Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end.

Linker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter.     

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.     

Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase.

This article describes the reconstitution in Escherichia coli of a heterologous protein secretion system comprising a gene for an extracellular protein together with its cognate secretion genes. The protein concerned, pullulanase, is a secreted lipoprotein of the Gram-negative bacterium Klebsiella pneumoniae. It is initially localized to the cell surface before being specifically released into the medium. E. coli carrying the cloned pullulanase structural gene (pulA) produces pullulanase but does not expose or secrete it. Secretion genes were cloned together with pulA in an 18.8 kbp fragment of K. pneumoniae chromosomal DNA. E. coli carrying this fragment exhibited maltose-inducible production, exposition and specific secretion of pullulanase. Transposon mutagenesis showed that the secretion genes are located on both sides of pulA. Secretion genes located 5' to pulA were transcribed in the opposite orientation to pulA under the control of the previously identified, malT-regulated malX promoter. Thus these secretion genes are part of the maltose regulon and are therefore co-expressed with pulA. Transposon mutagenesis suggested that secretion genes located 3' of pulA are not co-transcribed with pulA, raising the possibility that some secretion functions are not maltose regulated.     

Protein secretion by gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion by Escherichia coli K-12

Pullulanase secretion in Escherichia coli depends on the expression of a MalT-regulated operon called pulC. Characterization of the first two genes of this operon showed that they encode, respectively, a 31,000-Da protein (PulC) and a 70,600-Da protein (PulD) which has a putative signal peptide and that these two proteins are required for pullulanase secretion. The analysis of alkaline phosphatase hybrid proteins generated by TnphoA mutagenesis of pulC and pulD showed that both PulC and PulD contain export signals which can direct the alkaline phosphatase segment of the hybrids across the inner membrane. A representative PulC-PhoA hybrid protein fractionated mainly with the inner membrane upon isopycnic sucrose gradient centrifugation of membrane vesicles. This, together with sequencing data, suggests that PulC is an inner membrane protein. Antibodies raised against a purified PulD-PhoA hybrid protein were used to show that PulD was enriched in low density outer membrane vesicles.     

Klebsiella pneumoniae strain K21: evidence for the rapid secretion of an unacylated form of pullulanase

Klebsiella pneumoniae strain PAP996 was previously shown to secrete fatty acylated, aggregated (micellar) pullulanase only after the end of exponential growth. Here we show that the closely related strain K21 secretes large amounts of unacylated, non-aggregated (monomeric) pullulanase during exponential growth. Only a small amount (less than 10%) of the secreted pullulanase was initially retained by the exponentially growing cells to be subsequently secreted in a fatty acylated, aggregated form. Despite the absence of fatty acids in secreted monomeric pullulanase, the effects of the antibiotic globomycin on pullulanase maturation indicated that all of the enzyme synthesized by strain K21 is processed by lipoprotein signal peptidase.     

Stable periplasmic secretion intermediate in the general secretory pathway of Escherichia coli.

The secretion of the Klebsiella oxytoca cell surface lipoprotein pullulanase involves translocation across the cytoplasmic and outer membranes of the Gram-negative bacterial cell envelope. A variant of pullulanase was created by fusing the signal peptide-encoding 5' region of the Escherichia coli gene for periplasmic MalE protein to the 3' end of the pulA gene encoding almost the entire mature part of pullulanase. When produced in E. coli carrying the malE-pulA gene fusion on a high copy number plasmid and the complete set of genes specifically required for pullulanase secretion on a second plasmid, the hybrid protein differed from wild-type pullulanase as follows: (i) it was not fatty-acylated; (ii) it was apparently processed by LepB signal peptidase rather than by LspA lipoprotein signal peptidase; (iii) it was released into the periplasm and was only slowly transported across the outer membrane, and (iv) it was released directly into the medium rather than via the usual surface-anchored intermediate. The hybrid protein was secreted more rapidly when malE-pulA was expressed from a low copy number plasmid. The two steps in the secretion pathway could be totally uncoupled by expressing first the malE-pulA gene fusion and then the cognate secretion genes. These results show that fatty-acylation of wild-type PulA is not essential for secretion but may improve its efficiency when large amounts of the protein are produced, that the two steps in secretion can occur quite independently and that the periplasmic intermediate can persist for long periods under certain circumstances.     

