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.     

Construction, expression, and localization of a CycA::PhoA fusion protein in Rhodobacter sphaeroides and Escherichia coli.

We demonstrated the utility of Escherichia coli alkaline phosphatase, encoded by phoA, as a reporter molecule for genetic fusions in Rhodobacter sphaeroides. A portion of the R. sphaeroides cycA gene was fused to phoA, yielding a fusion protein comprising the putative signal sequence and first 10 amino acids of the cytochrome c2 apoprotein joined to the sixth amino acid of alkaline phosphatase. The fusion protein was efficiently transported to the periplasm of R. sphaeroides as determined by enzyme activity, Western immunoblot analysis, and immunogold electron microscopy. We also documented the ability of an R. sphaeroides mutant, RS104, with gross defects in photosynthetic membrane morphology to efficiently recognize and translocate the fusion protein to the periplasmic compartment. The inclusion of 500 base pairs of R. sphaeroides DNA in cis to the cycA structural gene resulted in a 2.5-fold increase in alkaline phosphatase activity in photosynthetically grown cells compared with the activity in aerobically grown cells, demonstrating that the fusion protein is regulated in a manner similar to that of cytochrome c2 regulation. We also constructed two pUC19-based plasmids suitable for the construction of translational fusions to phoA. In these plasmids, translational fusions of phoA to the gene under consideration can be made in all three reading frames, thus facilitating construction and expression of fusion protein systems utilizing phoA.     

A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli

A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.     

A system for the inducible secretion of proteins from Bacillus subtilis during logarithmic growth

Abstract A Bacillus subtilis-Escherichia coli shuttle vector was constructed containing the B. subtilis levansucrase gene promoter and region encoding its signal sequence.
A site for the restriction enzyme Nae I was included to facilitate precise translational fusions to the DNA encoding the levansucrase signal sequence. Fusions of TEM β-lactamase to this construct displayed sucrose-inducible expression and secretion of B. subtilis .     

Chloramphenicol acetyltransferase, a cytoplasmic protein is incompatible for export from Bacillus subtilis.

Bacillus subtilis cells expressing a hybrid protein (Lvsss-Cat) consisting of the B. amyloliquefaciens levansucrase signal peptide fused to B. pumilus chloramphenicol acetyltransferase (Cat) are unable to export Cat protein into the growth medium. A series of tripartite protein fusions was constructed by inserting various lengths of the Cat sequences between the levansucrase signal peptide and staphylococcal protein A or Escherichia coli alkaline phosphatase. Biochemical characterization of the various Cat protein fusions revealed that multiple regions in the Cat protein were causing the export defect.     

Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus subtilis

Two different hybrid genes were constructed which fuse the Bacillus amyloliquefaciens alkaline protease gene (apr[BamP]) promoter and signal peptide coding region to a synthetic bpr gene coding for the mature bovine pancreatic RNase A. The first gene fusion (apr-bpr1) contained the apr[BamP] signal peptide coding region fused to mature bpr through a linker coded 3-amino acid region and retained the signal processing site ala-ala of the alkaline protease. The second fusion (apr-bpr2) joined the end of the apr[BamP] signal peptide coding sequence to the mature bpr resulting in a hybrid signal processing site ala-lys. B. subtilis strains harboring these gene fusions secreted bovine pancreatic RNase A into the growth medium. Cleavage at the hybrid signal processing site ala-lys resulted in the secretion of bovine pancreatic RNase A from B. subtilis which had an N-terminal amino acid sequence that was identical to the native RNase A. Bovine pancreatic RNase A contains four disulfide bonds and the proper formation of these bonds is required for activity. RNase activity could be detected in the culture supernatants of strains carrying the apr-bpr gene fusions, which suggests that the proper disulfide bonds have formed spontaneously.     

Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase.

The structural gene (appA) for the periplasmic acid phosphatase (optimum pH 2.5) of Escherichia coli was cloned into a plasmid by using a combination of in vivo and in vitro techniques. The position and orientation of the appA gene within the cloned DNA fragment were identified by using fusions to the alkaline phosphatase gene (phoA) generated by Tn5 IS50L::phoA (TnphoA) insertions. For TnphoA-generated hybrid proteins to have high enzymatic activity, it appears that the phoA gene must be fused to a target gene coding for a signal which promotes protein export. The approach used to identify the appA gene thus appears to provide a simple general means of selectively identifying genes encoding membrane and secreted proteins.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process

The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents. Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids. The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein. To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively. All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity. The most N-terminal fusion joint is at amino acid 19 of Tsr. Tsr-lacZ fusions were found throughout the tsr gene. The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint. Fusions with low activity have fusion joints within the periplasmic loop of Tsr. The expression of these fusions is most likely reduced at the level of translation. In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB. A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature. The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain. We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins. Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.     

