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FliO Regulation of FliP in the Formation of the Flagellum


The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the ΔfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO43–125 or FliO1–95 was able to rescue motility of the ΔfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO43–125, should contain beta-structure and alpha-helices. FliO43–125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.


Vyšlo v časopise: FliO Regulation of FliP in the Formation of the Flagellum. PLoS Genet 6(9): e32767. doi:10.1371/journal.pgen.1001143
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1001143

Souhrn

The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the ΔfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO43–125 or FliO1–95 was able to rescue motility of the ΔfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO43–125, should contain beta-structure and alpha-helices. FliO43–125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.


Zdroje

1. BlockerA

KomoriyaK

AizawaS-I

2003 Type III secretion systems and bacterial flagella: Insights into their function from structural similarities. Proc Natl Acad Sci U S A 100 3027 3030

2. MacnabRM

2003 How bacteria assemble flagella. Annu Rev Microbiol 57 77 100

3. MacnabRM

2004 Type III flagellar protein export and flagellar assembly. Biochim Biophys Acta 1694 207 217

4. FanF

OhnishiK

FrancisNR

MacnabRM

1997 The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body. Mol Microbiol 26 1035 1046

5. KiharaM

MinaminoT

YamaguchiS

MacnabRM

2001 Intergenic suppression between the flagellar MS ring protein FliF of Salmonella and FlhA, a membrane component of its export apparatus. J Bacteriol 183 1655 1662

6. Van ArnamJS

McMurryJL

KiharaM

MacnabRM

2004 Analysis of an engineered Salmonella flagellar fusion protein, FliR-FlhB. J Bacteriol 186 2495 2498

7. MinaminoT

MacnabRM

1999 Components of the Salmonella flagellar export apparatus and classification of export substrates. J Bacteriol 181 1388 1394

8. ChevanceFFV

HughesKT

2008 Coordinating assembly of a bacterial macromolecular machine. Nat Rev Microbiol 6 455 465

9. MinaminoT

ImadaK

NambaK

2008 Mechanisms of type III protein export for bacterial flagellar assembly. Mol BioSyst 4 1105 1115

10. MinaminoT

NambaK

2008 Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export. Nature 451 485 488

11. PaulK

ErhardtM

HiranoT

BlairDF

HughesKT

2008 Energy source of flagellar type III secretion. Nature 451 489 492

12. Saijo-HamanoY

ImadaK

MinaminoT

KiharaM

ShimadaM

2010 Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export. Mol Microbiol 76 260 268

13. MooreSA

JiaY

2010 Structure of the cytoplasmic domain of the flagellar secretion apparatus component FlhA from Helicobacter pylori. J Biol Chem 285 21060 21069

14. WorrallLJ

VuckovicM

StrynadkaNC

2010 Crystal structure of the C-terminal domain of the Salmonella type III secretion system export apparatus protein InvA. Protein Sci 19 1091 1096

15. ZarivachR

DengW

VuckovicM

FeliseHB

NguyenHV

2008 Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS. Nature 453 124 127

16. DeaneJE

GrahamSC

MitchellEP

FlotD

JohnsonS

2008 Crystal structure of Spa40, the specificity switch for the Shigella flexneri type III secretion system. Mol Microbiol 69 267 276

17. LountosGT

AustinBP

NallamsettyS

WaughDS

2009 Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion. Prot Sci 18 467 474

18. MinaminoT

MacnabRM

2000 Interactions among components of the Salmonella flagellar export apparatus and its substrates. Mol Microbiol 35 1052 1064

19. FraserGM

González-PedrajoB

TameJRH

MacnabRM

2003 Interactions of FliJ with the Salmonella type III flagellar export apparatus. J Bacteriol 185 5546 5554

20. McMurryJL

Van ArnamJS

KiharaM

MacnabRM

2004 Analysis of the cytoplasmic domains of Salmonella FlhA and interactions with components of the flagellar export machinery. J Bacteriol 186 7586 7592

21. Saijo-HamanoY

MinaminoT

MacnabRM

NambaK

2004 Structural and functional analysis of the C-terminal cytoplasmic domain of FlhA, an integral membrane component of the type III flagellar protein export apparatus in Salmonella. J Mol Biol 343 457 466

