How proteins secure the pole position!

Everyone following Formula 1 knows that securing the pole position is not simple. The same is true for many proteins in rod-shaped bacteria that localize to the cell poles to function properly. I study how proteins that are important for motility in Myxococcus xanthus become localized to the cell poles.

Go to the profile of Dobromir Szadkowski
May 21, 2019
1
0

We use M. xanthus as a wonderful model organism to study how bacteria adapt and differentiate in response to changes in their environment. M. xanthus cells form spreading colonies in the presence of nutrients. In the absence of nutrients, they change motility behavior and initiate a developmental program, which culminates in the formation of spore-filled fruiting bodies1. Ultimately, we would like to understand how M. xanthus is able to respond to starvation with the formation of the spore-filled fruiting bodies. Because motility and its regulation are essential for this to happen, I investigate how M. xanthus cells move and regulate their motility.

M. xanthus cells are rod-shaped and have two motility systems, one for type IV pili-dependent motility and one for gliding. The two motility machineries are polarized and only assemble at the leading cell pole2. Sometimes M. xanthus cells reverse their direction of movement and after a reversal, the two motility machineries assemble at the new leading cell pole. When I started to work on motility, we knew that polarity of the motility machineries is regulated by three proteins that are all polarly localized3-7: MglA is a small Ras-like GTPase, which stimulates motility in its GTP-bound form. MglB is a GTPase activating protein (GAP) of MglA. Finally, we knew that RomR is important for polar localization of MglA-GTP. MglA-GTP localizes to the leading cell pole while MglB and RomR are asymmetrically localized to the cell poles but mostly at the lagging cell pole.

The inspiration for my work was the observation that small GTPases in eukaryotes not only depend on a GAP for efficient GTP hydrolysis but also on a guanine nucleotide exchange factor (GEF) to efficiently exchange GDP for GTP8. We already had some hints that MglA would also depend on a GEF for nucleotide exchange when I started my PhD project. To identify GEF candidates, Kristin Wuichet, a former postdoc in the lab, used a phylogenomics approach. Because MglA and MglB homologs are widespread in prokaryotes 9 while RomR has a more limited distribution7, Kristin decided to search for proteins that have the same genomic distribution as RomR. This approach turned out to be very successful and lead to the identification of the protein that we now call RomX. As expected for a GEF of MglA, RomX is essential for correct motility. We also found that RomX localizes dynamically to the cell poles and this localization depends on RomR (Figure 1).

 

Figure 1. M. xanthus cells expressing a RomX-YFP fusion illustrates the polar localization of RomX.

 

Next, I turned to in vitro analyses using purified proteins to determine whether RomX would interact with MglA, MglB or RomR. This work demonstrated that RomX interacts with RomR as well as with MglA-GTP, and the three proteins form a RomR/RomX/MglA-GTP complex. Importantly, we also found that RomX has MglA GEF activity and that RomR enhances this activity in the RomR/RomX complex. Putting all our data together, we suggest that in an M. xanthus cell, RomR recruits RomX to the cell poles and the RomR/RomX complex, in turn, recruits MglA-GTP to the leading cell pole via two mechanisms. First, RomR/RomX stimulates accumulation of MglA-GTP using its GEF activity; and, second, RomR/RomX binds MglA-GTP at the leading cell pole. Both activities contribute to a high local concentration of MglA-GTP at this pole with the stimulation of motility. MglA-GTP is kept away from the lagging cell pole because MglB with its GAP activity localizes to this pole. So, the spatially separated activities of the RomR/RomX GEF complex and the MglB GAP at opposite poles help to establish front-rear polarity for gliding motility The RomR/RomX complex is the first bacterial nucleotide exchange factor shown to be involved in regulating bacterial cell polarity.

Interestingly, the GEF and GAP of MglA have a spatial organization that is strikingly similar to the GEFs and GAPs of small GTPases involved in regulation of cell polarity and/or motility in eukaryotes. It is also interesting to note that MglA is found widespread in prokaryotes; however, RomR and RomX have a much more limited distribution suggesting that that there might be other GEFs acting on MglA-like proteins.

References

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

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

3          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).

4          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).

5          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).

6          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).

7          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).

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

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

Go to the profile of Dobromir Szadkowski

Dobromir Szadkowski

Postdoctoral Fellow , Max Planck Institute for Terrestrial Microbiology, Department of Ecophysiology

No comments yet.