Fig. 5.

Activity of WbbY and WbbZ proteins encoded by a bacteriophage belonging to the Caudovirales. (A) Western immunoblots demonstrating synthesis of the O1a and O1b antigens in E. coli DH5α. The Caudovirales wbbYZ^Cv sequences were identified from accession BK049963.1 in the BLAST search. The genes were synthesized, cloned in plasmid pWQ1115, and transformed into E. coli DH5α, together with plasmid pWQ288 carrying the rfb^2a locus (52). LPS in whole-cell lysates was probed using the identified antibodies. Silver staining and western blotting were performed twice with the same results. (B) Cell-free lysates containing K. pneumoniae WbbY^E318A and WbbY^Cv were combined and reacted with compound 1 in the presence of UDP-Galp and PEP. The identities of the reaction product (and control reaction products) shown were confirmed by MS (SI Appendix,Fig. S9). The same PAGE profile was obtained in three independent experiments, products shown were confirmed by MS. The tight band observed with the PAGE migration of 3 is a result of comigration with the loading dye.