Extracellular pullulanase of Klebsiella pneumoniae is a lipoprotein

Abstract

Pullulanase is a starch-debranching enzyme produced by the gram-negative bacterium Klebsiella pneumoniae. In this organism, the enzyme is first exported to the outer membrane and is subsequently released into the growth medium, Evidence reported here indicates that pullulanase is a lipoprotein. It is apparently synthesized as a precursor with a 19-residue-long signal sequence and modified by the covalent attachment of palmitate to the cysteine residue which becomes the amino terminus after cleavage of the signal sequence. In this respect, pullulanase is similar to some penicillinases produced by gram-positive bacteria which are initially exported to the cell surface and subsequently released into the medium. However, pullulanase and the penicillinases differ in one important aspect, namely, that the extraceliular pullulanase still carries the covalently attached fatty acyls, whereas extracellular pepicillinases lack the modified amino-terminal cysteine together with a limited number of other residues from the amino terminus.

Activation of reconstituted Escherichia coli outer‐membrane phospholipase A by membrane‐perturbing peptides results in an increased reactivity towards the affinity label hexadecanesulfonyl fluoride

“…It has been proposed that the detergent action of the lysophospholipids which result from the activation of OM PLA by the bacteriocin release protein will create 'permeable zones' in the otherwise impermeable outer membrane (Pugsley and Schwartz, 1984). This would also explain the observation that significant amounts of periplasmic proteins leak to the cell medium during this process (Pugsley and Schwartz, 1983). Moderate expression of the plasmid-encoded bacteriocin release protein can even be used to promote the release of heterologous proteins, produced in E. coli, from the periplasmic space (Hsiung et al, 1989).…”

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

“…The role of fatty acids in anchoring PulA to the cell envelope is well established (Pugsley et al, 1986), but it is not clear whether they actively participate in the translocation of the enzyme through the cell envelope. In previous studies, site-directed mutagenesis was used to replace the modified cysteine in PulA produced by Klebsiella aerogenes (Murooka and Ikeda, 1989) or by E. coli expressing the cloned Klebsiella oxytoca pul genes .…”

“…We are using a model system based on the amylolytic lipoprotein pullulanase (PulA) of Klebsiella oxytoca. The entire PulA polypeptide is extracellular, but it remains anchored to the cell surface by fatty acids attached to the N-terminal cysteine of the processed precursor, and is slowly and probably spontaneously released into the growth medium only after the end of exponential growth (Pugsley et al, 1986). The secretion mechanism has been studied in E. coli carrying the cloned pulA structural gene (d 'Enfert et al, 1987).…”

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

“…The second type is exemplified by the secretion of Klebsiella pneumoniae pullulanase and E. chrysanthemi pectic enzymes and cellulases. Secreted proteins of this group possess amino-terminal signal peptides which apparently function in E. coli in facilitating translocation of the proteins across the bacterial inner membrane (2,7,15,27,40,41,45). Extracellular secretion of these proteins, however, requires the function of accessory secretion genes, such as the out genes of E. chrysanthemi and Erwinia carotovora (1,26,37,50), the xcp genes of Pseudomonas aerguinosa (14), and the pul genes of K. pneumoniae (12).…”

“…For example, K. pneumoniae secretes a single lipoprotein, pullulanase, which is acylated after the removal of the signal peptide (41). Further secretion of pullulanase across the outer membrane requires secretion genes pulC-O and pulS (10,11,44,46).…”