Spatial control of the GTPase MglA by localized RomR–RomX GEF and MglB GAP activities enables Myxococcus xanthus motility

Abstract

The rod-shaped Myxococcus xanthus cells move with defined front–rear polarity using polarized motility systems. A polarity module consisting of the small GTPase MglA, its cognate GTPase activating protein (GAP) MglB and RomR establishes this polarity. Agl–Glt gliding motility complexes assemble and disassemble at the leading and lagging pole, respectively. These processes are stimulated by MglA-GTP at the leading and MglB at the lagging pole. Here, we identify RomX as an integral component of the polarity module. RomX and RomR form a complex that has MglA guanine nucleotide exchange factor (GEF) activity and also binds MglA-GTP. In vivo RomR recruits RomX to the leading pole forming the RomR–RomX complex that stimulates MglA-GTP formation and binding, resulting in a high local concentration of MglA-GTP. The spatially separated and opposing activities of the RomR–RomX GEF at the leading and the MglB GAP at the lagging cell pole establish front–rear polarity by allowing the spatially separated assembly and disassembly of Agl–Glt motility complexes. Our findings uncover a regulatory system for bacterial cell polarity that incorporates a nucleotide exchange factor as well as an NTPase activating protein for regulation of a nucleotide-dependent molecular switch and demonstrate a spatial organization that is conserved in eukaryotes.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: RomX is important for reversals in the T4P-dependent motility system and essential for gliding motility.
Fig. 2: RomX acts in the same pathway as MglA, MglB and RomR.
Fig. 3: RomX is polarly localized.
Fig. 4: RomR–RomX complex interacts with MglA-GTP and has GEF activity.
Fig. 5: RomR and RomX are important for Agl–Glt complex formation and are incorporated into these complexes.
Fig. 6: Model for front–rear polarity in M. xanthus.

Data availability

The authors declare that all data supporting this study are available within the article and its Supplementary Information files or are available from the corresponding author on request.

Code availability

The MATLAB script used in this study is available from the corresponding author upon request.

References

  1. 1.

    Rafelski, S. & Marshall, W. Building the cell: design principles of cellular architecture. Nat. Rev. Mol. Cell Biol. 9, 593–602 (2008).

    CAS  Article  Google Scholar 

  2. 2.

    Shapiro, L., McAdams, H. H. & Losick, R. Why and how bacteria localize proteins. Science 326, 1225–1228 (2009).

    CAS  Article  Google Scholar 

  3. 3.

    Treuner-Lange, A. & Søgaard-Andersen, L. Regulation of cell polarity in bacteria. J. Cell Biol. 206, 7–17 (2014).

    CAS  Article  Google Scholar 

  4. 4.

    Schumacher, D. & Søgaard-Andersen, L. Regulation of cell polarity in motility and cell division in Myxococcus xanthus. Annu. Rev. Microbiol. 71, 61–78 (2017).

    CAS  Article  Google Scholar 

  5. 5.

    Zhang, Y., Ducret, A., Shaevitz, J. & Mignot, T. From individual cell motility to collective behaviors: insights from a prokaryote, Myxococcus xanthus. FEMS Microbiol. Rev. 36, 149–164 (2012).

    CAS  Article  Google Scholar 

  6. 6.

    Blackhart, B. D. & Zusman, D. R. ‘Frizzy’ genes of Myxococcus xanthus are involved in control of frequency of reversal of gliding motility. Proc. Natl Acad. Sci. USA 82, 8771–8774 (1985).

    Article  Google Scholar 

  7. 7.

    Konovalova, A., Petters, T. & Søgaard-Andersen, L. Extracellular biology of Myxococcus xanthus. FEMS Microbiol. Rev. 34, 89–106 (2010).

    CAS  Article  Google Scholar 

  8. 8.

    Sun, H., Zusman, D. R. & Shi, W. Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system. Curr. Biol. 10, 1143–1146 (2000).

    CAS  Article  Google Scholar 

  9. 9.

    Merz, A. J., So, M. & Sheetz, M. P. Pilus retraction powers bacterial twitching motility. Nature 407, 98–102 (2000).

