Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca

Abstract

Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of beta-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.

FIG. 1.

(A) Cells of strain PAP5114(pCHAP1106) producing MalEsp-PulA and PAP5113(pCHAP4260) producing PelBsp-PulA were pulse-labeled for 3 min and chased for the indicated times (minutes) as described in Materials and Methods. Immunoprecipitated PulA proteins from cell-bound fractions (CELLS) and the total supernatant fractions (SN) were analyzed by SDS-PAGE and fluorography. Only the relevant parts of the fluorograms are shown. The positions of PulA, PulA′, and BlaM are indicated. (B) For each time point, PulA bands were quantified by densitometry, and the sum of cell and supernatant fractions was calculated as total protein. Secretion efficiency for each time point was calculated as the ratio of secreted versus total protein for MalEsp-PulA (□) and PelBsp-PulA (•). Data are presented graphically using Kaleidagraph software.

FIG. 2.

Secretion of different AB-reporter protein chimeras. Strain PAP7336 (no Pul secreton factors) or PAP7336(pCHAP710) (producing Pul secreton factors) was grown in the absence (−) or in the presence (+) of 1 mM IPTG as inducer. Proteins from equivalent amounts of cell (C) and supernatant (S) fractions were analyzed by SDS-PAGE and immunoblotting. (A) Secretion analysis of cells carrying plasmid pCHAP4267 developed with anti-BlaM and anti-MalE antisera. (B) Secretion analysis of cells carrying pCHAP4263 developed with anti-CelZ and anti-MalE antisera. (C) Secretion analysis of cells carrying pCHAP4262 developed with anti-NucB antiserum. (D) Secretion analysis of cells carrying pCHAP4266 developed with anti-TbpB antiserum.

FIG. 3.

Nonsecretion of AB peptide derived from PulA. Cells of strain PAP7336(pCHAP710) (Pul⁺) carrying pCHAP4293 or pCHAP4260 were grown in the absence (−) or presence (+) of 1 mM IPTG. Equivalent amounts of cell (C) and supernatant (S) fractions were analyzed by SDS-PAGE and immunoblotting using anti-AB antibodies. The positions of PulA and AB polypeptides and molecular mass markers (kDa) are indicated. Asterisks indicate the nonspecific bands that cross-react with the antiserum.

FIG. 4.

Effects of deletions in or of regions A and B on export (panel A) and secretion (panels B to E) of PelBsp-PulA (PrePulA). (A) Equivalent amounts of total (T), spheroplast (S), and periplasmic (P) fractions of E. coli K-12 carrying pCHAP4260 (PulA), pCHAP4458 (PulAΔA2-78), or pCHAP4459 (PulAΔA11-78) were analyzed by SDS-PAGE and immunoblotting using anti-PulA antibodies. The positions of PulA and PulAΔA proteins are indicated. (B to E) SDS-PAGE and anti-PulA immunoblot analysis of cell-bound (C) and supernatant (S) fractions of strain PAP7336(pCHAP710) (Pul⁺) and PAP7336 (Pul⁻) carrying pCHAP4260 (B), pCHAP4459 (C), pCHAP4375 (D), and pCHAP4470 (E). An asterisk indicates the position of the relevant full-length PulA variants, and molecular size markers are indicated in kDa.

FIG. 5.

TnTAP mutagenesis of PelBsp-PulA. (A) Schematic representation of the PulA protein with the signal peptide (SP) and regions A, B, and C indicated as shaded boxes. Positions of conserved protein domains in the primary structure of PulA identified using CDART are indicated: PUD, bacterial pullulanase domain; ISO, isoamylase N domain; and AMY, alpha-amylase domain. Arrows indicate positions of the 24-amino-acid TEV peptide insertions. Pullulanase secretion (SEC) was scored as normal (+), severely defective (−/+), or abolished (−). The presence (+) or absence (−) of pullulanase activity (ACT) is indicated for each variant. Positions of amino acid residues immediately upstream of the TEV peptide insertion are indicated where determined by DNA sequence analysis. N, position of insertion not determined. (B) Secretion of PulA::TEV variants. Equivalent amounts of cell (C) and supernatant (S) fractions were analyzed by SDS-PAGE and immunoblotting using anti-PulA antiserum. The positions of PulA and PulA′ polypeptides are indicated.

FIG. 6.

(A) Analysis of cell-bound (C) and supernatant (S) fractions of strains PAP7336(pCHAP710) (Pul⁺) and PAP7336 (Pul⁻) producing PulA₆₅-NucB, PulA₁₅₀-NucB, PulA₄₁₀-NucB, and PulA₈₁₅-NucB (PulA′-NucB) chimeras. Proteins were analyzed by SDS-PAGE and immunoblotting using anti-NucB antibodies. (B) Schematic representation of the location of the PulA-NucB constructs showing the position of region C. SEC, secretion; RES, residue in PulA immediately upstream from the fusion point.

FIG. 7.

Secretion in strains PAP7460(pCHAP710) (Pul⁺) or PAP7460(pCHAP40) (Pul⁻) carrying plasmids pCHAP4260 (PulA) or pCHAP4455 (PulAΔ199-432). Cell-bound (C) and supernatant (S) fractions were separated by SDS-PAGE and analyzed by immunoblotting using anti-PulA antiserum. (B) Proteinase K accessibility of wild-type lipoPulA encoded by pCHAP511, the lipoPulAΔ199-432 variant (encoded by pCHAP8058), and the lipoPulA variant containing the TEV peptide insertion 15-1 after residue K235 (encoded by pCHAP8063) in strain PAP7336 (Pul⁻) or strain PAP5114 (Pul⁺). The proteinase K accessibility of PulA in whole or sonicated cells was assayed as described previously (50).