22. MinaminoT

ShimadaM

OkabeM

Saijo-HamanoY

ImadaK

2010 Role of the C-terminal cytoplasmic domain of FlhA in bacterial flagellar type III protein export. J Bacteriol 192 1929 1936

23. MinaminoT

González-PedrajoB

YamaguchiK

AizawaS-I

MacnabRM

1999 FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly. Mol Microbiol 34 295 304

24. MinaminoT

MacnabRM

2000 Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol 182 4906 4914

25. FraserGM

HiranoT

FerrisHU

DevganLL

KiharaM

2003 Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB. Mol Microbiol 48 1043 1057

26. FerrisHU

FurukawaY

MinaminoT

KroetzMB

KiharaM

2005 FlhB regulates ordered export of flagellar components via autocleavage mechanism. J Biol Chem 280 41236 41242

27. MinaminoT

FerrisHU

MoriyaN

KiharaM

NambaK

2006 Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus. J Mol Biol 362 1148 1158

28. MoriyaN

MinaminoT

HughesKT

MacnabRM

NambaK

2006 The type III flagellar export specificity switch is dependent on FliK ruler and a molecular clock. J Mol Biol 359 466 477

29. ErhardtM

HiranoT

SuY

PaulK

WeeDH

2010 The role of the FliK molecular ruler in hook-length control in Salmonella enterica. Mol Micro 75 1272 1284

30. MalakootiJ

KomedaY

MatsumuraP

1989 DNA sequence analysis, gene product identification, and localization of flagellar motor components of Escherichia coli. J Bacteriol 171 2728 2734

31. MalakootiJ

ElyB

MatsumuraP

1994 Molecular characterization, nucleotide sequence, and expression of the fliO, fliP, fliQ, and fliR genes of Escherichia coli. J Bacteriol 176 189 197

32. OhnishiK

FanF

SchoenhalsGJ

KiharaM

MacnabRM

1997 The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly. J Bacteriol 179 6092 6099

33. SchoenhalsGJ

KiharaM

MacnabRM

1998 Translation of the flagellar gene fliO of Salmonella typhimurium from putative tandem starts. J Bacteriol 180 2936 2942

34. LiuR

OchmanH

2007 Stepwise formation of the bacterial flagellar system. Proc Natl Acad Sci U S A 104 7116 7121

35. PallenMJ

PennCW

ChaudhuriRR

2005 Bacterial flagellar diversity in the post-genomic era. Trends Microbiol 13 143 149

36. ManoilC

BeckwithJ

1986 A genetic approach to analyzing membrane protein topology. Science 233 1403 1408

37. EhrmannM

BoydD

BeckwithJ

1990 Genetic analysis of membrane protein topology by a sandwich gene fusion approach. Proc Natl Acad Sci U S A 87 7574 7578

38. BoydD

TraxlerB

BeckwithJ

1993 Analysis of the topology of a membrane protein by using a minimum number of alkaline phosphatase fusions. J Bacteriol 175 553 556

39. FeilmeierBJ

IsemingerG

SchroederD

WebberH

PhillipsGJ

2000 Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J Bacteriol 182 4068 4076

40. RichardsonJS

RichardsonDC

2002 Natural β-sheet proteins use negative design to avoid edge-to-edge aggregation. Proc Natl Acad Sci U S A 99 2754 2759

41. ShigenobuS

WatanabeH

HattoriM

SakakiY

IshikawaH

2000 Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407 81 86

42. KostyukovaAS

PyatibratovMG

FilimonovVV

FederovOV

1988 Flagellin parts acquiring a regular structure during polymerization are disposed on the molecule ends. FEBS Lett 241 141 144

43. KostyukovaAS

TiktopuloEI

MaédaY

2001 Folding properties of functional domains of tropomodulin. Biophys J 81 345 351

44. TokerAS

KiharaM

MacnabRM

1996 Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium. J Bacteriol 178 7069 7079

45. DatsenkoKA

WannerBL

2000 One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97 6640 6645

46. KarlinseyJE

2007 λ-Red genetic engineering in Salmonella enterica serovar Typhimurium. Methods Enzymol 421 199 209

47. EthierJ

BoydJM

2000 Topological analysis and role of the transmembrane domain in polar targeting of PilS, a Pseudomonas aeruginosa sensor kinase. Mol Microbiol 38 891 903

48. ColeC

BarberJD

BartonGJ

2008 The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36 W197 W201

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