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

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)-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 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).