Analyzing the Modification of the Shewanella oneidensis MR-1 Flagellar Filament

Abstract

The unsheathed flagellar filament of Shewanella oneidensis MR-1 is composed of two highly homologous flagellins, FlaA, and the major structural unit, FlaB. We identified a gene cluster, SO_3261-SO_3265 (now sfmABCDE), that is required for the formation of a fully functional filament and for motility. The predicted function of the corresponding gene products strongly indicated a role in flagellin modification. Accordingly, loss of sfmABCDE results in a significant mass shift of both FlaA and FlaB. Mass spectroscopy analysis and single residue substitutions identified five serine residues in both flagellins that are modified via O-linkage. Modeling of the flagellin structures strongly suggests that at least four of the modified residues are exposed to the filament’s surface. However, none of the five serine residues solely is crucial for function and assembly. Structural analysis of the flagellin modification revealed that it likely contains a nonulosonic acid (274 Da) linked to each glycosylated serine. The putative nonulosonic acid is further substituted with a 236 Da moiety which can carry additional methyl groups (250 Da, 264 Da). In addition, at least 5 lysine residues in FlaB and one in FlaA were found to be methylated. Based on homology comparisons we suggest that smfABCDE is required for species-specific flagellin modification in S. oneidensis MR-1.

Spatial arrangement of several flagellins within bacterial flagella improves motility in different environments

“…This is higher than the masses deduced from the amino acid sequence (~28.5 kDa) 23 , indicating modification of both flagellins, e.g., by glycosylation. To determine if the flagellins of S. putrefaciens are glycosylated, we introduced in-frame deletions into the genes encoding PseG (Sputcn32_2626) and Maf-1 (Sputcn32_2630), as the deletion of the orthologs in S. oneidensis results in completely non-glycosylated flagellins 26,27 . Immunoblotting analysis of PAGE-separated crude extracts (Supplementary Fig.…”

Two Residues Predominantly Dictate Functional Difference in Motility between Shewanella oneidensis Flagellins FlaA and FlaB

“…These data indicate that the L 5a loop is more critical in general than L 5b and L 5c in terms of motility. Because mutations of T178A and S180A did not cause reduced motility and Thr 179 is unlikely to be glycosylated (28,29), we speculate that the non-motile phenotype of FFM carrying T179A may result from the loss of the polar hydroxyl group in threonine. To test this, Thr 178 , Thr 178 , and Ser 180 were replaced by cysteine.…”

“…These flagellins are rather small, given that the minimum length for any flagellin is ϳ250 aa (30). Interestingly, FlaA and FlaB differ substantially in their ability to propel cells, although they share a sequence identity of ϳ89% (13,29) (Fig. 1).…”

“…These data suggest that mutations in FlaB cause a decrease in motility by mainly influencing the efficacy of flagellin assembly into the filament. ) within FlaB that are glycosylated (28,29), residue 106, which is replaced by threonine in FlaA, is the only difference (Fig. 1).…”

Partially Reciprocal Replacement of FlrA and FlrC in Regulation of Shewanella oneidensis Flagellar Biosynthesis

“…With respect to the flagellar system, extensive studies have been carried out, revealing a number of surprising findings that provide important insights into the assembly of the locomotive organelle and its regulation. These include the biology of two stator systems (13,20,21), flagellins (14,16,18,38), flagellar number and location (19), flagellum and protein secretion (15), and flagellar regulation (17). Additionally, studies of dual flagellar systems in other Shewanella species have elucidated the role played by these systems in acquiring a fitness gain in various environments (39)(40)(41)(42).…”