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Applied and Environmental Microbiology, May 2009, p. 3077-3085, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.00087-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Kristof Zarschler, Bettina Janesch, Sonja Zayni, Christina Schäffer,* and Paul Messner*

Department für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1180 Vienna, Austria

Received 14 January 2009/ Accepted 17 March 2009

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)-β-D-Galp-(1[{alpha}-D-Glcp-(1->6)]->4)-β-D-ManpNAc-(1->] repeating units that is linked by an adaptor of -[GroA-2->OPO2->4-β-D-ManpNAc-(1->4)]->3)-{alpha}-L-Rhap-(1->3)-{alpha}-L-Rhap-(1->3)-{alpha}-L-Rhap-(1->3)-β-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.

* Corresponding author. Mailing address: Department für NanoBiotechnologie, Universität für Bodenkultur Wien, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria. Phone: 43-1-47654, ext. 2202. Fax: 43-1-4789112. E-mail for Paul Messner: paul.messner{at}boku.ac.at. E-mail for Christina Schäffer: christina.schaeffer{at}boku.ac.at

{triangledown} Published ahead of print on 20 March 2009.

Applied and Environmental Microbiology, May 2009, p. 3077-3085, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.00087-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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