Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.     

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.     

Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria.

Type-4 fimbriae (pili) are associated with a phenomenon known as twitching motility, which appears to be involved with bacterial translocation across solid surfaces. Pseudomonas aeruginosa mutants which produce fimbriae, but which have lost the twitching motility function, display altered colony morphology and resistance to fimbrial-specific bacteriophage. We have used phenotypic complementation of such mutants to isolate a region of DNA involved in twitching motility. This region was physically mapped to a SpeI fragment around 20 min on the P. aeruginosa PAO chromosome, remote from the major fimbrial locus (around 75 min) where the structural subunit-encoding gene (fimA/pilA) and ancillary genes required for fimbrial assembly (pilB, C and D) are found. A gene, pilT, within the twitching motility region is predicted to encode a 344-amino acid protein which has strong homology to a variety of other bacterial proteins. These include the P. aeruginosa PilB protein, the ComG ORF-1 protein from the Bacillus subtilis comG operon (necessary for competence), the PulE protein from the Klebsiella oxytoca (formerly K. pneumoniae) pulC-O operon (involved in pullulanase export), and the VirB-11 protein from the virB operon (involved in virulence) which is located on the Agrobacterium tumefaciens Ti plasmid. We have also identified other sets of homologies between P. aeruginosa fimbrial assembly (Pil) proteins and B. subtilis Com and K. oxytoca Pul proteins, which suggest that these are all related members of a specialised protein export pathway which is widespread in the eubacteria.     

The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex

It has been proposed that the four type IV pilin-like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans-periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin-like proteins required for pullulanase secretion in Klebsiella oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X-100 + EDTA or at high pH, but not by Triton X-100 atone or by 8M urea, 6M guanidine hydrochloride or 1 M NaCl. Like type IV pilins, non-sedimentable PuiG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross-linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N-methylates the PulG precursor, and PulE, a putative ATP-binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution.     

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.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.     

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.     

Role of the mature protein sequence of maltose-binding protein in its secretion across the E. coli cytoplasmic membrane

Secretion of amber fragments of an E. coli periplasmic protein, the maltose-binding protein, was studied to determine if the mature portion of the protein is required for its export across the cytoplasmic membrane. A fragment lacking 25–35 amino acid residues at the C terminus is secreted at normal levels, suggesting that this sequence is not required for secretion. This is in contrast to the results obtained with the periplasmic protein β-lactamase. In studying another fragment of one-third the molecular weight of the intact protein, we found that the majority of the fragment is not recovered from the periplasmic fraction. However, a small amount of secretion of this polypeptide was observed. This fragment is synthesized as a larger molecular weight form when cells are induced for the synthesis of a maltose-binding protein-β-galactosidase hybrid protein, which was previously shown to block the proper localization and processing of envelope proteins. This result is consistent with the idea that the larger form is a precursor with an unprocessed signal sequence, whereas in the absence of the hybrid protein the fragment is a processed mature form. Thus secretion of the smaller fragment may be occurring up to the point where the signal sequence is removed. That this fragment has passed through the cytoplasmic membrane is further supported by its accessibility to externally added trypsin. We suggest that the fragment may be secreted to the periplasm, but cannot assume a water-soluble conformation; the majority of the polypeptide may be associated with the external surface of the cytoplasmic membrane. Thus the mature sequence of maltose-binding protein, at least its C-terminal two thirds, may not be required for its export across the cytoplasmic membrane.     

Regulation of a membrane component required for protein secretion in Escherichia coli

We have previously described a gene, secA, which may code for a component of the secretion machinery of E. coli. Temperature-sensitive mutations in this gene lead to the cytoplasmic accumulation of precursors to a number of secreted proteins. In this paper, we describe the use of antibody to the SecA protein to characterize the cellular location and regulation of the protein. The antibody was elicited in response to a SecA-LacZ hybrid protein, produced by a strain carrying a secA-lacZ gene fusion. The secA gene product is a 92 kd polypeptide that is present in small amounts in the cell and that fractionates as a peripheral cytoplasmic membrane protein. The synthesis of the SecA protein is greatly derepressed (at least tenfold) when secretion in E. coli is blocked either in a secAts mutant or in the presence of a MalE-LacZ hybrid protein. We suggest that components of the secretion machinery of E. coli, such as the SecA protein, may be regulated in response to the secretion needs of the cell. When suppression of a secAam mutant is eliminated, leading to the absence of SecA protein, the synthesis of maltose-binding protein is greatly reduced. These results support a mechanism in which secretion and translation are coupled.     

