Flashy flagella: flagellin modification is relatively common and highly versatile among the Enterobacteriaceae

Abstract

Background: Post-translational glycosylation of the flagellin protein is relatively common among Gram-negative bacteria, and has been linked to several phenotypes, including flagellar biosynthesis and motility, biofilm formation, host immune evasion and manipulation and virulence. However to date, despite extensive physiological and genetic characterization, it has never been reported for the peritrichously flagellate Enterobacteriaceae.

Results: Using comparative genomic approaches we analyzed 2,000 representative genomes of Enterobacteriaceae, and show that flagellin glycosylation islands are relatively common and extremely versatile among members of this family. Differences in the G + C content of the FGIs and the rest of the genome and the presence of mobile genetic elements provide evidence of horizontal gene transfer occurring within the FGI loci. These loci therefore encode highly variable flagellin glycan structures, with distinct sugar backbones, heavily substituted with formyl, methyl, acetyl, lipoyl and amino groups. Additionally, an N-lysine methylase, FliB, previously identified only in the enterobacterial pathogen Salmonella enterica, is relatively common among several distinct taxa within the family. These flagellin methylase island loci (FMIs), in contrast to the FGI loci, appear to be stably maintained within these diverse lineages.

Conclusions: The prevalence and versatility of flagellin modification loci, both glycosylation and methylation loci, suggests they play important biological roles among the Enterobacteriaceae.

Keywords: Enterobacteriaceae; Flagellin glycosylation; Flagellum; Methylation; N-lysine methylase.

Fig. 1

Family-wide distribution of FGI and FMI loci. A circularized, topology-only neighbour-joining phylogeny was constructed on the basis of the concatenated amino acid sequences of the house-keeping markers GyrB, InfB, RecA and RpoB. Bootstrap analysis was performed (n = 100) and bootstrap values above 50 % are shown for the major clades. The strains were incorporated in twenty deeper-branching clades (A-T). FGI⁺ Enterobacteriaceae are indicated by green dots and branch lines, while FMI⁺ strains are indicated in blue

Fig. 2

Flagellin methylase phylogeny versus house-keeping marker phylogeny. Neighbour-joining phylogenies were constructed on the basis of alignments of the concatenated house-keeping markers GyrB, InfB, RecA and RpoB (left) and the flagellin methylases FliB and SmtA (right). Bootstrap analyses were performed (n = 1,000) and bootstrap support values above 50 % are shown. The red branch indicates the distinct methylase (SmtA) encoded in the FMI locus of Pectobacterium wasabiae

Fig. 3

Flagellin glycosylation typing dendrogram. A typing dendrogram was constructed on the basis of a distance matrix representing the presence/absence of orthologs of each of the 218 distinct proteins encoded within the FGIs (right). FGI types were distinguished on the basis of 50 % distance cut-off values. The FGI typing dendrogram was compared against a neighbour-joining phylogeny on the basis of the amino acid sequences of the house-keeping markers GyrB, InfB, RecA and RpoB (left) for all the FGI⁺ Enterobacteriaceaea. Bootstrap analyses were performed for the latter phylogeny and bootstrap values above 50 % are shown

Fig. 4

Alignment of the type 12 FGI loci of P. ananatis AJ1335 and C. sakazakii SP291 and the type 27 FGI locus of P. stewartii M009. Glycosyltransferase and sugar biosynthetic genes are indicated by dark and light green arrows, respectively. Formyltransferases, methyltransferases, acetyltransferases and aminotransferases are encoded by genes represented by dark blue, purple, light blue and yellow arrows, respectively. Pink arrows indicates genes involved in fatty acid biosynthesis. Flanking genes are indicated by grey arrows, genes coding for hypothetical proteins by white arrows and black arrows indicate endonuclease (edn2) genes. The grey blocks indicate the regions of homology between the compared strains

Fig. 5

Alignment of the FGI loci of three distinct Plesiomonas shigelloides strains with homologous loci in other bacteria. The sugar biosynthetic genes are indicated by light green arrows, while the glycosyltransferases are represented by dark green arrows. Putative fatty acid biosynthesis, acetyltransferase, aminotransferase and methyltransferase genes are depicted by pink, light blue, yellow and purple arrows, respectively. The black arrows indicate transposase genes, while flanking genes are coloured in grey. The grey blocks indicate the regions of homology between the compared strains