Analysis of protein localization by use of gene fusions with complementary properties.

This report describes a new transposon designed to facilitate the combined use of beta-galactosidase and alkaline phosphatase gene fusions in the analysis of protein localization. The transposon, called TnlacZ, is a Tn5 derivative that permits the generation of gene fusions encoding hybrid proteins carrying beta-galactosidase at their C termini. In tests with plasmids, TnlacZ insertions that led to high cellular beta-galactosidase activity were restricted to sequences encoding either cytoplasmic proteins or cytoplasmic segments of a membrane protein. The fusion characteristics of TnlacZ are thus complementary to those of TnphoA, a transposon able to generate alkaline phosphatase fusions whose high-activity insertion sites generally correspond to periplasmic sequences. The structure of TnlacZ allows the conversion of a TnlacZ fusion into the corresponding TnphoA fusion (and vice versa) through recombination or in vitro manipulation in a process called fusion switching. Fusion switching was used to generate the following two types of fusions with unusual properties: a low-specific-activity beta-galactosidase-alkaline phosphatase gene fusion and two toxic periplasmic-domain serine chemoreceptor-beta-galactosidase gene fusions. The generation of both beta-galactosidase and alkaline phosphatase fusions at exactly the same site in a protein permits a comparison of the two enzyme activities in evaluating the subcellular location of the site, such as in studies of membrane protein topology. In addition, fusion switching makes it possible to generate gene fusions whose properties should facilitate the isolation of mutants defective in the export or membrane anchoring of different cell envelope proteins.     

A gene fusion approach to the study of pullulanase export and secretion in Eschenchia coli

A series of fusions between the gene for the Klebsiella pneumoniae secreted lipoprotein pullulanase (pulA) and the genes for cytoplasmic beta-galactosidase (lacZ) or periplasmic alkaline phosphatase (phoA) were created by transposon mutagenesis using mini-MudII1681 or TnphoA, respectively. The hybrid genes were expressed in Escherichia coli K-12 with or without the K. pneumoniae genes that promote pullulanase secretion in E. coli. We characterized seven different pulA-lacZ gene fusions encoding hybrid polypeptides containing from 14 to c. 1060 residues of pro-pullulanase. All but the smallest hybrid were fatty acylated and were toxic to producing cells, causing the accumulation of precursors of other exported proteins. Four different pulA-phoA gene fusions encoded hybrids with alkaline phosphatase activity. All four hybrids were fatty acylated, but were not toxic. Although the hybrids were apparently membrane-associated, they were not secreted into the medium either by E. coli carrying pullulanase secretion genes or by K. pneumoniae. Immunofluorescence tests indicated that the pullulanase secretion genes promoted the localization of one of these hybrids to the outer face of the E. coli outer membrane, which may have important implications for the design of live vaccine strains and of immobilized enzymes.     

In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli.

Using recombinant DNA techniques, we have constructed phoA-lacZ gene fusions. Two of the fusions encode hybrid proteins containing approximately half of alkaline phosphatase at the amino terminus joined to beta-galactosidase. For the one fusion strain analyzed in detail, it was shown that the hybrid protein is found in the membrane fraction of cells. In its membrane location, the beta-galactosidase activity of the hybrid is not sufficient to support cell growth on lactose. Unexpectedly, fusions containing phoA and lacZ joined in the wrong translational reading frame were also obtained. These fusions direct the phosphate-regulated synthesis of beta-galactosidase, apparently via a translation restart mechanism. Thus, when gene fusions are constructed, the presence of properly regulated beta-galactosidase activity does not necessarily indicate that a hybrid protein is being produced.     

Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.     

Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.     

Use of gene fusions to determine a partial signal sequence of alkaline phosphatase.

We have isolated strains of Escherichia coli in which an amino-terminal portion of the cytoplasmic enzyme beta-galactosidase is replaced by an amino-terminal portion of the periplasmic enzyme alkaline phosphatase. The synthesis of these hybrid proteins is regulated by inorganic phosphate and they are located in the cytoplasm. One of these proteins was purified, and 14 amino acids of the amino-terminal sequence were determined. The first five amino acids, Met-Lys-Gln-Ser-Thr, appear to represent a portion of the signal sequence of the precursor of alkaline phosphatase, and the remaining sequence corresponds to that of beta-galactosidase, beginning at amino acid residue 20. The approach described here could be used for the analysis of signal sequences of exported proteins and for partial amino acid sequence determination of certain of certain other proteins.     

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.     

A molecular, genetic and immunological approach to the functioning of colicin A, a pore-forming protein

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.     

Topology and function of the integral membrane protein conferring immunity to colicin A

The topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore-forming colicin A, was analysed using fusions to alkaline phosphatase. The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds. The protein was found to contain four transmembrane sequences and its N- and C-terminal regions were found to be directed towards the cytoplasm. Oligonucleotide-directed mutagenesis and sequence comparisons between Cai, Cbi (colicin B immunity protein), and Cni (colicin N immunity protein) were carried out to determine the functional regions of Cai. The possible roles of the various regions of Cai in its protective function and in its topological organization are discussed.     

Release of colicin E2 from Escherichia coli.

Treatment of Escherichia coli K-12(ColE2.P9) with 500 ng of mitomycin C per ml resulted in rapid and almost synchronous colicin E2 production. Colicin accumulated outside the cytoplasmic membrane, most probably in the periplasmic space. Colicin release occurred during a period in which the turbidity of the culture declined markedly. Periplasmic alkaline phosphatase was released during the same period, but cytoplasmic beta-galactosidase release was delayed.     

