Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA.

Lambda transducing phages containing portions of the phoA gene have been isolated and used to construct a deletion map of the phoA gene. The isolation of a plaque-forming lambda transducing phage carrying the entire phoA gene is also described. Two new methods for screening or selection of mutants that have altered levels of alkaline phosphatase activity are reported.     

Cloning and expression in Escherichia coli of a phoA gene encoding a phosphate-irrepressible alkaline phosphatase of Zymomonas mobilis

The Zymomonas mobilis phoA gene, encoding a phosphate-irrepressible alkaline phosphatase (ZAPase), was cloned and its expression was studied in phoA mutants of Escherichia coli. The ZAPase was recovered in the soluble fraction of E. coli. The enzyme was synthesized constitutively and its synthesis not repressed by phosphate, unlike the phoA gene of E. coli. The phoA gene of Z. mobilis was mutagenized by Mini Mu PR13 and the mutated gene crossed into Z. mobilis in order to obtain phoA mutants by reverse genetics. Although Z. mobilis mutants with Mini Mu PR13 integrated in the chromosome were obtained, none had an allele replacement for none was defective in ZAPase.     

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.     

Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli

Summary Restriction maps of several recombinant plasmids representing a section of the E. coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared. The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase. From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived.     

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.     

Production of extracellular alkaline phosphatase by Escherichia coli K-12 periplasmic-leaky mutants carrying phoA + Plasmids

Summary Col E1 hybrid plasmids carrying the phoA ⁺ structural gene of alkaline phosphatase, a periplasmic enzyme of Escherichia coli K-12, were identified from the Clarke and Carbon genomic bank. Wild-type (lky ⁺) phoA ⁺ plasmid-bearing strains synthesized 14 times more intracellular enzyme than the haploid lky ⁺ strain. Phosphate-induced repression was maintained in transformed strains. PhoA ⁺ plasmids carrying the phoB and phoR regulatory genes were introduced into a periplasmic-leaky (lky) recipient strain able to release alkaline phosphatase into the extracellular medium. Transformed lky mutants excreted up to 90% of total enzyme activity which corresponded to 3.5 times the amount of intracellular alkaline phosphatase made by the haploid lky ⁺ strain. The protein composition analysis of periplasmic and extracellular fractions showed that: (i) wild-type phoA ⁺ hybrid plasmidbearing clones did not excrete alkaline phosphatase but had a modified periplasmic content; (ii) alkaline phosphatase was the major excreted protein by transformed lky mutants. The use of periplasmic-leaky phoA ⁺ hybrid plasmid-bearing mutants for an easier production and purification of alkaline phosphatase is discussed.     

The nucleotide sequence of the promoter and the amino-terminal region of alkaline phosphatase structural gene (phoA) of Escherichia coli

The promoter and the amino-terminal region of phoA, the structural gene for alkaline phosphatase of Escherichia coli K12, was cloned by using a promoter cloning vector pMC1403. The nucleotide sequence of the cloned fragment has been determined. A sequence encoding the amino-terminal portion of mature alkaline phosphatase is found and it is preceded by a sequence encoding the signal peptide. The signal peptide consists of 21 amino acids; Met-Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala. The translation initiation codon is GUG, which is preceded by the Shine-Dalgarno sequence GGAG. Upstream to these sequences, there is a typical procaryotic promoter. TATAGTC for the Pribnow box. Around the Pribnow box, there are several dyad symmetrical sequences, which may probably be concerned with the regulation of this gene.     

Depletion of phosphatidylethanolamine affects secretion of Escherichia coli alkaline phosphatase and its transcriptional expression

In this report we demonstrate that depletion of the major phospholipid phosphatidylethanolamine, a single non-bilayer forming phospholipid of Escherichia coli, significantly reduces the secretion efficiency of alkaline phosphatase in vivo. Secretion, however, is correlated with the content in membranes of cardiolipin, which in combination with selected divalent cations has a strong tendency to adopt a non-bilayer state indicating the possible involvement of lipid polymorphism in efficient protein secretion. Depletion of this zwitterionic phospholipid also inhibits expression of the protein controlled by the endogenous P(PHO) promoter but not the P(BAD) promoter, which is suggested to be due to the effect of unbalanced phospholipid composition on the orthophosphate signal transduction system (Pho regulon) through an effect on its membrane bound sensor.     

Membrane phospholipid composition of Escherichia coli affects secretion of periplasmic alkaline phosphatase into the medium

Secretion of alkaline phosphatase (PhoA) encoded by a gene constituent of plasmids has been studied in Escherichia coli strains with controlled synthesis of anionic phospholipids (phosphatidylglycerol and cardiolipin, strain HDL11) and zwitterionic phospholipid (phosphatidylethanolamine, strain AD93). Changing the phospholipid composition of the membrane of these strains leads to an increase in secretion of PhoA, which is usually localized in the periplasm, into the culture medium. This correlates with a higher secretion of exopolysaccharides and lower content of lipopolysaccharide in the outer membrane. The results show the possibility of coupling protein secretion into the medium with biogenesis of cell envelope components in which phospholipids are involved.     

Repression of alkaline phosphatase in Salmonella typhimurium carrying a phoA+ phoR- episome from Escherichia coli.

