Prof. Dr. Lotte Sogaard-Andersen - Research Area

Bacterial adaption and differentiation

Intercellular communication

Self-organization and pattern formation

Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Motility

Regulation of motility & cell polarity

Cell cycle regulation with an emphasis on chromosome replication & cell division

Synthetic (micro)biology & cell polarity modularity

The genetic basis underlying differences in fruiting body morphology


Intercellular communication

Over the past decades the perception of bacterial cells as autonomous individuals each following their own agenda and not interacting with each other has been replaced by the view that bacteria interact extensively both within and between species by means of intercellular signal molecules. Each of these signal molecules constitutes part of an information processing system that is constructed of four parts: The donor cell synthesizing the signal, the signal molecule, the recipient cell, and the output response. Like in any other information processing system, the signal must be tailored to the talents of the recipient. A clear example of a tailor-made signal molecule is the C-signal molecule in M. xanthus. Most intercellular signals identified in bacteria are small, i.e. with sizes < 1000 Da, freely diffusible molecules that are part of quorum sensing systems, which helps bacterial cells to assess population size. However, that is not the case for the intercellular C-signal in M. xanthus. This signal is a 17 kDa cell surface-associated protein and non-diffusible, and C-signal transmission depends on direct contacts between cells.

<strong> In vegetative cells PopC interacts with PopD and, consequently, PopC secretion is inhibited.</strong> In response to starvation, the (p)ppGpp synthase RelA is activated and by an unknown mechanism (p)ppGpp results in PopD degradation in a process that depends on the FtsH<sup>D</sup> protease. Subsequently, PopC is secreted and cleaves p25 to generate p17. Zoom Image
In vegetative cells PopC interacts with PopD and, consequently, PopC secretion is inhibited. In response to starvation, the (p)ppGpp synthase RelA is activated and by an unknown mechanism (p)ppGpp results in PopD degradation in a process that depends on the FtsHD protease. Subsequently, PopC is secreted and cleaves p25 to generate p17. [less]

The C-signal is one of two functionally and biochemically characterized intercellular signals required for fruiting body formation. The C-signal is the intercellular signal that induces and coordinates aggregation of cells into fruiting bodies and sporulation of those cells that have accumulated inside fruiting bodies. C-signal synthesis depends on the csgA gene that encodes the full-length 25 kDa CsgA protein (p25), which is anchored in the outer membrane. p25 accumulates in vegetative cells as well as in starving cells. During starvation p25 is cleaved to a 17 kDa protein, which is also anchored in the outer membrane and exposed on the cell surface. This 17 kDa protein (p17) is the actual C-signal. So, unlike other intercellular signaling molecules identified in bacteria, the C-signal is not a small diffusible molecule but a relatively large cell-surface associated protein. Consistently, C-signal transmission depends on direct cell-cell contacts. Recently, we have figured out how p17 synthesis is restricted to starving cells by regulated proteolysis involving an FtsH protease and the secreted protease PopC. Our current research focuses on understanding how PopC and p25 secretion is regulated and the identification of the p17 receptor.


Some of our recent publications on intercellular signaling:


Lobedanz, S. & Søgaard-Andersen, L. (2003). Identification of the C-signal, a contact-dependent morphogen coordinating multiple developmental responses in Myxococcus xanthus. Genes Dev. 17, 2151-2161.

Rolbetzki, A., Ammon, M., Jakovljevic, V., Konovalova, A. & Søgaard-Andersen, L. (2008) Regulated secretion of a protease activates intercellular signaling during fruiting body formation in M. xanthus. Dev. Cell. 15, 627-634.

Konovalova, A., Löbach, S. & Søgaard-Andersen, L. (2012) A RelA-dependent two-tiered regulated proteolysis cascade controls synthesis of a contact-dependent intercellular signal in Myxococcus xanthus. Mol. Microbiol. 84, 260-275.

Konovalova, A., Wegener-Feldbrügge, S. & Søgaard-Andersen, L. (2012). Two intercellular signals required for fruiting body formation in Myxococcus xanthus act sequentially but non-hierarchically. Mol. Microbiol. 86, 65-81.


