Genotype-to-phenotype analysis of flagella and its Oglycosylation genes in Campylobacter jejuni NCTC11168

Campylobacter jejuni was the first microorganism in which a bacterial N-linked glycosylation system was discovered. Since then, Campylobacters have become the focus in the study of another carbohydrate dependent post translational modification system called Olinked glycosylation responsible for flagellin glycosylation. Flagellin glycosylation by maf glycotransferases increases the mass of the protein by ∼10% and pseudaminic acid is the primary substrate. Consequently, mutations in pseudaminic acid biosynthetic genes result in nonmotile aflagellate bacteria.

However, a reversible nonmotile phenotype has been observed in Campylobacters with pseudaminic acid and legionaminic acid but not in isolates with pseudaminic acid alone. Therefore, motility is rescued by some mechanism, not yet fully elucidated, in the presence of an additional glycan (legionaminic acid) and maf glycotransferases activity.

In this study, genome analysis of the relationship between pseudaminic acid, legionaminic acid and glycotransferase genes showed that legionaminic acid biosynthetic genes were the main driver of variations in the number of glycotransferase genes available across isolates. Absence of these genes led to absence of 6 out of 7 maf glycotransferase genes in several clonal complexes. A correlation that was significant. Further analysis of maf glycotransferase genes revealed that the maf gene family was made of a core gene set, maf3, maf4 and maf5. Mutation of maf3 in C. jejuni NCTC11168 produced a nonmotile phenotype that led to motility pseudorevertants similar to maf5 mutation in Karlyshev’s work. Treatment of C. jejuni 81116 and C. jejuni NCTC11168 with a pseudaminic acid synthesis enzyme F (pseF) inhibitor showed that motility was only reduced in C. jejuni 81116, a strain possessing pseudaminic acid alone. In addition, site directed mutagenesis of serine residue 425, important for filament formation in C. jejuni 81-176, had no effect on motility in C. jejuni NCTC11168. Therefore, presence of legionaminic acid increases structural fitness of the flagella in Campylobacters.