    CAS  Article  Google Scholar 

  10. 10.

    Skerker, J. M. & Berg, H. C. Direct observation of extension and retraction of type IV pili. Proc. Natl Acad. Sci. USA 98, 6901–6904 (2001).

    CAS  Article  Google Scholar 

  11. 11.

    Mignot, T., Shaevitz, J. W., Hartzell, P. L. & Zusman, D. R. Evidence that focal adhesion complexes power bacterial gliding motility. Science 315, 853–856 (2007).

    CAS  Article  Google Scholar 

  12. 12.

    Sun, M., Wartel, M., Cascales, E., Shaevitz, J. W. & Mignot, T. Motor-driven intracellular transport powers bacterial gliding motility. Proc. Natl Acad. Sci. USA 108, 7559–7564 (2011).

    CAS  Article  Google Scholar 

  13. 13.

    Nan, B., Mauriello, E. M. F., Sun, I.-H., Wong, A. & Zusman, D. R. A multi-protein complex from Myxococcus xanthus required for bacterial gliding motility. Mol. Microbiol. 76, 1539–1554 (2010).

    CAS  Article  Google Scholar 

  14. 14.

    Jakobczak, B., Keilberg, D., Wuichet, K. & Søgaard-Andersen, L. Contact- and protein transfer-dependent stimulation of assembly of the gliding motility machinery in Myxococcus xanthus. PLoS Genet. 11, e1005341 (2015).

    Article  Google Scholar 

  15. 15.

    Treuner-Lange, T. et al. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions. J. Cell Biol. 210, 243–256 (2015).

    CAS  Article  Google Scholar 

  16. 16.

    Luciano, J. et al. Emergence and modular evolution of a novel motility machinery in bacteria. PLoS Genet. 7, e1002268 (2011).

    CAS  Article  Google Scholar 

  17. 17.

    Faure, L. M. et al. The mechanism of force transmission at bacterial focal adhesion complexes. Nature 539, 530–535 (2016).

    CAS  Article  Google Scholar 

  18. 18.

    Nan, B. et al. Myxobacteria gliding motility requires cytoskeleton rotation powered by proton motive force. Proc. Natl Acad. Sci. USA 108, 2498–2503 (2011).

    CAS  Article  Google Scholar 

  19. 19.

    Miertzschke, M. et al. Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity. EMBO J. 30, 4185–4197 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    Zhang, Y., Franco, M., Ducret, A. & Mignot, T. A bacterial Ras-like small GTP-binding protein and its cognate GAP establish a dynamic spatial polarity axis to control directed motility. PLoS Biol. 8, e1000430 (2010).

    Article  Google Scholar 

  21. 21.

    Leonardy, S. et al. Regulation of dynamic polarity switching in bacteria by a Ras-like G-protein and its cognate GAP. EMBO J. 29, 2276–2289 (2010).

    CAS  Article  Google Scholar 

  22. 22.

    Mauriello, E. M. F. et al. Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA. EMBO J. 29, 315–326 (2010).

    CAS  Article  Google Scholar 

  23. 23.

    Patryn, J., Allen, K., Dziewanowska, K., Otto, R. & Hartzell, P. L. Localization of MglA, an essential gliding motility protein in Myxococcus xanthus. Cytoskeleton 67, 322–337 (2010).

    CAS  PubMed  Google Scholar 

  24. 24.

    Leonardy, S., Freymark, G., Hebener, S., Ellehauge, E. & Søgaard-Andersen, L. Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus. EMBO J. 26, 4433–4444 (2007).

    CAS  Article  Google Scholar 

  25. 25.

    Keilberg, D., Wuichet, K., Drescher, F. & Søgaard-Andersen, L. A response regulator interfaces between the Frz chemosensory system and the MglA/MglB GTPase/GAP module to regulate polarity in Myxococcus xanthus. PLoS Genet. 8, e1002951 (2012).

    CAS  Article  Google Scholar 

  26. 26.

    Zhang, Y., Guzzo, M., Ducret, A., Li, Y.-Z. & Mignot, T. A dynamic response regulator protein modulates G-protein–dependent polarity in the bacterium Myxococcus xanthus. PLoS Genet. 8, e1002872 (2012).