Some of our recent publications on cell-cell contact-dependent activities and regulated proteolysis in bacteria:


Konovalova, A. & Søgaard Andersen, L. (2011). Close encounters: Contact-dependent interactions in bacteria. Mol. Microbiol. 91, 297-301.

Konovalova, A, Søgaard-Andersen, L. & Lee Kroos (2014) Regulated proteolysis in bacterial development. FEMS Microbiol. Rev. 38, 493-522.

Bacterial adaption and differentiation

Intercellular communication

Self-organization and pattern formation

Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Motility

Regulation of motility & cell polarity

Cell cycle regulation with an emphasis on chromosome replication & cell division

Synthetic (micro)biology & cell polarity modularity

The genetic basis underlying differences in fruiting body morphology


Self-organization and pattern formation

Formation of spatial patterns of cells from a mass of initially identical cells is a recurring theme in developmental biology. The dynamics that direct pattern formation in biological systems often involve morphogenetic cell movements. Fruiting body formation in M. xanthus is also such an example. Here, an initially unstructured population of identical cells self-organizes into an asymmetric, stable pattern of multicellular fruiting bodies. This pattern formation process depends on the ability of cells to move and regulate their motility behavior. Fruiting body formation is a self-organizing pattern formation process as it starts from a homogeneous and symmetric population of starving cells, and occurs without the contribution of external cues to help direct the movements of cells.

<strong>The x, y positions of wt cells (green) and csgA cells (orange) that had been starved for 3 hrs and 15 hrs.</strong> Each trajectory represents the x, y positions of a representative cell recorded every 15 sec for 900 sec. Zoom Image
The x, y positions of wt cells (green) and csgA cells (orange) that had been starved for 3 hrs and 15 hrs. Each trajectory represents the x, y positions of a representative cell recorded every 15 sec for 900 sec. [less]

M. xanthus cells are rod-shaped and move in the direction of their long axis when placed on a solid surface. Occasionally cells stop and then reverse their direction of movement. So, over time, M. xanthus cells display irregular oscillations. The C-signal (=p17) is essential for aggregation of cells into fruiting bodies and changes the motility behavior of cells after 6 hrs of starvation. To understand how the C-signal directs cells into fruiting bodies, we analyzed how C-signaling affects cell behavior. In total, the outcome of the C-signal-dependent changes in motility is a switch from an oscillatory to a unidirectional type of motility behavior in which the net-distance traveled by a cell per minute is increased (compare the green trajectories to the orange trajectories at 15 hrs of starvation). Based on these findings, we have started to develop mathematical models that describe pattern formation in M. xanthus. Probably the most important effect of the C-signal on cell behavior is the inhibition of cellular reversals. In our current research we focus on understanding at the molecular level how this inhibition is brought about.


Some of our recent publications on pattern formation:


Jelsbak, L. & Søgaard-Andersen, L. (1999). The cell-surface associated, intercellular C-signal induces behavioral changes in individual Myxococcus xanthus cells during fruiting body morphogenesis. Proc. Natl. Acad. Sci. USA. 96, 5031-5036.

Jelsbak, L. & Søgaard-Andersen, L. (2002). Pattern formation by a cell surface-associated morphogen in M. xanthus. Proc. Natl. Acad. Sci. USA. 99, 2032-2037.

Starruß, J., Bley, T., Søgaard­Andersen, L. & Deutsch; A. (2007). A new mechanism for collective migration of Myxococcus xanthus. J. Stat. Phys. 128, 269-286.

Lenz, P. & Søgaard-Andersen, L. (2011) Temporal and spatial oscillations in bacteria. Nat. Rev. Microbiol. 9, 565-577.

Peruani, F., Starruß, J., Jakovljevic, V., Søgaard-Andersen, L., Bär, M. & Deutsch, A. (2012). Collective motion and nonequilibrium cluster formation in colonies of gliding bacteria. Phys. Rev. Lett. 108, 098102.

Starruß, J., Peruani, F., Jakovljevic, V., Søgaard-Andersen, L., Deutsch, A. & Bär, M., (2012) Pattern formation mechanisms in motility mutants of Myxococcus xanthus. R. Soc. Interface Focus. 2, 774-785.