Homology between endoglucanase Z of Erwinia chrysanthemi and endoglucanases of Bacillus subtilis and alkalophilic Bacillus

Nucleotide sequencing of the celZ gene encoding the extracellular endoglucanase Z of Erwinia chrysanthemi indicated the presence of an open reading frame encoding 428 amino acids. The mature protein appeared to be extended by a signal peptide of 43 amino acids; this sequence is unusually long and positively charged (+5). It was shown to function as a signal peptide by fusing it to a truncated phoA gene encoding Escherichia coli alkaline phosphatase. Comparison of the encoded sequence with those of the endoglucanases of Bacillus subtilis and alkalophilic Bacillus revealed the existence of a region of extensive homology occurring in all three proteins at about the same distance from the NH2-terminal end. These regions may be involved in substrate binding and/or catalytic sites.     

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.     

Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences.

Secretion vectors based on the genes from Bacillus amyloliquefaciens P for alkaline protease (aprBamP) and neutral protease (nprBamP) were constructed. With both aprBamP and nprBamP, a unique restriction site was introduced 3' of the predicted signal coding region by using the technique of oligonucleotide-directed mutagenesis. The new sites enabled us to fuse a heterologous gene to the expression and secretion elements. We used the protein A gene (spa) from Staphylococcus aureus as a heterologous gene. Bacillus subtilis cells carrying the resulting apr-spa or npr-spa gene fusions synthesized the fusion protein. B. subtilis cells were also capable of removing the signal peptide from the fusion protein, as indicated by the appearance of processed protein A into the growth medium. In addition, these gene fusions allowed us to identify the signal processing site of both the APR-SPA and NPR-SPA proteins.     

Expression of Bacillus amyloliquefaciens amylase and Vibrio alginolyticus protease A fusion genes.

Previously we reported [Deane, S. M., Maharaj, R., Robb, F. T. & Woods, D. R. (1987) Journal of General Microbiology 133, 2295-2302] that the production of a Vibrio alginolyticus SDS-resistant alkaline serine protease (Pro A) cloned in Escherichia coli was characterized by a 12 h delay between the synthesis of an inactive precursor and secretion of active Pro A. Replacement of the V. alginolyticus promoter region by the alpha-amylase promoter region from Bacillus amyloliquefaciens resulted in the simultaneous synthesis and secretion of Pro A in E. coli. The V. alginolyticus pro A gene cloned on a shuttle vector did not produce active Pro A in Bacillus subtilis. Although Pro A has a typical Gram-positive signal sequence, it was not functional in B. subtilis. Replacement of the Pro A signal sequence with the alpha-amylase signal sequence resulted in the production of active Pro A in B. subtilis.     

A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm of Escherichia coli.

A vector encoding the Staphylococcal protein A was modified by cloning the spa gene, including its signal peptide-encoding sequence, downstream of the translation initiation sites of the phage lambda cro gene and under the control of the temperature-inducible phage lambda pR promoter. The expression from this construct was studied using the Escherichia coli phoA gene as a reporter gene after fusion to the spa gene. Determination of alkaline phosphatase activity, 1 h after temperature induction of expression at 42 degrees C, revealed an 800-fold increase over host strain background level. The presence of the alternating selectable markers on the described vector, pHEMa153, which are essential for efficient oligonucleotide-directed construction of mutations by the gapped duplex DNA method, allows the construction of recombinant and mutated forms of Staphylococcal protein A fusion proteins and efficient expression of spa gene fusions without changing the vector system.     

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.     

Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli

We isolated a collection of mutants defective in the export of alkaline phosphatase to the periplasm. Two classes of mutants were obtained: one class with lesions unlinked to the phoA gene and a second class harboring linked mutations. Among the former class, one mutant is cold sensitive for growth and may be defective in a component of the Escherichia coli secretory apparatus. Included in the latter class are 47 mutants which are characterized in detail in this report. To facilitate DNA sequence analysis of these mutants, we devised a convenient method that relies on homologous recombination in vivo to transfer phoA mutations from the bacterial chromosome directly onto the genome of a single-stranded M13 phage vector. DNA sequence analysis revealed that our collection of mutants comprises six unique mutations, all of which reside in the phoA signal sequence coding region and lend further support to the notion that the length of the hydrophobic core of the signal sequence is crucial for its function in protein export. Kinetic studies showed that in these mutants, the small fraction of alkaline phosphatase which succeeds in reaching a periplasmic location, despite a defective signal sequence, is translocated across the membrane in a slow, posttranslational fashion.     

Cloning and nucleotide sequence of phoP, the regulatory gene for alkaline phosphatase and phosphodiesterase in Bacillus subtilis.

Two DNA fragments which complement the alkaline phosphatase-negative mutation phoP of Bacillus subtilis were cloned from a B. subtilis chromosome with the prophage vector phi CM (a derivative of phi 105). One of the fragments contained the regulatory gene phoR in addition to phoP. Nucleotide sequence analysis of the phoP region revealed that the phoP gene product consists of 241-amino-acid residues and that the sequence of these amino acids is extensively homologous with the sequence of the phoB gene product. This protein is the positive regulator for the phosphate regulon in Escherichia coli. It therefore appears that phoP is a regulatory gene for alkaline phosphatase synthesis in B. subtilis.     

Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.     

Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase.

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.