    CAS  Article  Google Scholar 

  27. 27.

    Hodgkin, J. & Kaiser, D. Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): two gene systems control movement. Mol. Gen. Genet. 171, 177–191 (1979).

    Article  Google Scholar 

  28. 28.

    Yang, R. et al. AglZ is a filament-forming coiled-coil protein required for adventurous motility of Myxococcus xanthus. J. Bacteriol. 186, 6168–6178 (2004).

    CAS  Article  Google Scholar 

  29. 29.

    Bos, J. L., Rehmann, H. & Wittinghofer, A. GEFs and GAPs: critical elements in the control of small G proteins. Cell 129, 865–877 (2007).

    CAS  Article  Google Scholar 

  30. 30.

    Zhou, T. & Nan, B. Exopolysaccharides promote Myxococcus xanthus social motility by inhibiting cellular reversals. Mol. Microbiol. 103, 729–743 (2017).

    CAS  Article  Google Scholar 

  31. 31.

    Guzzo, M. et al. Evolution and design governing signal precision and amplification in a bacterial chemosensory pathway. PLoS Genet. 11, e1005460 (2015).

    Article  Google Scholar 

  32. 32.

    Guzzo, M. et al. A gated relaxation oscillator mediated by FrzX controls morphogenetic movements in Myxococcus xanthus. Nat. Microbiol. 3, 948–959 (2018).

    CAS  Article  Google Scholar 

  33. 33.

    Simões, S. et al. Compartmentalisation of Rho regulators directs cell invagination during tissue morphogenesis. Development 133, 4257–4267 (2006).

    Article  Google Scholar 

  34. 34.

    Horiuchi, H. et al. A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 90, 1149–1159 (1997).

    CAS  Article  Google Scholar 

  35. 35.

    Lutkenhaus, J. The ParA/MinD family puts things in their place. Trends Microbiol. 20, 411–418 (2012).

    CAS  Article  Google Scholar 

  36. 36.

    Schumacher, D. et al. The PomXYZ proteins self-organize on the bacterial nucleoid to stimulate cell division. Dev. Cell 41, 299–314 (2017).

    CAS  Article  Google Scholar 

  37. 37.

    Inclán, Y. F., Vlamakis, H. C. & Zusman, D. R. FrzZ, a dual CheY-like response regulator, functions as an output for the Frz chemosensory pathway of Myxococcus xanthus. Mol. Microbiol. 65, 90–102 (2007).

    Article  Google Scholar 

  38. 38.

    Kaimer, C. & Zusman, D. R. Phosphorylation-dependent localization of the response regulator FrzZ signals cell reversals in Myxococcus xanthus. Mol. Microbiol. 88, 740–753 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    McLoon, A. L. et al. MglC, a paralog of Myxococcus xanthus GTPase-activating protein MglB, plays a divergent role in motility regulation. J. Bacteriol. 198, 510–520 (2016).

    CAS  Article  Google Scholar 

  40. 40.

    Pogue, C. B., Zhou, T. & Nan, B. PlpA, a PilZ-like protein, regulates directed motility of the bacterium Myxococcus xanthus. Mol. Microbiol. 107, 214–228 (2018).

    CAS  Article  Google Scholar 

  41. 41.

    Wittinghofer, A. & Vetter, I. R. Structure–function relationships of the G domain, a canonical switch motif. Annu. Rev. Biochem. 80, 943–971 (2011).

    CAS  Article  Google Scholar 

  42. 42.

    Cherfils, J. & Zeghouf, M. Regulation of small GTPases by GEFs, GAPs and GDIs. Physiol. Rev. 93, 269–309 (2013).

    CAS  Article  Google Scholar 

  43. 43.

    Hodgkin, J. & Kaiser, D. Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus. Proc. Natl Acad. Sci. USA 74, 2938–2942 (1977).

    CAS  Article  Google Scholar 

  44. 44.

    Shi, X. et al. Bioinformatics and experimental analysis of proteins of two-component systems in Myxococcus xanthus. J. Bacteriol. 190, 613–624 (2008).