Bacterial adaption and differentiation

Intercellular communication

Self-organization and pattern formation

Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Motility

Regulation of motility & cell polarity

Cell cycle regulation with an emphasis on chromosome replication & cell division

Synthetic (micro)biology & cell polarity modularity

The genetic basis underlying differences in fruiting body morphology


Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Despite the multitude of cues that bacteria need to monitor and respond to, the signal transduction schemes involved center on just a few types. One of the most important of these schemes is that of two component systems. A standard two component system consists of a histidine protein kinase and a partner response regulator. In response to a specific stimulus, the kinase autophosphorylates on His residue and then the phosphoryl-group is transferred to a Asp residue in the receiver domain of the response regulator.

<strong>Phosphate flow in a standard two component system.</strong> Zoom Image
Phosphate flow in a standard two component system.

The M. xanthus genome encodes more than 200 proteins of two component systems and several of these proteins are important for fruiting body formation. Intriguingly, these genes are organized in a highly unusual manner with 55% being orphan and 16% in complex gene clusters whereas only 29% display the standard paired gene organization. The large number of orphan proteins is peculiar and it represents a major challenge to understand how they are wired together. Our bioinformatics analyses have shown that a major output of signaling by two component systems is the regulation of signaling by the second messenger cyclic-di-GMP regulation. In general, c-di-GMP controls the switch from a planktonic, motile lifestyle to a surface-associated, sessile biofilm lifestyle.

A major goal in our research is to understand how proteins of two component systems are connected and to identify their function. Also, we have a strong interest in understanding the mechanism of structurally unusual proteins of two component systems. In addition to identifying several proteins of two component systems that are important for fruiting body formation or motility, we have recently shown that the orphan histidine protein kinase SgmT is a c-di-GMP receptor and functions together with the orphan response regulator DigR to regulate motility and fruiting body formation. In our current research we focus on understanding how c-di-GMP signaling regulates motility and fruiting body formation.


Some of our recent publications on signal transduction by two component systems & the second messenger c-di-GMP:


Rasmussen, A. Aa., Wegener-Feldbrügge, S., Porter S., Armitage, J.P. & Søgaard-Andersen, L. (2006). Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity. Mol. Microbiol. 60, 525-534.

Overgaard; M, Wegener-Feldbrügge, S. & Søgaard-Andersen, L. (2006). The orphan response regulator DigR is required for synthesis of extracellular matrix fibrils in Myxococcus xanthus. J. Bacteriol. 188, 4384-4394.

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

Shi, X., Wegener-Feldbrügge, S., Huntley, S., Hamann, N., Hedderich, R. & Søgaard-Andersen, L. (2008) Bioinformatics and experimental analysis of proteins of two-component systems in Myxococcus xanthus. J. Bacteriol. 190, 613-624.

Wegener-Feldbrügge, S. & Søgaard-Andersen, L. (2009) The atypical hybrid histidine protein kinase RodK in Myxococcus xanthus: Spatial proximity supersedes kinetic preference in phosphotransfer reactions. J. Bacteriol. 191, 1765-1776.

Petters, T., Zhang, X., Nesper, J., Treuner-Lange, A., Gomez-Santos, N., Hoppert, M., Jenal, U. & Søgaard-Andersen, L. (2012). The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus. Mol. Microbiol. 84, 147-165.

Keilberg, D., Wuichet, K., Drescher, F. & Søgaard-Andersen, L. (2012). A response regulator interfaces between the Frz chemosensory system and the MglA/MglB GTPase/GAP module to regulate polarity in Myxococcus xanthus. PLoS Genet. 9, e1002951.

Skotnicka, D., Petters, T., Heering, J., Hoppert, M., Kaever, V. & Søgaard-Andersen, L. (2015) c-di-GMP regulates type IV pili-dependent-motility in Myxococcus xanthus. J. Bacteriol. In press.

Bacterial adaption and differentiation

Intercellular communication

Self-organization and pattern formation

Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Motility

Regulation of motility & cell polarity

Cell cycle regulation with an emphasis on chromosome replication & cell division

Synthetic (micro)biology & cell polarity modularity

The genetic basis underlying differences in fruiting body morphology


Motility

Motility significantly contributes to the fitness of bacteria. Generally, bacteria can move using one or more of three different types of motility machineries. These nanomachines include flagella, type IV pili (T4P) and gliding machineries. While the flagella and T4P machineries are highly conserved the machineries for gliding are not highly conserved.

<strong><em>M. xanthus cells</em> have two motility systems.</strong> Type IV pili localize to the leading cell pole. Focal adhesion complexes are assembled at the leading cell pole and disassemble at the lagging cell pole. Reversals are induced by the Frz chemosensory system and a cell switch polarity during a reversal. Zoom Image
M. xanthus cells have two motility systems. Type IV pili localize to the leading cell pole. Focal adhesion complexes are assembled at the leading cell pole and disassemble at the lagging cell pole. Reversals are induced by the Frz chemosensory system and a cell switch polarity during a reversal. [less]

Motility is essential for formation of both types of cellular patterns or biofilms in M. xanthus. M. xanthus moves by means of T4P and gliding. The rod-shaped M. xanthus cells move in the direction of their long axis and have a leading cell pole and a lagging cell pole. Occasionally, cells reverse their direction of movement and during a reversal, the old leading pole becomes the new lagging cell pole. T4P assemble at the leading cell pole and during a reversal they disassemble at the old leading pole and reassemble at the new leading cell pole. In the case of the gliding motility system, the machinery (which is often referred to as focal adhesion complexes, FAC) assembles at the leading cell pole and disassemble at the lagging cell pole in moving cells. During a reversal, the cell poles at which FAC assemble and disassemble also switches. So, in total, during a reversal, the two motility machineries switch polarity.

In vegetative cells, cells at the edge of a colony efficiently explore the neighborhood by moving away from the colony and then reversing to return to the colony. During the aggregation stage of fruiting body formation, the C-signal induces a decrease in the cellular reversal frequency and switches cell behavior from an oscillatory mode to a unidirectional mode in which cells are moving towards nascent fruiting bodies. In our motility research we focus on elucidating how the two motility machineries assemble and function as well as on the mechanisms underlying polarity switching of the two motility systems. We also aim to understand at the molecular level how the C-signal inhibits reversals.

T4P are surface structures that are found on a large number of different bacterial species and they have important function in biofilm formation, virulence and motility. T4P function depends on the assembly of a macromolecular complex consisting of 10 proteins that localize to the outer membrane, periplasm, inner membrane and cytoplasm. We have shown that eight of these proteins localize to both cell poles and remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction of T4P, respectively, localize to opposite poles with PilB predominantly at the leading and PilT predominantly at the lagging pole, and these proteins switch poles during reversals. In other words, type IV pili pole-to-pole switchings involve the disassembly of the type IV pili machinery at the old leading pole and reassembly of this machinery at the new leading pole.

<strong>Type IV pili pole-to-pole switching depend on the disassembly and reassembly of the type IV pili molecular machine.</strong> Zoom Image
Type IV pili pole-to-pole switching depend on the disassembly and reassembly of the type IV pili molecular machine.

Currently, we are focusing on analyses to understand how T4P proteins are targeted to the poles. We are also focusing on analyses to understand the structure of FAC and how they assemble and disassemble at opposite poles.


Some of our recent publications on motility:


Jakovljevic, V., Leonardy, S., Hoppert, M. & Søgaard-Andersen, L. (2008) PilB and PilT are ATPases acting antagonistically in type IV pili function in Myxococcus xanthus. J. Bacteriol. 190, 2411-2421.

Leonardy, S., Bulyha, I. & Søgaard-Andersen, L. (2008) Reversing cells and oscillating proteins. Mol. BioSystems 4, 1009 - 1014.

Clausen, M., Jakovljevic, V., Søgaard-Andersen, L. & Maier, B. (2009) High force generation is a conserved property of type IV pilus systems. J. Bacteriol. 191, 4633-4638.