The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o terminal oxidase complex is a component of the aerobic respiratory chain of Escherichia coli. This enzyme catalyzes the oxidation of ubiquinol-8 to ubiquinone-8 within the cytoplasmic membrane and the concomitant reduction of O2 to H2O. The hydropathy profiles of the deduced amino acid sequences suggest that all five of the gene products of the cyo operon contain multiple membrane-spanning helical segments. The goal of this work was to obtain experimental evidence for the topology of the five gene products in the cytoplasmic membrane by using the technique of gene fusions. A number of random gene fusions were generated in vitro encoding hybrid proteins in which the amino-terminal portion was provided by the subunit of interest and the carboxyl-terminal portion by one of two sensor proteins, alkaline phosphatase lacking its signal sequence or beta-galactosidase. Results obtained are self-consistent, and topological models are proposed for all of the five gene products encoded by the cyo operon. Based on the sequence similarities with subunits of the aa3-type cytochrome c oxidases, the experimental evidence obtained here can be used to infer topological models for the mitochondrial encoded subunits of the eukaryotic cytochrome c oxidases.     

The normally periplasmic enzyme β-lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell-surface enzyme pullulanase

Hybrid proteins were constructed in which C-terminal regions of the bacterial cell surface and extracellular protein pullulanase were replaced by the mature forms of the normally periplasmic Escherichia coli proteins beta-lactamase or alkaline phosphatase. In E. coli strains expressing all pullulanase secretion genes, pullulanase-beta-lactamase hybrid protein molecules containing an N-terminal 834-amino-acid pullulanase segment were efficiently and completely transported to the cell surface. This hybrid protein remained temporarily anchored to the cell surface, presumably via fatty acids attached to the N-terminal cysteine of the pullulanase segment, and was subsequently specifically released into the medium in a manner indistinguishable from that of pullulanase itself. These results suggest that the C-terminal extremity of pullulanase lacks signal(s) required for export to the cell surface. When beta-lactamase was replaced by alkaline phosphatase, the resulting hybrid also became exposed at the cell surface, but exposition was less efficient and specific release into the medium was not observed. We conclude that proteins that do not normally cross the outer membrane can be induced to do so when fused to a permissive site near the C-terminus of pullulanase.     

The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface.

The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic membrane independently of other products of the Ti plasmid. VirB4 was resistant to extraction from membranes of wild-type strain A348 or a Ti-plasmidless strain expressing virB4 from an IncP replicon. To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and 'phoA, which encodes a periplasmically active alkaline phosphatase (AP) deleted of its signal sequence. VirB4::PhoA hybrid proteins exhibiting AP activity in Escherichia coli and A. tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and between residues 450 and 514 (region 2). Conversely, VirB4::beta-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low beta-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high beta-galactosidase activities. Enzymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein. Proteinase K treatment of A. tumefaciens spheroplasts resulted in the disappearance of the 87-kDa VirB4 protein and the concomitant appearance of two immunoreactive species of approximately 35 and approximately 45 kDa. Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains.     

Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase.

Several unknown Escherichia coli genes for different species of acid phosphatase were cloned in vivo with the plasmid Mu dII4042. When present in a multicopy state, each gene promoted hydrolysis of p-nitrophenyl-phosphate at acidic pH. Among seven recombinant clones that encoded periplasmic acid phosphatase activities, five different genes could be distinguished by the pH optimum and substrate preference for the enzyme and by the restriction enzyme pattern. A 1.7-kilobase recombinant DNA fragment, common to two clones, was inserted into plasmid pBR322 and shown to contain a new gene, agp, which leads to the overexpression of the periplasmic acid glucose-1-phosphatase, a dimer of a 44-kilodalton polypeptide. Fusions of agp to gene phoA deprived of its own signal sequence conferred an alkaline phosphatase-positive phenotype to bacteria, showing the presence of an export signal on agp. The resulting hybrid proteins were characterized by immunoprecipitation with an antiserum directed against purified acid phosphatase or against alkaline phosphatase, showing that agp is the structural gene of the acid phosphatase. The beginning, the orientation, and the end of gene agp on the cloned DNA fragment were determined by the characteristics of such hybrid proteins.     

A hybrid toxin from bacteriophage f1 attachment protein and colicin E3 has altered cell receptor specificity.

A hybrid protein was constructed in vitro which consists of the first 372 amino acids of the attachment (gene III) protein of filamentous bacteriophage f1 fused, in frame, to the carboxy-terminal catalytic domain of colicin E3. The hybrid toxin killed cells that had the F-pilus receptor for phage f1 but not F- cells. The activity of the hybrid protein was not dependent upon the presence of the colicin E3 receptor, BtuB protein. The killing activity was colicin E3 specific, since F+ cells expressing the colicin E3 immunity gene were not killed. Entry of the hybrid toxin was also shown to depend on the products of tolA, tolQ, and tolR which are required both for phage f1 infection and for entry of E colicins. TolB protein, which is required for killing by colicin E3, but not for infection by phage f1, was also found to be necessary for the killing activity of the hybrid toxin. The gene III protein-colicin E3 hybrid was released from producing cells into the culture medium, although the colicin E3 lysis protein was not present in those cells. The secretion was shown to depend on the 18-amino-acid-long gene III protein signal sequence. Deletion of amino acids 3 to 18 of the gene III moiety of the hybrid protein resulted in active toxin, which remained inside producing cells unless it was mechanically released.     

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.     

Production of specific antibodies against protein A fusion proteins.

The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta-galactosidase, alkaline phosphatase and human insulin-like growth factor I (IGF-I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG-Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF-I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF-I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed.     

Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K12

Summary To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2–73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, (iii) that colicin N uses OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.