[Function of rmlB in the pathogenic Escherichia coli 44277 (O2: k1)]

To identify the role of rmlB in synthesizing L-rhamnose in the pathogenic Escherichia coli 44277 (O2:K1:H4).

The rmlB gene was expressed and the activity of the recombinant protein was assayed by measuring the quantity of reaction product. The rmlB gene was deleted by homologous recombination, then phenotypic changes of the delta rmlB mutant was analyzed by Electron Microscope, Tricine SDS-PAGE and immunological methods. Further, various methods including MALDI-TOF-MS/MS, GC-MS and NMR was used to investigate the O antigen structure of the delta rmlB mutant.

RmlB was confirmed to be a protein harboring the activity of dTDP-D-glucose 4,6-dehydratase through enzyme assay. The delta rmlB mutant was successfully constructed and no phenotypic change was observed after compared with the wild type strain. L-rhamnose still existed in the delta rmlB mutant, indicating that there may be isoenzyme of RmlB presenting in the mutant or there was a novel way synthesizing L-rhamnose in the mutant.

RmlB has the activity of dTDP-D-glucose 4,6-dehydratase but it is not essential for the synthesis of L-rhamnose.