Bulyha, I., Schmidt, C., Lenz, P., Jakovljevic, V., Höne, A., Maier, B., Hoppert, M., & Søgaard-Andersen, L. (2009) Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins. Mol. Microbiol. 74, 691-706.

Søgaard-Andersen, L. (2011) Directional intracellular trafficking in bacteria. Proc. Natl. Acad. Sci. USA 108, 7283-7284.

Bulyha, I., Lindow,S., Lin, L., Bolte, K., Wuichet, K., Kahnt, J., van der Does, C., Thanbichler, M. & Søgaard-Andersen, L. (2013) Two small GTPases act in concert with the bactofilin cytoskeleton to regulate dynamic bacterial cell polarity. Dev. Cell. 25, 119-131.

Friedrich, C., Bulyha, I. & Søgaard-Andersen, L. (2014) Outside-in assembly pathway of the type IV pili system in Myxococcus xanthus. J. Bacteriol. 196, 378-390.

Siewering, K., Jain, S., Friedrich, C., Webber-Birungi, M.T., Semchonok, D.A., Binzen, I., Wagner, A., Huntley, S., Kahnt, J., Klingl, A., Boekema, E.J., Søgaard-Andersen, L. & van der Does, C. (2014) Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili. Proc. Natl. Acad. Sci. USA. 111, E953-E961.

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

Bacterial adaption and differentiation

Intercellular communication

Self-organization and pattern formation

Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Motility

Regulation of motility & cell polarity

Cell cycle regulation with an emphasis on chromosome replication & cell division

Synthetic (micro)biology & cell polarity modularity

The genetic basis underlying differences in fruiting body morphology


Regulation of motility & cell polarity

Motility in M. xanthus depends on the polar localization of motility proteins. Some of these protein localize in a stationary manner at the poles and others (such as PilB and PilT) localize dynamically to the cell poles and switch poles during reversals. At the cellular level, these localization patterns reflect the underlying polarity of the rod-shaped M. xanthus cells with a leading and lagging cell pole.

To understand regulation of motility and the underlying cell polarity, we have focused on the dynamic localization of PilB and PilT to opposite cell poles. It turns out that this localization depends on an intricate interplay between two small Ras-like GTPases and the cytoskeleton: We and others have shown that the small GTPase MglA functions as a nucleotide-dependent molecular switch to stimulate motility and that MglB represents a novel GTPase-activating protein (GAP) family and is the cognate GAP of MglA. MglA-GTP represents the active form, which stimulates motility. Lately, we have shown that MglA-GTP is targeted to the leading cell pole by the response regulator RomR, whereas MglA-GDP, the inactive form of MglA, localizes diffusely throughout the cell. MglB localizes to the lagging pole together with RomR. MglB together with RomR excludes MglA-GTP from the lagging pole by converting MglA-GTP to MglA-GDP and, thus, set up the MglA-GTP asymmetry. During reversals MglA, MglB and RomR switch poles resulting in an inversion of the leading/lagging cell polarity axis. Recently, we were able to show that MglA-GTP stimulates T4P-dependent motility by sorting PilB and PilT to opposite cell poles, i.e. MglA in the absence of MglA, PilB and PilT localize to the same cell pole.

<strong>Left panel, MglA, MglB and RomR establish the leading/lagging cell polarity axis.</strong> Right panel, the Frz chemosensory system signals to the RomR response regulator for reversals. Zoom Image
Left panel, MglA, MglB and RomR establish the leading/lagging cell polarity axis. Right panel, the Frz chemosensory system signals to the RomR response regulator for reversals. [less]

We have also shown that the RomR response regulator interfaces with FrzZ, the output response regulator of the Frz chemosensory system, to regulate reversals. Thus, RomR serves at the interface to connect a classic bacterial signaling module (Frz) to a classic eukaryotic polarity module (MglA/MglB). Our bioinformatics analyses have shown that this modular design is paralleled by the phylogenetic distribution of the proteins suggesting an evolutionary scheme in which RomR was incorporated into the MglA/MglB module to regulate cell polarity followed by the addition of the Frz system to dynamically regulate cell polarity.

MglA is important for sorting PilB and PilT to opposite cell poles but not for polar localization per se of the two proteins. Motivated by the observation that cell polarity and motility in eukaryotic cells often depend on two or more small GTPases that act in parallel or in a cascade, we hypothesized that polar localization of PilB and PilT could involve an additional small GTPase. Indeed, we have shown that the small Ras-like GTPase SofG is important for polar localization of PilB and PilT. Moreover, we have shown that SofG directly interacts with BacP, a bactofilin cytoskeletal protein, and that BacP is also required for polar localization of PilB and PilT. Using a variety of different cell imaging approaches we found that polymeric BacP localizes in both subpolar regions. SofG associates with one of these patches forming a subpolar cluster that shuttles to the pole to establish polar localization of PilB and PilT at the same pole. Following the SofG- and BacP-dependent localization of PilB and PilT to the same pole, a second event follows in which MglA sorts PilB and PilT to opposite poles to set up their correct polar localization in this way enabling T4P-dependent motility. Thus, the two small GTPases SofG and MglA function in a cascade-like manner to regulate PilB and PilT polarity. During reversals, the Frz chemosensory system causes the inversion of the leading/lagging polarity axis by inducing the relocation of MglA, MglB and RomR. Thus, three regulatory systems function in a cascade to regulate the dynamic localization of PilB and PilT.

<strong>Hierarchical regulation of dynamic PilB &amp; PilT polarity.</strong> For simplicity only a single BacP filament is shown in the two subpolar regions. (i-iv) BacP and SofG establish the localization of PilB and PilT to the same pole by SofG shuttling on BacP. (v) MglA sorts PilB and PilT to opposite poles. (vi) During a cellular reversal induced by the Frz chemosensory system with an inversion of the leading/lagging polarity axis, MglA, MglB and RomR switch poles, and, therefore, the polarity of PilB and PilT is also inverted. Zoom Image
Hierarchical regulation of dynamic PilB & PilT polarity. For simplicity only a single BacP filament is shown in the two subpolar regions. (i-iv) BacP and SofG establish the localization of PilB and PilT to the same pole by SofG shuttling on BacP. (v) MglA sorts PilB and PilT to opposite poles. (vi) During a cellular reversal induced by the Frz chemosensory system with an inversion of the leading/lagging polarity axis, MglA, MglB and RomR switch poles, and, therefore, the polarity of PilB and PilT is also inverted. [less]

One way to think about the MglA, MglB and RomR system is that it functions as a spatial toggle switch that is either locked in one or the other of two possible states. In response to signaling by the Frz system, the switch is flipped. This unusual system is amenable to mathematical modeling and we are intensively pursuing this approach to understand the minimal requirements for the system to work.

In our current research we are aiming at understanding how MglA, MglB and RomR localize to the cell poles and to understand in details how the Frz system "communicates" with MglA/MglB/RomR. We are also interested in understanding how MglA helps to sort PilB and PilT to opposite cell poles.


Some of our recent publications on regulation of motility & cell polarity:


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

Leonardy, S., Bulyha, I. & Søgaard-Andersen, L. (2008) Reversing cells and oscillating proteins. Mol. BioSystems 4, 1009 - 1014.

Bulyha, I., Schmidt, C., Lenz, P., Jakovljevic, V., Höne, A., Maier, B., Hoppert, M., & Søgaard-Andersen, L. (2009) Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins. Mol. Microbiol. 74, 691-706.

Leonardy, S., Miertzschke, M., Bulyha, I., Sperling, E., Wittinghofer, A. & Søgaard-Andersen, L. (2010) Regulation of dynamic polarity switching in bacteria by a Ras-like G-protein and its cognate GAP. EMBO J. 29, 2276-2289.

Lenz, P. & Søgaard-Andersen, L. (2011) Temporal and spatial oscillations in bacteria. Nat. Rev. Microbiol. 9, 565-577.

Miertzschke M., Koerner C., Vetter I.R., Keilberg D., Hot E., Leonardy S., Søgaard-Andersen L. & Wittinghofer A. (2011) Mechanistic insights into bacterial polarity from structural analysis of the Ras-like G protein MglA and its cognate GAP MglB. EMBO J. 30, 4185- 4197.

