Purification of extracellular alkaline phosphatase released by Escherichia coli excretory mutants

Summary Alkaline phosphatase (APase) is the major protein released into the extracellular medium by strain 706, a periplasmic-excretory (lky) mutant of Escherichia coli K12. We developed a rapid three step procedure for APase purification from culture supernatants of lky mutants. Two ultrafiltration stages and an heat treatment were sufficient to obtain a 99% pure enzyme preparation. Batch culture conditions of strain 706 in a 15 l fermentor leading to an extracellular APase yield of 1250 U/ml were determined.Abbreviation APase E. coli alkaline phosphatase     

Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli.

The intracellular nucleotide pool of Escherichia coli W3110 reproducibly changes from conditions of growth in phosphate excess to phosphate starvation, with at least two nucleotides appearing under starvation conditions and two nucleotides appearing only under excess phosphate conditions. Strains bearing a deletion of the phoA gene show the same pattern, indicating that dephosphorylation by alkaline phosphatase is not responsible for the changes. Strains with mutations in the phoU gene, which result in constitutive expression of the pho regulon, show the nucleotide pattern of phosphate-starved cells even during phosphate excess growth. These changes in nucleotides are therefore due to phoU mutation but not to alkaline phosphatase constitutivity. In fact, a phoR (phoR68) mutant strain has the patterns of the wild type in spite of being constitutive for alkaline phosphatase. That these nucleotides might be specific signals for pho regulon expression was supported by the fact that the two nucleotides appearing under phosphate starvation induced the synthesis of alkaline phosphatase in repressed permeabilized wild-type cells under conditions of phosphate excess.     

Enzyme secretion in Escherichia coli: synthesis of alkaline phosphatase and acid hexose phosphatase in the absence of phospholipid synthesis.

De novo synthesis of two periplasmic enzymes in Escherichia coli, alkaline phosphatase and acid hexose phosphatase, have been studied in the presence and absence of new phospholipid synthesis. Alkaline phosphatase synthesis was initiated by a temperature shift in a strain carrying a phoA amber mutation and a temperature-sensitive suppressor mutation; acid hexose phosphatase was studied after relief of catabolite repression. Glycerol auxotrophs (gpsA) were used to control phospholipid synthesis. Synthesis of both enzymes proceeded at a normal rate for 0.5 to 1.0 generation of growth, although it was then curtailed. It is concluded that secretion of these enzymes is not obligatorily coupled to new net phospholipid synthesis.     

Co-regulation of the phosphate-binding protein and alkaline phosphatase synthesis in Escherichia coli.

In phosphate-starved cells of Escherichia coli, the synthesis of alkaline phosphatase and some additional periplasmic proteins is derepressed. One of these proteins, which does not appear in a phoS- constitutive strain, has been identified as well the periplasmic phosphate-binding protein.     

Production of alkaline phosphatase by immobilized growing cells of Escherichia coli excretory mutants

Summary In continuous cultures, alkaline phosphatase was synthesised and excreted for more than 250 h by immobilized growing cells in contrast to free cells for which the excretion decreased after 150 h of culture. This observed increase in alkaline phosphatase synthesis and excretion by immobilized cells may have resulted from growing conditions within the gel beads.Offprint requests to: C. Manin     

Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis.

Elimination of plasmids from regenerating S. aureus protoplasts occurred when the regeneration medium contained sucrose but not when it contained sodium succinate. This difference was caused by the occurrence of cell division prior to regeneration of the cell wall on sucrose but not on succinate. Coexisting compatible plasmids were cured independently; coexisting incompatible plasmids were cured jointly. These results support the hypothesis that plasmid pools exist as physically sequestered units in protoplasts and that curing is a consequence of the segregation of such units during abnormal division of wall-less organisms.     

Excretion of alkaline phosphatase by Escherichia coli K-12 pho constitutive mutants transformed with plasmids carrying the alkaline phosphatase structural gene.

Escherichia coli alkaline phosphatase constitutive mutants carrying a pst or a phoS mutation and a plasmid-bearing gene phoA+ excreted into the growth medium up to 50% of the total alkaline phosphatase production. This excretion was pH dependent and did not involve drastic modifications of the cell envelope. Alkaline phosphatase accounted for 80% of total released proteins. Amplification of gene phoA+ was a necessary condition for excretion to occur. When the beta-lactamase structural gene bla+ was coamplified with gene phoA+, both enzymes were excreted. pst-transformed excretory strains did not show the pleiotrophic phenotype previously described for lky mutants.     

Optimized extracellular production of alkaline phosphatase by lky mutants of Escherichia coli K12

Summary Periplasmic-leaky (lky) Hfr mutant strains of Escherichia coli K12, grown in low-phosphate Tris medium, excreted alkaline phosphatase (AP) into the extracellular fluid. The lky207 mutation, which proved to induce the highest AP excretion rate, was transferred to an F^- host, carrying a phoS, T mutation allowing constitutive AP biosynthesis. Use of high-phosphate LB-rich medium for growing this F^- lky strain improved cell biomass, extracellular AP activity and excretion specificity in favour of the enzyme. Physiological studies helped us to develop a new culture medium (LB 8.3) giving higher enzyme and excretion yields. LB 8.3 medium also increased cell viability of lky mutants stored at 4° C.Using optimized culture conditions, the highest extracellular enzyme activity produced by lky mutant 706 was reached in the late stationary growth phase and was equal to 1,400 U/ml of culture medium (i.e., 6 times the intracellular AP content of wild-type strain, Ga15, developed in derepressed conditions); AP released into the extracellular fluid corresponded to 34% of total excreted proteins and was equivalent to a purified enzyme preparation.     

Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunological techniques, we have compared the synthesis of the phoA protein (alkaline phosphatase) and the phoS protein (phosphate-binding protein) in response to the level of phosphate in the medium in different genetic backgrounds containing the known alkaline phosphatase control mutations. Both proteins are produced in excess phosphate media in a phoR1a- strain, whereas neither protein is produced in a phoB- strain even under derepression conditions. In four different phoR1c- strains, however, the phoA product cannot be detected in extracts of cells obtained from any growth condition, whereas the phoS product is produced in both excess and limiting phosphate media. It is not yet known if phoR1c- mutants are a special class of mutations within the phoB gene or whether they occur in a separate cistron involved in alkaline phosphatase regulation. From these results we conclude that the expression of the phoA gene is not always co-regulated with expression of the phoS gene product. We have determined that the phoS protein is a component of periplasmic protein band P4 described by Morris et al. (1974). The phoS product lacks sulfur-containing amino acids and is extractable by treatment with polymyxin sulfate. The other component of band P4 contains methionine and/or cysteine and is not extracted by polymyxin sulfate treatment. Like the phoS and phoA proteins, its synthesis is sensitive to the concentration of phosphate in the growth medium. In addition, the existence of a new class of periplasmic proteins synthesized at maximum rate in high phosphate media is demonstrated.     

Escherichia coli mutants deficient in the production of alkaline phosphatase isozymes.

Escherichia coli K-12 mutants showing an altered isozyme pattern of alkaline phosphatase were isolated. Whereas wild-type strains synthesized all three isozymes in a synthetic medium supplemented with Casamino Acids or arginine but synthesized only isozyme 3 in a medium without supplement, the mutant strains synthesized isozyme 1 and a small amount (if any) of isozyme 2, but no isozyme 3, under all growth conditions. The mutation responsible for the altered isozyme pattern, designated iap, was mapped by P1 transduction in the interval between cysC and srl (at about 58.5 min on the E. coli genetic map). It was cotransducible with cysC and srl at frequencies of 0.54 and 0.08, respectively. The order of the genes in this region was srl-iap-cysC-argA-thyA-lysA. Three more independent mutations were also mapped in the same locus. We purified isozymes 1' and 3' from iap and iap+ strains and analyzed the sequences of four amino acids from the amino terminus of each polypeptide. They were Arg-Thr-Pro-Glu (or Gln) in isozyme 1' and Thr-Pro-Glu (or gln)-Met in isozyme 3', which were identical with those of corresponding isozymes produced by the wild-type phoA+ strain (P.M. Kelley, P.A. Neumann, K. Schriefer, F. Cancedda, M.J. Schlesinger, and R.A. Bradshaw, Biochemistry 12:3499-3503, 1973; M.J. Schlesinger, W. Bloch, and P.M. Kelley, p. 333-342, in Isozymes, Academic Press Inc., 1975). These results indicate that the different mobilities of isozymes 1, 2, and 3 are determined by the presence or absence of amino-terminal arginine residues in polypeptides.     

Effect of export-specific cytoplasmic chaperone, protein SecB, on secretion of Escherichia coli alkaline phosphatase

The efficiency of secretion of Escherichia coli alkaline phosphatase depends on the presence in cells of a cytoplasmic chaperone—protein SecB. Secretion increases in the presence of this chaperone at 30°C, which is the most favorable for the interaction of SecB with the export-initiation domain found previously in the N-terminal region of the mature enzyme. This interaction most likely occurs in the region of the export domain, which is located close to the signal peptide and in complex with a translocational ATPase—protein SecA.     

deoP1 promoter and operator mutants in Escherichia coli: isolation and characterization

Plasmid DNA containing deoP1, one of the two major promoters of the deo operon, has been mutagenized using hydroxylamine, and promoter down-mutations and operator mutations were selected. The isolated mutants are all located within a 16 bp palindromic sequence containing the -10 region of deoP1. The results show that RNA polymerase and DeoR repressor compete for the same DNA target. The deoP1 promotor activity is dependent on a TG motif one base pair upstream of the -10 consensus sequence. The sequence of the deo operator site was further verified by use of a synthetic linker.     

Inactivation of pgsA gene affects biosynthesis and secretion of Escherichia coli alkaline phosphatase

Inactivation of pgsA, which is responsible for biosynthesis of anionic phospholipid phosphatidylglycerol (PG), was shown to affect biosynthesis and secretion of alkaline phosphatase (PhoA) in Escherichia coli. A decrease in PG, but not in total anionic phospholipids, correlated with reduction of PhoA secretion, suggesting the role of PG in this process. A dramatic decrease in PG (from 18 to 3, but not 8, percent of the total phospholipids) inhibited not only secretion, but also synthesis of PhoA. In addition, pgsA inactivation expedited repression of PhoA synthesis by exogenous orthophosphate.     

Isolation and preliminary characterization of mutants of Escherichia coli K-12 overproducers of 2 exported proteins: beta-lactamase and alkaline phosphatase

Spontaneous mutants of Escherichia coli characterized by the overproduction of two periplasmic proteins, beta-lactamase and alkaline phosphatase were isolated. Such olp (Overproduction of beta-Lactamase and alkaline Phosphatase) mutants were selected for growth in the presence of ampicillin and were identified on the basis of their increased content in alkaline phosphatase activity. Phenotypic analysis of olp mutants (resistance to bacteriophages and colicins) suggest that the organisation of their envelope has been deeply modified. Analysis of their cell envelope protein composition indicated that most mutants have a decreased content of porin proteins OmpF and OmpC. These mutations were mapped near the mtl locus, at minute 81 of the bacterial genetic map.     

Osmoregulation of alkaline phosphatase synthesis in Escherichia coli K-12.

Alkaline phosphatase, the phoA product, is synthesized constitutively in phoR mutants. This constitutive synthesis, which is independent of phosphate control, varies with changes in the osmolarity of the growth medium; phoA expression increases with increasing osmolarity. Maximum expression of the osmoregulated genes phoA, ompC, and ompF was achieved by osmotic manipulation of minimal medium; complex media repressed their expression.