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Dynamic Measures of Flagellar Gene Expression

  • Santosh Koirala
  • Christopher V. RaoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1593)

Abstract

Many genes are required to assemble flagella. These genes encode not only the structural elements of the flagellum but also a number of regulators that control how the flagellar genes are temporally expressed during the assembly process. These regulators also specify the likelihood that a given cell will express the flagellar genes. In particular, not all cells express the flagellar genes, resulting in mixed populations of motile and non-motile cells. Nutrients provide one signal that specifies the motile fraction. In this chapter, we describe two methods for measuring flagellar gene expression dynamics using fluorescent proteins in Salmonella enterica. Both the methods can be used to investigate the mechanisms governing flagellar gene expression dynamics.

Key words

Salmonella Flagella Gene expression Fluorescence Flow cytometry 

References

  1. 1.
    Chilcott GS, Hughes KT (2000) Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiol Mol Biol Rev 64(4):694–708CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Chevance FF, Hughes KT (2008) Coordinating assembly of a bacterial macromolecular machine. Nat Rev Microbiol 6(6):455–465. doi: 10.1038/nrmicro1887 CrossRefPubMedGoogle Scholar
  3. 3.
    Kalir S, McClure J, Pabbaraju K, Southward C, Ronen M, Leibler S, Surette MG, Alon U (2001) Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science 292(5524):2080–2083. doi: 10.1126/science.1058758 CrossRefPubMedGoogle Scholar
  4. 4.
    Karlinsey JE, Tanaka S, Bettenworth V, Yamaguchi S, Boos W, Aizawa SI, Hughes KT (2000) Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol Microbiol 37(5):1220–1231CrossRefPubMedGoogle Scholar
  5. 5.
    Kalir S, Alon U (2004) Using a quantitative blueprint to reprogram the dynamics of the flagella gene network. Cell 117(6):713–720. doi: 10.1016/j.cell.2004.05.010 CrossRefPubMedGoogle Scholar
  6. 6.
    Kalir S, Mangan S, Alon U (2005) A coherent feed-forward loop with a SUM input function prolongs flagella expression in Escherichia coli. Mol Syst Biol 1(2005):0006. doi: 10.1038/msb4100010 PubMedGoogle Scholar
  7. 7.
    Saini S, Floess E, Aldridge C, Brown J, Aldridge PD, Rao CV (2011) Continuous control of flagellar gene expression by the sigma28-FlgM regulatory circuit in Salmonella enterica. Mol Microbiol 79(1):264–278. doi: 10.1111/j.1365-2958.2010.07444.x CrossRefPubMedGoogle Scholar
  8. 8.
    Brown JD, Saini S, Aldridge C, Herbert J, Rao CV, Aldridge PD (2008) The rate of protein secretion dictates the temporal dynamics of flagellar gene expression. Mol Microbiol 70(4):924–937. doi: 10.1111/j.1365-2958.2008.06455.x PubMedGoogle Scholar
  9. 9.
    Saini S, Brown JD, Aldridge PD, Rao CV (2008) FliZ Is a posttranslational activator of FlhD4C2-dependent flagellar gene expression. J Bacteriol 190(14):4979–4988. doi: 10.1128/JB.01996-07 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Koirala S, Mears P, Sim M, Golding I, Chemla YR, Aldridge PD, Rao CV (2014) A nutrient-tunable bistable switch controls motility in Salmonella enterica serovar Typhimurium. MBio 5(5):e01611–e01614. doi: 10.1128/mBio.01611-14 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Saini S, Koirala S, Floess E, Mears PJ, Chemla YR, Golding I, Aldridge C, Aldridge PD, Rao CV (2010) FliZ induces a kinetic switch in flagellar gene expression. J Bacteriol 192(24):6477–6481. doi: 10.1128/JB.00751-10 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Stewart MK, Cummings LA, Johnson ML, Berezow AB, Cookson BT (2011) Regulation of phenotypic heterogeneity permits Salmonella evasion of the host caspase-1 inflammatory response. Proc Natl Acad Sci U S A 108(51):20742–20747. doi: 10.1073/pnas.1108963108 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Stewart MK, Cookson BT (2014) Mutually repressing repressor functions and multi-layered cellular heterogeneity regulate the bistable Salmonella fliC census. Mol Microbiol 94(6):1272–1284. doi: 10.1111/mmi.12828 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Cummings LA, Wilkerson WD, Bergsbaken T, Cookson BT (2006) In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted. Mol Microbiol 61(3):795–809. doi: 10.1111/j.1365-2958.2006.05271.x CrossRefPubMedGoogle Scholar
  15. 15.
    Wada T, Morizane T, Abo T, Tominaga A, Inoue-Tanaka K, Kutsukake K (2011) EAL domain protein YdiV acts as an anti-FlhD4C2 factor responsible for nutritional control of the flagellar regulon in Salmonella enterica serovar typhimurium. J Bacteriol 193(7):1600–1611. doi: 10.1128/JB.01494-10 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Takaya A, Erhardt M, Karata K, Winterberg K, Yamamoto T, Hughes KT (2012) YdiV: a dual function protein that targets FlhDC for ClpXP-dependent degradation by promoting release of DNA-bound FlhDC complex. Mol Microbiol 83(6):1268–1284. doi: 10.1111/j.1365-2958.2012.08007.x CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wada T, Tanabe Y, Kutsukake K (2011) FliZ acts as a repressor of the ydiV gene, which encodes an anti-FlhD4C2 factor of the flagellar regulon in Salmonella enterica serovar typhimurium. J Bacteriol 193(19):5191–5198. doi: 10.1128/JB.05441-11 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1):87–90. doi: 10.1038/nbt0102-87 CrossRefPubMedGoogle Scholar
  19. 19.
    Hawley TS, Hawley RG (2011) Flow cytometry protocols, Methods in molecular biology, vol 699. Humana, New YorkGoogle Scholar
  20. 20.
    Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S (1998) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64(6):2240–2246PubMedPubMedCentralGoogle Scholar
  21. 21.
    Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12):1567–1572. doi: 10.1038/nbt1037 CrossRefPubMedGoogle Scholar
  22. 22.
    Haldimann A, Wanner BL (2001) Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J Bacteriol 183(21):6384–6393. doi: 10.1128/JB.183.21.6384-6393.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Leveau JH, Lindow SE (2001) Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria. J Bacteriol 183(23):6752–6762. doi: 10.1128/JB.183.23.6752-6762.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ronen M, Rosenberg R, Shraiman BI, Alon U (2002) Assigning numbers to the arrows: parameterizing a gene regulation network by using accurate expression kinetics. Proc Natl Acad Sci U S A 99(16):10555–10560. doi: 10.1073/pnas.152046799 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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