Salmonella typhimurium does not produce alkaline phosphatase (nor beta-galactosidase). Nevertheless, it has the function of the phoR+ regulatory gene but lacks the function of the lacI+ regulatory gene. Several periplasmic proteins are derepressed when cells of S. typhimurium are starved for inorganic phosphate. The role of phoR is discussed.     

Use of gene replacement to construct Escherichia coli strains carrying mutations in two genes required for stability of multicopy plasmids.

Escherichia coli mutants completely defective in ColE1 cer-mediated site-specific recombination have been mapped to two genes, xerA and xerB. In this study, xerA xerB double mutants were constructed by gene replacement with a lambda dv plasmid and were shown to be both viable and defective in ColE1 site-specific recombination.     

Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli

The nucleotide sequence of the alkaline phosphatase (APase) gene (phoA) of Escherichia coli strain 294 has been determined. Pre-APase has a total of 471 amino acids (aa) including a signal sequence of 21 aa. The derived aa sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at positions 16 and 36, and glutamic acid instead of glutamine at position 197. Two open reading frames (ORF1 and ORF2) located downstream from phoA or upstream from proC have been found. ORF1 encodes a putative presecretory protein of 106 aa with a signal sequence of 21 or 22 aa. If this protein is actually produced, it may be one of the smallest periplasmic proteins in E. coli.     

Processing of alkaline phosphatase precursor to the mature enzyme by an Escherichia coli inner membrane preparation.

An inner membrane preparation co-translationally cleaved both the alkaline phosphatase and bacteriophage f1 coat protein precursors to the mature proteins. Post-translational outer membrane proteolysis of pre-alkaline phosphatase generated a protein smaller than the authentic monomer.     

Effects of pho regulatory mutations and phoA gene amplification on alkaline phosphatase synthesis and release by lky mutants of Escherichia coli K12

lky mutants of Escherichia coli K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml-1. APase is a phosphate repressible periplasmic enzyme encoded by the gene phoA. With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic lky strains containing either a constitutive pho regulatory mutation, or a hybrid plasmid carrying the structural gene phoA+ and pho regulatory genes, or a transducing phi 80 phoA+ phage. In the presence of the phoS2333 mutation, F- lky strains lysogenized with phi 80 phoBin phoA+ phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml-1) than haploid strain 2336 (phoS+ lky) grown in low phosphate medium. Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release. Sufficient APase was released into the medium to facilitate its purification.     

Interaction of SecB and SecA with the N-terminal region of mature alkaline phosphatase on its secretion in Escherichia coli

Export-specific chaperone SecB and translocational ATPase SecA catalyze the cytoplasmic steps of Sec-dependent secretion in Escherichia coli. Their effects on secretion of periplasmic alkaline phosphatase (PhoA) were shown to depend on the N-terminal region of the mature PhoA sequence contained in the PhoA precursor. Amino acid substitutions in the vicinity of the signal peptide (positions +2, +3) not only dramatically inhibited secretion, but also reduced its dependence on SecB and SecA. Immunoprecipitation reported their impaired binding with mutant prePhoA. The results testified that SecB and SecA interact with the mature PhoA region located close to the signal peptide in prePhoA.     

Aeromonas spp. can secrete Escherichia coli alkaline phosphatase into the culture supernatant, and its release requires a functional general secretion pathway

Aerolysin is a channel-forming protein secreted by Aeromonas hydrophila. To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E. coli, Aeromonas salmonicida, and A. hydrophila. We were surprised to find that secretion of the enzyme by both Aeromonas spp. was independent of the aerolysin segments fused to it. The smallest fusion product contained only the signal sequence and two amino acids of aerolysin. The largest had more than 90% of the aerolysin molecule. The fusion proteins were found in the periplasms of E. coli and A. salmonicida grown in LB medium containing glucose, as well as in the shocked cells. Aerolysin itself was secreted by A. salmonicida under these conditions. In contrast, when A. salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas β-lactamase remained in its normal periplasmic location. Similar results were obtained with A. hydrophila. The change in location of the enzyme in A. salmonicida appeared to be related to the pH of the growth medium. A. salmonicida and A. hydrophila also secreted native E. coli alkaline phosphatase, but A. hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme. We conclude that the Aeromonas secretion system can recognize the E. coli enzyme as an extracellular protein and direct it outside the cell.     

Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro

The structural gene for alkaline phosphatase (phoA) of Escherichia coli was cloned into the PstI site of pBR322, from a transducing bacteriophage, lambda p(phoA-proC). The restriction map of the plasmid was established. Based upon this information, several phoA deletion plasmids as well as a smaller phoA+ plasmid were constructed. The genetic map and restriction map were correlated by recombination analysis. Cells carrying one of the phoA+ plasmids overproduce alkaline phosphatase 10-fold upon phosphate limitation. However, both regulation and processing of the enzyme were found to be normal.     

Secretion of human interferon-alpha induced by using secretion vectors containing a promoter and signal sequence of alkaline phosphatase gene of Escherichia coli

We constructed a new vector containing the promoter and the signal sequence of E. coli phoA gene, the structural gene for the periplasmic alkaline phosphatase. One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence. This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide. Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene. Human alpha-interferon gene was inserted into this HindIII site. When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.