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 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.