    CAS  Article  Google Scholar 

  45. 45.

    Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory Press, 2001).

  46. 46.

    Shi, W. & Zusman, D. R. The two motility systems of Myxococcus xanthus show different selective advantages on various surfaces. Proc. Natl Acad. Sci. USA 90, 3378–3382 (1993).

    CAS  Article  Google Scholar 

  47. 47.

    Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  Article  Google Scholar 

  48. 48.

    Paintdakhi, A. et al. Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis. Mol. Microbiol. 99, 767–777 (2016).

    CAS  Article  Google Scholar 

  49. 49.

    Ducret, A., Théodely, O. & Mignot, T. Single cell microfluidic studies of bacterial motility. Methods Mol. Biol. 966, 97–107 (2013).

    CAS  Article  Google Scholar 

  50. 50.

    de Chaumont, F. et al. Icy: an open bioimage informatics platform for extended reproducible research. Nat. Methods 9, 690–696 (2012).

    Article  Google Scholar 

  51. 51.

    Chenouard, N., Buisson, J., Bloch, I., Bastin, P. & Olivo-Marin, J. Curvelet analysis of kymograph for tracking bi-directional particles in fluorescence microscopy images. In 2010 IEEE International Conference on Image Processing 3657–3660 (IEEE, 2010).

  52. 52.

    Bulyha, I. et al. Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins. Mol. Microbiol. 74, 691–706 (2009).

    CAS  Article  Google Scholar 

  53. 53.

    Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. & Candia, O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95–97 (1979).

    CAS  Article  Google Scholar 

  54. 54.

    Lenzen, C., Cool, R. H. & Wittinghofer, A. Analysis of intrinsic and CDC25-stimulated guanine nucleotide exchange of p21ras-nucleotide complexes by fluorescence measurements. Methods Enzymol. 255, 95–109 (1995).

    CAS  Article  Google Scholar 

  55. 55.

    Wuichet, K. & Søgaard-Andersen, L. Evolution and diversity of the Ras superfamily of small GTPases in prokaryotes. Genome Biol. Evol. 7, 57–70 (2015).

    Article  Google Scholar 

  56. 56.

    Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank D. Skotnicka for construction of SA8802 as well as A. Treuner-Lange and T. Bender for construction of pMAT162 and pTB005, respectively. This work was funded by the Deutsche Forschungsgemeinschaft (project no. 269423233) within the framework of the Transregio 174 ‘Spatiotemporal dynamics of bacterial cells’ (to U.G. and L.S.-A.) and the German–Israeli Project Cooperation ‘Spatial and Temporal Regulation of Macromolecular Complex Formation in Bacteria’ (to L.S.-A.), as well as by the Max Planck Society.

Author information

Affiliations

Authors

Contributions

K.W., D.K., D.S. and L.S.-A. conceptualized the study. K.W., D.K., D.S., A.H., L.A.M.C. and A.P. conducted the experimental work. D.S., L.A.M.C., M.W., U.G. and L.S.-A. developed the methodology for quantification of microscopy images. D.S., A.H. and L.S.-A. analysed experimental data. D.S. and L.S.-A. wrote the original draft of the manuscript. D.S., L.S.-A., K.W., D.K., L.A.M.C., M.W., A.P. and U.G. reviewed and edited the manuscript. U.G. and L.S.-A. acquired funding. U.G. and L.S.-A. provided supervision.

Corresponding author

Correspondence to Lotte Søgaard-Andersen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Notes, Supplementary Figures 1–10, Supplementary Tables 1–4 and Supplementary References.

Reporting Summary

Supplementary Dataset

Original data for Figure 1e,f,h; Figure 2b,c; Figure 4d–f; and Supplementary Figure 5a,b.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Szadkowski, D., Harms, A., Carreira, L.A.M. et al. Spatial control of the GTPase MglA by localized RomR–RomX GEF and MglB GAP activities enables Myxococcus xanthus motility. Nat Microbiol 4, 1344–1355 (2019). https://doi.org/10.1038/s41564-019-0451-4

Download citation

Further reading

Search

Quick links

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing