulate in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into E. coli 188 which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for beta-galactosidase, a cytoplasmic marker. Abnormally large amounts of both alpha-amylase and beta-galactosidase were found in the culture fluid of cells grown in rich medium. Furthermore a severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by two types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. These results suggest that the synthesis and export of B. coagulans alpha-amylase is deleterious to E. coli, especially in media containing maltose or glycerol as sole carbon source.     

Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and beta-galactosidase fusions.

The mtr gene of Escherichia coli K-12 encodes an inner membrane protein which is responsible for the active transport of trypotophan into the cell. It has been proposed that the Mtr permease has a novel structure consisting of 11 hydrophobic transmembrane spans, with a cytoplasmically disposed amino terminus and a carboxyl terminus located in the periplasmic space (J.P. Sarsero, P. J. Wookey, P. Gollnick, C. Yanofsky, and A.J. Pittard, J. Bacteriol. 173:3231-3234, 1991). The validity of this model was examined by the construction of fusion proteins between the Mtr permease and alkaline phosphatase or beta-galactosidase. In addition to the conventional methods, in which the reporter enzyme replaces a carboxyl-terminal portion of the membrane protein, the recently developed alkaline phosphatase sandwich fusion technique was utilized, in which alkaline phosphatase is inserted into an otherwise intact membrane protein. A cluster of alkaline phosphatase fusions to the carboxyl-terminal end of the Mtr permease exhibited high levels of alkaline phosphatase activity, giving support to the proposition of a periplasmically located carboxyl terminus. The majority of fusion proteins produced enzymatic activities which were in agreement with the positions of the fusion sites on the proposed topological model of the permease. The synthesis of a small cluster of hybrid proteins, whose enzymatic activity did not agree with the location of their fusion sites within putative transmembrane span VIII or the preceding periplasmic loop, was not detected by immunological techniques and did not necessitate modification of the proposed model in this region. Slight alterations may need to be made in the positioning of the carboxyl-terminal end of transmembrane span X.     

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.     

Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase.

An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E. coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E. coli alkaline phosphatase (APase) gene. One of the transformants was named E15/pA alpha NE1. Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins. The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it. A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E. coli. Transportation of the chimeric proteins to periplasmic space was studied. All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space.     

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase.

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.     

Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.     

Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation

An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis. The intact gene could not be assembled in Escherichia coli and is presumed to be lethal. Therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect. A Gln-73 mutant gene was stable in E. coli but only produced low amounts of barnase antigen. Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for barnase antigen. None of the mutant proteins with alterations at aa residue 102 possessed RNase activity. The level of barnase (Asp-102) was higher in E. coli than in B. subtilis but the protein was not processed to the correct size in E. coli. To obtain correct processing, the barnase (Asp-102) structural gene was fused to the E. coli alkaline phosphatase promoter and signal sequence (phoA). Cells containing this construct secreted correctly processed barnase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/l. Barnase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active barnase (His-102). The cloning and expression of barnase in E. coli will allow detailed analysis of barnase protein folding by molecular genetic approaches.