Export and secretion of the lipoprotein pullulanase by Klebsiella pneumoniae

Pullulanase, a secreted lipoprotein of Klebsiella pneumoniae, is initially localized to the outer face of the outer membrane, as shown by protease and substrate accessibility and by immunofluorescence tests. Freeze-thaw disruption of these cells released both membrane-associated and apparently soluble forms of pullulanase. Membrane-associated pullulanase co-fractionated with authentic outer membrane vesicles upon isopycnic sucrose-gradient centrifugation, whereas the quasi-soluble form had the same equilibrium density as inner membrane vesicles and extracellular pullulanase aggregates. The latter also contained outer membrane maltoporin, but were largely devoid of other membrane components including LPS and lipids. K. pneumoniae carrying multiple copies of the pullulanase structural gene (pulA) produced increased amounts of cell-associated and secreted pullulanase, but a large proportion of the enzyme was neither exposed on the cell surface nor released into the medium, even after prolonged incubation. This suggests that factors necessary for pullulanase secretion were saturated by the over-produced pullulanase. When pulA was expressed under lacZ promotor control, the pullulanase which was produced was not exposed on the cell surface at any time, suggesting that pullulanase secretion genes are not expressed constitutively, and raising the possibility that they, like pulA, may be part of the maltose regulon.     

Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae.

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.     

Molecular characterization of pulA and its product, pullulanase, a secreted enzyme of Klebsielia pneumoniae UNF5023

The determined nucleotide sequence of the Klebsiella pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090-residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Klebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen-like sequence present at the N-terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C-terminus. Pullulanases with or without the collagen-like sequence could not be separated by gel electrophoresis under denaturing or non-denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso-amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino-terminal 656 residues of prepullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti-pullulanase serum in immuno-fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase-specific extension of the secretion pathway are located near the N-terminus of the pullulanase polypeptide.     

Secretion of the cell surface lipoprotein pullulanase in Escherichia coli. Cooperation or competition between the specific secretion pathway and the lipoprotein sorting pathway.

The fatty acid-acylated enzyme pullulanase is normally found in either of two locations in Escherichia coli, depending on whether or not the producing strains also express the genes specifically required for the second step in pullulanase secretion. When they are expressed, the enzyme is localized to the cell surface, while in their absence, it is directed to an unidentified location in the cell envelope which, upon lysis, forms vesicles whose density is intermediate between those of outer and cytoplasmic membrane vesicles. In order to test the role of the putative lipoprotein sorting signal, Asp2, in pullulanase sorting and secretion, the structural gene (pulA) was subjected to site-directed mutagenesis. Replacement of the Asp2 residue by Asn, Glu, or Ser caused the enzyme to fractionate with outer membrane-derived vesicles rather than with intermediate density vesicles from E. coli cells devoid of pullulanase secretion genes. A pronounced secretion defect was observed in a two-step secretion assay in which the first (sec gene-dependent) and second (pul gene-dependent) secretion steps were uncoupled. We propose that the Asp residue increases the efficiency of pullulanase secretion by allowing the enzyme to be initially sorted to a region of the cell envelope wherein most of the pullulase-specific secretion factors are located.     

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.     

PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA

To enable effective use of phoA gene fusions in Legionella pneumophila, we constructed MudphoA, a derivative of the mini-Mu phage Mu dII4041, which is capable of generating gene fusions to the Escherichia coli alkaline phosphatase gene (EC 3.1.3.1). Although an existing fusion-generating transposon, TnphoA, has been a useful tool for studying secreted proteins in other bacteria, this transposon and other Tn5 derivatives transpose inefficiently in Legionella pneumophila, necessitating the construction of a more effective vector for use in this pathogen. Using MudphoA we generated fusions to an E. coli gene encoding a periplasmic protein and to an L. pneumophila gene encoding an outer membrane protein; both sets of fusions resulted in alkaline phosphatase activity. We have begun to use MudphoA to mutate secreted proteins of L. pneumophila specifically, since this subset of bacterial proteins is most likely to be involved in host-bacterial interactions. This modified transposon may be useful for studies of other bacteria that support transposition of Mu, but not Tn5, derivatives.     

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.     

Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein

The previously uncharacterized third and fourth genes (pulE and pulF) of the pullulanase secretion gene operon of Klebsiella oxytoca strain UNF5023 are, respectively, predicted to encode a 55 kDa polypeptide with a putative nucleotide-binding site, and a highly hydrophobic 44 kDa polypeptide that probably spans the cytoplasmic membrane several times. Expression of pulE in minicells or under the control of a strong bacteriophage T7 promoter resulted in the production of a c. 58 kDa cytoplasmic protein. A representative PulE-beta-galactosidase hybrid protein created by Tnlac mutagenesis was also found mainly in the cytoplasm. These results are in line with the predicted absence from PulE of a region of sufficient hydrophobicity to function as a signal sequence. The PulF polypeptide could not be detected either in minicells or when the gene was transcribed from the T7 promoter, but the acquirement of three pulF-lacZ gene fusions that encoded hybrid proteins with relatively high levels of beta-galactosidase activity indicates that this gene can be transcribed and translated. Gene disruption experiments indicated that both pulE and pulF are required for pullulanase secretion in Escherichia coli K-12. Both proteins exhibit considerable homology throughout their entire lengths with other proteins involved in protein secretion, pilin assembly, conjugation and transformation competence in a variety of bacteria. In addition, PulE protein has consensus sequences found in a wide variety of nucleotide-binding proteins. This study completes the initial characterization of the pullulanase secretion gene operon, which comprises 13 genes that are all essential for the transport of pullulanase across the outer membrane.     

Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae.

Some strains of Klebsiella pneumonia secrete pullulanase, a debranching enzyme which produces linear molecules (maltodextrins, amylose) from amylopectin and glycogen. pulA, the structural gene for pullulanase, was introduced into Escherichia coli, either on a multiple-copy-number plasmid or as a single copy in the chromosome. When in E. coli, pulA was controlled by malT, the positive regulatory gene of the maltose regulon. Indeed, pulA expression was undetectable in a malT-negative mutant and constitutive in a malTc strain. Furthermore, the plasmid carrying pulA titrated the MalT protein. When produced in E. coli, pullulanase was not localized in the same way as in K. pneumoniae. In the latter case it was first exported to the outer membrane, with which it remained loosely associated, and was then released into the growth medium. In E. coli the enzyme was distributed both in the inner and the outer membranes and was never released into the growth medium.     

Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane.

The plant pathogenic enterobacterium Erwinia chrysanthemi EC16 secretes several extracellular, plant cell wall-degrading enzymes, including pectate lyase isozyme PelE. Secretion kinetics of 35S-labeled PelE indicated that the precursor of PelE was rapidly processed by the removal of the amino-terminal signal peptide and that the resulting mature PelE remained cell bound for less than 60 s before being secreted to the bacterial medium. PelE-PhoA (alkaline phosphatase) hybrid proteins generated in vivo by TnphoA insertions were mostly localized in the periplasm of E. chrysanthemi, and one hybrid protein was observed to be associated with the outer membrane of E. chrysanthemi in an out gene-dependent manner. A gene fusion resulting in the substitution of the beta-lactamase signal peptide for the first six amino acids of the PelE signal peptide did not prevent processing or secretion of PelE in E. chrysanthemi. When pelE was overexpressed, mature PelE protein accumulated in the periplasm rather than the cytoplasm in cells of E. chrysanthemi and Escherichia coli MC4100 (pCPP2006), which harbors a functional cluster of E. chrysanthemi out genes. Removal of the signal peptide from pre-PelE was SecA dependent in E. coli MM52 even in the presence of the out gene cluster. These data indicate that the extracellular secretion of pectic enzymes by E. chrysanthemi is an extension of the Sec-dependent pathway for general export of proteins across the bacterial inner membrane.     

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.     

Klebsiella pneumoniae pulS gene encodes an outer membrane lipoprotein required for pullulanase secretion.

The product of the Klebsiella pneumoniae gene pulS, which is located downstream from the pullulanase structural gene (pulA), is essential for the cell surface localization and extracellular release of pullulanase in Escherichia coli K-12. pulS is transcribed in the opposite direction to pulA, from which it is separated by a region of 624 nucleotides. Although this latter region contains a new component of the maltose regulon, pulB, which is transcribed from the pulA promoter, it is not required for pullulanase synthesis or secretion. Unlike pulA and all other pullulanase secretion genes characterized so far, the expression of pulS is not induced by growth in the presence of maltose and is unaffected by mutations in the maltose regulator gene malT. The pulS gene product was identified as a ca. 12-kilodalton outer membrane lipoprotein. The characterization of PulS brings to three the number of identified proteins which are known to be required for pullulanase secretion in addition to the components of the signal sequence-dependent general protein export pathway.     

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.     

Analysis of the subcellular location of pullulanase produced by Escherichia coli carrying the pulA gene from Klebsiella pneumoniae strain UNF5023

Three different techniques, protease accessibility, cell fractionation and in situ immunocytochemistry, were used to study the location of the lipoprotein pullulanase produced by Escherichia coli K12 carrying the cloned pullulanase structural gene (pulA) from Klebsiella pneumoniae, with or without the K. pneumoniae genes required to transport pullulanase to the cell surface (secretion-competent and secretion-incompetent, respectively). Pullulanase produced by secretion-competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked the N-terminal fatty-acylated cysteine residue (and probably also a short N-terminal segment of the pullulanase polypeptide), confirming that the N-terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion-incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electronmicroscopy of vesicles labelled with antipullulanase serum and protein A-gold confirmed that pullulanase was associated with both types of vesicles. When thin-sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion-competent cells and mainly in the proximity of the inner membrane in the secretion-incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility and in situ immunocytochemistry) show that pullulanase is transported to the cell surface of secretion-competent cells, this could not be confirmed by cell-fractionation techniques. Possible explanations for this discrepancy are discussed.