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Query: UNIPROT:P06889 (Mol)
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The PulO protein required for extracellular secretion of pullulanase by Klebsiella oxytoca is known to be highly homologous to two type IV prepilin peptidases, namely XcpA(PilD) (Pseudomonas aeruginosa) and TcpJ (Vibrio cholerae). The predicted prepilin peptidase activity of PulO was confirmed by showing that it could correctly process the product of the cloned pilE.1 type IV pilin structural gene from Neisseria gonorrhoeae in Escherichia coli. The P. aeruginosa prepilin peptidase and another putative prepilin peptidase, ComC from Bacillus subtilis, also processed prePilE. Subcellular fractionation showed that the pilE gene product that had been processed by PulO remained associated with the cytoplasmic membrane, as did the unprocessed precursor. PulO was also shown to process three of the four prePilE-PhoA hybrids tested. Southern hybridization experiments suggest that a pulO homologue is present in the N. gonorrhoeae chromosome.
Mol Microbiol 1992 Jul
PMID:PulO, a component of the pullulanase secretion pathway of Klebsiella oxytoca, correctly and efficiently processes gonococcal type IV prepilin in Escherichia coli. 135 33

The out genes of Erwinia chrysanthemi are required for the translocation across the outer membrane of pectate lyases and cellulases. We present the characterization and the nucleotide sequence of five genes of the out cluster. The products of outS, B, C, D and E have significant homology with the PulS, B, C, D and E proteins necessary to the secretion of pullulanase in Klebsiella pneumoniae. An open reading frame, outT, located between outB and outC has no homology with the pul cluster but is involved in secretion. outC, outD and outE form an operon while outS, outB and outT constitute independent transcription units. outT and the outCDE operon are regulated by kdgR, the negative regulatory gene controlling pectinase production. outB and outS seem to be expressed constitutively.
Mol Microbiol 1992 Nov
PMID:Some of the out genes involved in the secretion of pectate lyases in Erwinia chrysanthemi are regulated by kdgR. 145 58

Klebsiella pullulanase is a lipoprotein synthesized as a precursor with a signal peptide, which is processed by lipoprotein signal peptidase. To clarify the role of lipid modification of pullulanase, we purified lipid-modified wild-type and the unmodified (mutant) pullulanases and compared their properties. The Km and Vmax values of both pullulanases for pullulan were the same. The optimal pH and temperature, the stabilities over pH and temperature ranges, the specificity of substrates, and the patterns of inhibition of the lipid-modified and unmodified pullulanases were also the same. However, we found that the wild-type pullulanase formed trimers whereas the unmodified enzyme did not, and that the migrations of the two enzymes on sodium dodecyl sulphate/electrophoresis were different when the samples were applied on the gel without heating. The results presented in this paper and in previous work show that the correct processing and translocation of pullulanase in K. aerogenes require modification of lipid. However, the enzymatic properties and physical stabilities of pullulanase were not affected by the lipid modification.
Mol Microbiol 1992 Feb
PMID:Role of lipid modification on a starch-debranching enzyme, Klebsiella pullulanase: comparison of properties of lipid-modified and unmodified pullulanases. 155 52

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.
Mol Microbiol 1992 Mar
PMID:An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca. 157 4

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.
Mol Microbiol 1992 Jan
PMID:Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein. 173 17

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.
Mol Microbiol 1991 Apr
PMID:Two distinct steps in pullulanase secretion by Escherichia coli K12. 185 8

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.
Mol Microbiol 1991 Feb
PMID:The general protein-export pathway is directly required for extracellular pullulanase secretion in Escherichia coli K12. 204 72

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.
Mol Gen Genet 1990 Jul
PMID:Five additional genes in the pulC-O operon of the gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion. 212 43

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.
Mol Microbiol 1990 Mar
PMID:Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion. 216 63

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.
Mol Microbiol 1990 Jan
PMID:Analysis of the subcellular location of pullulanase produced by Escherichia coli carrying the pulA gene from Klebsiella pneumoniae strain UNF5023. 218 Dec 41


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