Posttranslational modification of flagellin FlaB in Shewanella oneidensis

Abstract

Shewanella oneidensis is a highly motile organism by virtue of a polar, glycosylated flagellum composed of flagellins FlaA and FlaB. In this study, the functional flagellin FlaB was isolated and analyzed with nano-liquid chromatography-mass spectrometry (MS) and tandem MS. In combination with the mutational analysis, we propose that the FlaB flagellin protein from S. oneidensis is modified at five serine residues with a series of novel O-linked posttranslational modifications (PTMs) that differ from each other by 14 Da. These PTMs are composed in part of a 274-Da sugar residue that bears a resemblance to the nonulosonic acids. The remainder appears to be composed of a second residue whose mass varies by 14 Da depending on the PTM. Further investigation revealed that synthesis of the glycans initiates with PseB and PseC, the first two enzymes of the Pse pathway. In addition, a number of lysine residues are found to be methylated by SO4160, an analogue of the lysine methyltransferase of Salmonella enterica serovar Typhimurium.

Fig 1

FlaB amino acid sequence coverage obtained from the nano-LC-MS/MS analysis. The peptide (in black) and glycopeptide (underlined) coverage of the FlaB protein amino sequence is indicated. Peptides (in gray) were missed from the analysis. The glycosylation and methylated lysine residues (italicized) that have been confirmed by MS are shown here also. Approximately 89% of the FlaB amino acid sequence has been mapped in this MS study.

Fig 2

Nano-LC-MS and -MS/MS of the modified FlaB tryptic glycopeptide, ⁹⁴DLTVQSENGANSSADLSALK¹¹³. (A) Nano-LC-MS mass spectrum showing a group of modified peptide ions separated by 14 Da, a pattern observed frequently throughout this analysis. Expanded views of the mass spectrum showing the doubly (separated by 7 m/z in the spectrum) and triply (separated by 4.7 m/z in the spectrum) protonated ions are presented in the insets. Rel. Int., relative intensity. (B) CID-MS/MS spectrum of the MH₃³⁺ ion at m/z 852.4. The b and y fragment ions identify the T^94-113 peptide, while the underlined ions in the low m/z region arise from a 538.3-Da modification. (C and D) CID-MS/MS of the other triply charged ions in this grouping (m/z 848.8 and m/z 844.1, respectively), indicating that they are composed of the same peptide sequence but modified with a 524.3- and 510.3-Da modification, respectively. The fragment ion at m/z 275.1 was observed in all three MS/MS spectra, indicating that a portion of these modifications is constant and common to all. Rel. Int., relative intensity.

Fig 3

FeCID-MS/MS of unknown glycan modifications. FeCID/MS/MS spectra of the glycan fragment ions arising from the PTM, namely, the ion at m/z 521.3 corresponding to the dehydrated modification (A), as well as the constant fragment ion at m/z 275.2 (B). The lack of abundant ions in the upper half of the MS/MS spectrum in panel A indicates that the m/z 521.3 glycan is composed of two residues of roughly equal mass. Many of the fragment ions (underlined) observed in the MS/MS spectrum of the m/z 275.2 ion (B) are also found in the fragment ion spectra of pseudaminic acid (23, 32) and other nonulosonic acids, suggesting that this residue is a similar glycan.

Fig 4

Glycopeptide detection using ETD-MS/MS and stepped-collision offset LC-MS. (A) ETD-MS/MS analysis of the ion at m/z 852.7, corresponding to the MH₃³⁺ precursor ion of the modified T^94-113 peptide. The fragmentation observed identified the linkage site as Ser¹⁰⁶. Therefore, even though this peptide contains a consensus sequon for N-glycosylation, it is actually O-glycosylated. (B) Glycopeptide detection using stepped-collision offset LC-MS. (a) Total ion LC-MS chromatogram acquired for the FlaB tryptic digest using low-collision offset (10 V). (b) Extracted ion chromatogram (EIC) acquired for the m/z 275.1 fragment ion using high-collision offset (35 V). This fragment ion is common to all glycan modifications. (c to e) EICs of the intact glycan fragment ions at m/z 539.3, 525.3, and 511.3, respectively. Glycopeptides are to be found where the peaks in the intact glycan fragment ion EICs align with those in the m/z 275.1 trace. Most glycopeptides elute between 26.5 and 32.0 min (hashed lines). TIC, total ion current.

Fig 5

Nano-LC-MS and -MS/MS of the FlaB tryptic peptide, T^147-163, containing methylated lysines. (A) Mass spectrum showing a series of doubly and triply protonated T^147-163 ions, each separated by 14 Da. (B) MS/MS analysis of the MH₂²⁺ ion at m/z 931.5 corresponding to methylated T^147-163. The peptide fragment ions confirm the peptide contains a single methylated lysine residue, K(Me)¹⁵⁹. (C) MS/MS analysis of the MH₂²⁺ ion at m/z 939.0, corresponding to T^147-163 containing two methylated lysine residues, K(Me)¹⁵⁹ and K(Me)¹⁶². These peptides are not glycosylated, although others are both glycosylated and methylated (data not shown).

Fig 6

Characteristics of S. oneidensis cells expressing mutant flagellins. (A) Relative motility of S. oneidensis cells expressing each of the mutant flagellins. Relative motility was obtained as the ratio of the diameter of the area of motility from mutant flagellin to that from wild-type flagellin. (B) Mutant flagellins that introduced significant reduction in motility or that carried the mutation at the glycosylation sites. FFM with FlaB^WT (top) and FFM with each of the mutated flagellins (bottom) were compared to each other in a single assay. FFM carrying the empty vector pHG101 was used as the negative control (FFM/V^e). (C) Western blot analyses of flagellins carrying mutations at glycosylation sites. Flagellar filaments from FFM containing one of the mutant flagellins were purified and applied to SDS-PAGE for detecting the migration shift. Those migrating differently on SDS-PAGE were examined using antibodies against S. oneidensis flagellins and are presented here.

Fig 7

Nano-LC-MS and -MS/MS of the FlaB tryptic peptide, T^147-163, containing methylated lysines. (A) MS/MS analysis of the MH₂²⁺ ion at m/z 717.9 corresponding to T^147-159 containing the single methylated lysine residue K(Me)¹⁵⁹. (B) MS/MS analysis of the MH₃³⁺ ion at m/z 626.1 corresponding to T^147-163 containing two methylated lysine residues, K(Me)¹⁵⁹ and K(Me)¹⁶². These peptides are not methylated in the SO4160 mutant.

Fig 8

SO3270 is required for flagellar assembly in S. oneidensis. Motility and TEM of the indicated strains. ΔSO3270^C indicates that the mutant is complemented in trans.

Fig 9

MS analysis of reactions catalyzed by SO3271 and SO3270. The MS graphs are lined with the corresponding chemicals shown on the left (upper, UDP-GlcNac; middle, UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose; lower, UDP-4-amino-4,6-dideoxy-β-l-AltNAc). In reactions A and B, UDP-d-GlcNAc is possibly converted to UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose by SO3271; in reaction C, no conversion; in reaction D, UDP-d-GlcNAc is possibly converted to UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose by SO3271, followed by UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose possibly being converted to UDP-4-amino-4,6-dideoxy-β-l-AltNAc by SO3271. y axis represents relative intensity.