Construction of a gene knockout system for application in Paenibacillus alvei CCM 2051T, exemplified by the S-layer glycan biosynthesis initiation enzyme WsfP

Abstract

The gram-positive bacterium Paenibacillus alvei CCM 2051T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [-->3)-beta-D-Galp-(1[alpha-D-Glcp-(1-->6)]-->4)-beta-D-ManpNAc-(1-->] repeating units that is linked by an adaptor of -[GroA-2-->OPO2-->4-beta-D-ManpNAc-(1-->4)]-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-Galp-(1--> to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the sgsE S-layer gene promoter of Geobacillus stearothermophilus NRS 2004/3a and on the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. As a target gene, wsfP, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional P. alvei CCM 2051T wsfP mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of wsfP in the glycan-deficient P. alvei mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism P. alvei CCM 2051T.

FIG. 1.

Schematic drawing of the construction of the shuttle plasmid pTT_wsfP1176, containing the wsfP targetron.

FIG. 2.

Determination of optimal electroporation parameters for wild-type cells (•, ▪, and ▴) and wsfP mutant cells (○, □, and ▵) of P. alvei CCM2051^T. The relationship between the numbers of transformants obtained per μg of DNA (pNW33N) and per 10⁶ competent cells and the applied voltage is shown. Electroporation experiments were performed with cultures from the early growth phase (OD₆₀₀, ∼0.2 to 0.3) at voltages ranging from 5 to 20 kV/cm and at resistance levels of 100 Ω (•/○), 200 Ω (▪/□), or 400 Ω (▴/▵).

FIG. 3.

Predicted topology of the WsfP protein of P. alvei CCM 2051^T. Shown are the five transmembrane helices (boxed), the central extracellular loop, and the carboxy-terminal cytosolic tail. Black amino acid residues are identical to corresponding amino acids in the functional WsaP homologue of G. stearothermophilus NRS 2004/3a.

FIG. 4.

Bacterial mobile group II intron-mediated gene disruption of wsfP in P. alvei CCM 2051^T. (A) Screening of Cm-resistant P. alvei CCM 2051^T colonies for intron insertion by in situ PCR using primers KO_wsfP_control_for_1 (→) and KO_wsfP_control_rev_1 (←). A PCR product obtained from a wild-type colony (lane 1), a PCR fragment obtained from a wsfP mutant (lane 2), and PCR products obtained from a colony containing both wild-type and intron-inserted wsfP (lane 3) are shown. (B) Schematic drawing of the wsfP gene with (bottom) and without (top) intron insertion, indicating the positions of primers KO_wsfP_control_for_1 (→) and KO_wsfP_control_rev_1 (←).

FIG. 5.

SDS-PAGE gels showing the S-layer glycosylation profile of P. alvei CCM 2051^T wild-type cells (lanes 1 and 5), wsfP mutant cells (lanes 2 and 6), and wsfP mutant cells after reconstitution with WsfP (lanes 3 and 7) and WsaP (lanes 4 and 8) upon plasmid-based expression. Results are shown for Coomassie brilliant blue G250 staining (A) and PAS staining for carbohydrate (B). Nonglycosylated (N), monoglycosylated (M) and diglycosylated (D) S-layer SpaA proteins are indicated on the left. SDS-PAGE was performed using a 10% gel, and 10 μg and 20 μg of protein were loaded for Coomassie and PAS staining, respectively.

FIG. 6.

Dionex carbohydrate analysis of S-layer extracts from P. alvei CCM 2051^T wild-type and wsfP mutant cells. (A) Standards (1 nmol each); (B) S-layer from wild-type cells (30 μg); (C) S-layer from wsfP::Ll.LtrB cells (175 μg).