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.     

Cloning and sequence analysis of a novel hemolysin gene (vllY) from Vibrio vulnificus.

A gene (vllY) encoding a novel hemolysin of Vibrio vulnificus CKM-1 has been cloned and sequenced. When the vllY gene was expressed in minicells, a unique peptide of approximately 40 kDa was identified. Subcellular fractionation of Escherichia coli cells carrying the vllY gene indicated that the VllY protein was distributed in both the cytoplasmic and the periplasmic fractions, with the notable ability to appear in the latter compartment. Nucleotide sequence analysis predicted a single open reading frame of 1,071 bp encoding a 357-amino acid polypeptide with an estimated pI of 5.02. The deduced amino acid sequence of VllY showed high similarity to the sequence of legiolysin, responsible for hemolysis, pigment production, and fluorescence in Legionella pneumophila. The enzyme also exhibited sequence homology to the MelA protein sequence of Shewanella colwelliana and the sequences of 4-hydroxyphenylpyruvate dioxygenase family proteins from various organisms. PCR screening and Southern blotting of V. vulnificus strains revealed that all of the 41 V. vulnificus clinical isolates contained vllY-like genes.     

An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca

The last gene (pulO) of the pulC-O pullulanase secretion gene operon of Klebsiella oxytoca codes for a protein that is 52% identical to the product of the pilD/xcpA gene required for extracellular protein secretion and type IV pilus biogenesis in Pseudomonas aeruginosa. The PilD/XcpA protein is known to remove the first six amino acids of the signal sequence of the type IV pilin precursor by cleaving after the glycine residue in the conserved sequence GF(M)XXXE (where X represents hydrophobic amino acids). This prepilin peptidase cleavage site is present in the products of four genes in the pulC-O operon (PulG, PulH, Pull and PulJ proteins). It is shown here that PulO processes the pulG gene product in vivo. Processing was maximal within 15 seconds, but experiments in which the expression of pulO was uncoupled from that of the other genes in the secretion operon suggest that processing can also occur post-translationally. The products of two pulG derivatives with internal inframe deletions were also processed by PulO, but the three PulG-PhoA hybrids, two PulJ-PhoA hybrids and the single PulH-PhoA hybrid tested did not appear to be processed. Sucrose gradient fraction experiments showed that both precursor and mature forms of PulG appear to be associated with low-density, outer membrane vesicles prepared by osmotic lysis of sphaeroplasts. Neither the xcpA gene nor the Bacillus subtilis gene comC, which is also homologous to pulO and codes for a protein with type IV prepilin peptidase activity, can correct the pullulanase secretion defect in an Escherichia coli strain carrying all of the genes required for secretion except pulO. Furthermore, neither XcpA nor ComC is able to process prePulG protein in vivo.     

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.     

The conserved tetracysteine motif in the general secretory pathway component PulE is required for efficient pullulanase secretion

The PulE component of the pullulanase secretion pathway, a typical main terminal branch of the general secretory pathway, has a tetracysteine motif (4Cys) that is also present in almost all of the many PulE homologues, including those involved in type-IV piliation and conjugal DNA transfer. The 4Cys resembles a zinc-binding motif found in other proteins such as adenylate kinases, which may be pertinent in view of the fact that PulE has a consensus ATP-binding motif and since at least one PulE homologue has been reported to have kinase activity. In PulE, the Cys residues of this motif form scrambled intra- and intermolecular disulfide bonds when cells are disrupted. Replacement of one or more Cys of this motif by Ser reduces PulE function, but at least two adjacent Cys must be replaced to prevent intramolecular disulfide bond formation.     

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.     

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.     

Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.