Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051 T , Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP

Abstract

The gram-positive bacterium Paenibacillus alvei CCM 2051 ^T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β- d -Gal p -(1[α- d -Glc p -(1→6)]→4)-β- d -Man p NAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO ₂ →4-β- d -Man p NAc-(1→4)]→3)-α- l -Rha p -(1→3)-α- l -Rha p -(1→3)-α- l -Rha p -(1→3)-β- d -Gal p -(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 2051 ^T 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 2051 ^T .