Pullulanase Secretion Research Articles

Outer membrane translocation of the extracellular enzyme pullulanase in Escherichia coli K12 does not require a fatty acylated N-terminal cysteine

Site-directed mutagenesis was used to construct three mutant derivatives of the extracellular, cell surface lipoprotein pullulanase (PulA) in which the normally fatty acylated cysteine of the signal peptide-bearing precursor was replaced by other amino acids. When produced in Escherichia coli expressing all genes required for pullulanase secretion, approximately 90% of the PulA derivatives persisted as cell-associated precursors, indicating inefficient signal peptide processing. Processed (intermediate-sized) forms of the two derivatives that were studied in detail were found to result from proteolytic cleavage at different sites within the signal peptide. Both were further processed to smaller polypeptides by cleavage at an undetermined site that is presumably close to their C termini. The intermediate-sized pullulanase derived from prepullulanase in which Cys+1 had been replaced by Leu and Gly-1 by Glu (PulA:C1L/G-1E) appeared rapidly, was apparently entirely extracellular, and accounted for approximately 10% of synthesized PulA. Prolonged incubation did not result in further conversion of the precursor to the intermediate form, and the precursor remained anchored to the cytoplasmic membrane. The smaller processed form was also found extracellularly. The active form of the extracellular enzyme was monomeric, which is again in contrast to the fatty acylated, wild-type enzyme. Taken together, these results indicate that replacement of Cys+1 of prePulA eliminates processing by lipoprotein signal peptidase and does not permit processing by leader peptidase, but allows inefficient, aberrant processing by an unknown peptidase and immediate secretion of the resulting polypeptide, which retains most of its signal peptide. Processing and secretion only occur when the pullulanase secretion functions are expressed.

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.

Two distinct steps in pullulanase secretion by Escherichia coli K12

Two distinct steps in the secretion of the extracellular, cell-surface-anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide/sec-gene-dependent general export pathway, and then inducing the pulC-O operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate-density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell-surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher-ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.

The general protein‐export pathway is directly required for extracellular pullulanase secretion in Escherichia coli k12

Pullulanase is an extracellular, cell surface-anchored lipoprotein produced by Gram-negative bacteria belonging to the genus Klebsiella. Its correct localization in recombinant Escherichia coli requires the products of 14 genes that are linked to the enzyme structural gene in the Klebsiella chromosome. In addition, we show here that six sec genes (secA, secB, secD, secE, secF and secY) are all required for processing of the prepullulanase signal peptide to occur. This implies that pullulanase crosses the cytoplasmic membrane via the general export pathway of which the sec gene products are essential components. Removal or drastic alteration of the prepullulanase signal peptide cause the enzyme to remain cytoplasmic. We propose that pullulanase secretion occurs in two steps, the first of which is common to all signal peptide-bearing precursors of exported and secreted proteins, whereas the second is specifically involved in translocating pullulanase to the cell surface.

Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged.

Extracellular Production of Klebsiella Pullulanase by Escherichia coli That Carries the Pullulanase Secretion Genes.

The pullulanase gene, pulA, and the genes for pullulanase secretion factors (called the pullulanase secretion genes) from Klebsiella aerogenes were cloned into a cosmid vector, pHC79, in Escherichia coli. Four of one thousand clones isolated secreted pullulanase into their culture broth when the cells were grown with pullulan or maltose. One of the four clones produced much pullulanase extracellularly in a short time, with a level of production eightfold that of the maximum production of the wild-type K. aerogenes W70. Deletion analysis of the recombinant plasmid showed that the pullulanase secretion genes and pulA were all in a 22-kb DNA segment derived from the chromosomal DNA of K. aerogenes.

Protein secretion in Pseudomonas aeruginosa: the xcpA gene encodes an integral inner membrane protein homologous to Klebsiella pneumoniae secretion function protein PulO

xcp mutations have pleiotropic effects on the secretion of proteins in Pseudomonas aeruginosa PAO. The nucleotide sequence of a 1.2-kb DNA fragment that complements the xcp-1 mutation has been determined. Sequence analysis shows the xcpA gene product to be a 31.8-kDa polypeptide, with a highly hydrophobic character. This is consistent with a localization in the cytoplasmic membrane in P. aeruginosa, determined after specific expression of the xcpA gene under control of the T7 phi 10 promoter. A very strong homology was found between XcpA and PulO, a membrane protein required for pullulanase secretion in Klebsiella pneumoniae. This suggests the existence of a signal sequence-dependent secretion process common to these two unrelated gram-negative bacteria.

Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria.

The xcp genes are required for protein secretion by Pseudomonas aeruginosa. They are involved in the second step of the process, i.e. the translocation across the outer membrane, after the exoproteins have reached the periplasm in a signal peptide dependent fashion. The nucleotide sequence of a 2.5 kb DNA fragment containing xcp genes showed at least two complete open reading frames, potentially encoding proteins with molecular weights of 41 and 19 kd. Products with these apparent molecular weights were identified after expression of the DNA fragment in vitro and in vivo. Subcloning and complementation experiments showed that both proteins are required for secretion. The two products are located in the inner membrane and share highly significant homologies with the PulL and PulM proteins which are required for the specific secretion of pullulanase in Klebsiella pneumoniae. These homologies reveal the existence of a common mechanism for protein secretion in Pseudomonas aeruginosa and Klebsiella pneumoniae.

Five additional genes in thepulC-O operon of the gram-negative bacteriumKlebslella oxytoca UNF5023 which are required for pullulanase secretion

DNA sequence analysis, Tnpho and Tntac-1, mutagenesis, deletion analysis, expression under bacteriophage T7 gene 10 promoter control, subcellular fractionation and complementation tests were used to study the function of DNA located in the centre of the pulC-O operon from Klebsiella oxytoca strain UNF5023. The characterized region of the operon includes five genes (pulG, pulH, pulI, pulJ and pulK) coding for apparently integral inner membrane proteins which are required for pullulanase secretion. The results presented here and previously show that the pulC-O operon contains at least 11 pullulanase secretion genes.

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.

Five genes at the 3′ end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion

The nucleotide sequence of a 5082bp fragment of chromosomal DNA from Klebsiella pneumoniae strain UNF5023 is reported. The sequence includes the last four genes of an operon of genes specifically required for the secretion of the enzyme pullulanase. All four genes (pulL, pulM, pulN and pulO) are shown to be required for pullulanase secretion, as is a fifth gene (pulK) which extends beyond the 5' end of the sequenced DNA. The products of the pulL, pulM, pulN and pulO genes (44 kD, 18 kD, 27 kD and 24 kD, respectively) are all predicted to have one or more hydrophobic domains typical of signal sequences and/or membrane anchors, and were all found mainly associated with the inner membranes of subfractionated cells in which the corresponding genes had been expressed from the bacteriophage T7 gene 10 promoter. The results of this study increase the number of genes which have been identified as required for pullulanase secretion to eight, in addition to genes coding for components of the general export pathway.

Biosynthesis and Secretion of Pullulanase, a Lipoprotein from Klebsiella aerogenes

We constructed, by site-directed mutagenesis, a mutant pullulanase gene in which the cysteine residue in a pentapeptide sequence, Leu16-Leu-Ser-Gly-Cys20 within the NH2-terminal region of pullulanase from Klebsiella aerogenes, is replaced by serine (Ser20). The modification, processing, and subcellular localization of the mutant pullulanase were studied. Labeling studies with [3H]palmitate and immunoprecipitation with mouse antiserum raised against pullulanase showed that the wild form of both the extracellular and intracellular pullulanases contained lipids, whereas the mutant enzyme was not modified with lipids. Only the Cys20 was modified with glyceryl lipids. The bulk of the mutant pullulanase was located in the periplasm, but a portion of the unmodified, mutant pullulanase was secreted into the medium. Mutant pullulanases from the extracellular and the periplasm were purified and their NH2-terminal sequences were determined. Both the mutant pullulanases were cleaved between residues of Ser13 and Leu14 which is 6-amino acid residues upstream of the lipid modified pullulanase cleavage site. This new cleavage was resistant to globomycin, an inhibitor of the prolipoprotein signal peptidase of Escherichia coli. These results indicate that the pentapeptide sequence plays an important role in maturation and translocation of pullulanase in K. aerogenes. However, the modification of pullulanase with lipids seems to be not essential for export of the enzyme across the outer membrane.

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 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.

A new regulatory locus of the maltose regulon in Klebsiella pneumoniae strain K21 identified by the study of pullulanase secretion mutants.

This study has shown that Klebsiella pneumoniae strain K21 differs from the previously characterized and closely related K. pneumoniae strain PAP996 in that expression of the pullulanase gene (pulA) and other genes of the maltose regulon is partially independent of exogenous inducer (maltose/maltotriose). Mutants of strain K21 which are defective in pullulanase synthesis and/or secretion were isolated following Tn10 mutagenesis. Three phenotypic classes of mutants were identified. Class I mutants were defective in the surface localization and secretion of pullulanase. Class II mutants did not secrete detectable levels of pullulanase but were able to export pullulanase to the cell surface. Class II mutants also expressed pullulanase and other maltose-regulated genes at markedly lower levels than those found in the parent strain under non-inducing conditions. The single class III mutant was intermediate between K21 and class I mutants; most of the cell-associated pullulanase was localized at the cell surface whilst a significant amount was secreted into the medium. Mapping indicated that all but three of the Tn10 insertions were adjacent to, and at either side of, pulA. One class II mutant carried a Tn10 insertion in or close to malT whereas in the remaining class II mutants the insertions were located at least 4 kb upstream of pulA in a region which may define a new regulatory locus of the maltose operon.

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.

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.

A gene fusion approach to the study of pullulanase export and secretion in Escherichia 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.