Bulyha, I., Hot, E., Huntley, S. & Søgaard-Andersen, L. (2011). GTPases in bacterial cell polarity and signalling. Curr. Opin. Microbiol. 14, 726-733.

Rashkov, P., Schmitt, B.A., Søgaard-Andersen, L., Lenz, P. & Dahlke, S. (2012). A model of oscillatory protein dynamics in bacteria. Bull. Math. Biol. 74, 2183-2203.

Keilberg, D., Wuichet, K., Drescher, F. & Søgaard-Andersen, L. (2012). A response regulator interfaces between the Frz chemosensory system and the MglA/MglB GTPase/GAP module to regulate polarity in Myxococcus xanthus. PLoS Genet. 9, e1002951.

Bulyha, I., Lindow,S., Lin, L., Bolte, K., Wuichet, K., Kahnt, J., van der Does, C., Thanbichler, M. & Søgaard-Andersen, L. (2013) Two small GTPases act in concert with the bactofilin cytoskeleton to regulate dynamic bacterial cell polarity. Dev. Cell. 25, 119-131.

Rashkov, P., Schmitt, B.A., Søgaard-Andersen, L., Lenz, P. & Dahlke, S. (2013). A model for antagonistic protein dynamics. Int. J. Biomath. Biostat. 2, 75-85.

Keilberg, D. & Søgaard-Andersen, L. (2014) Regulation of bacterial cell polarity by small GTPases. Biochemistry. 53, 1899-1907.

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

Wuichet, K. & Søgaard-Andersen, L.(2014) Evolution and diversity of the Ras superfamily of small GTPases in prokaryotes. Genome Biol. Evol. 7, 57-70.

Treuner-Lange, A., Macia, E., Guzzo, M., Hot, E., Faure, L., Jakobczak, B., Espinosa, L., Alcor, D., Ducret, A., Keilberg, D., Castaing, J.P., Gervais, S.L., Franco, M., Søgaard-Andersen, L. & Mignot, T. (2015) The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions. J. Cell Biol. 210, 243-256.

McLoon, A.L., Wuichet, K., Häsler, M., Keilberg,D., Szadkowski, D. & Søgaard-Andersen, L. (2015) MglC, a paralog of Myxococcus xanthus GTPase activating protein MglB, plays a divergent role in motility regulation. J. Bacteriol. In press.

Bacterial adaption and differentiation

Intercellular communication

Self-organization and pattern formation

Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Motility

Regulation of motility & cell polarity

Cell cycle regulation with an emphasis on chromosome replication & cell division

Synthetic (micro)biology & cell polarity modularity

The genetic basis underlying differences in fruiting body morphology


Cell cycle regulation with an emphasis on chromosome replication & cell division

In all cells, accurate positioning of the cell division site is essential for generating appropriately-sized daughter cells with a correct chromosome number. In bacteria, cell division generally occurs at mid-cell and initiates with assembly of the tubulin homologue FtsZ into a circumferential ring-like structure, the Z-ring, at the incipient division site. Subsequently, FtsZ recruits the remaining components of the cell division machinery needed to carry out cytokinesis. Thus, the position of Z-ring formation dictates the cell division site. Accordingly, all known systems that regulate positioning of the division site in bacteria control Z-ring positioning. In principle, specification of the cell division site could depend on positively acting systems that precisely define the site of cell division, on negatively acting systems that inhibit cell division everywhere in a cell except at the incipient division site, or a combination of both. In other bacteria, regulators of Z-ring formation act negatively to inhibit Z-ring formation at the cell poles and over the nucleoid, leaving only mid-cell free for Z-ring formation. Our data suggest that Z-ring formation is positively regulated in M. xanthus.

<strong>Spatial regulation of Z-ring formation and cell division in bacteria</strong>. Left cell, regulators (red) inhibit Z-ring (green) formation at the cell poles and over the nucleoid (light blue) leaving only midcell free for Z-ring formation; right cell, regulators (red) directly stimulate Z-ring formation at midcell. Zoom Image
Spatial regulation of Z-ring formation and cell division in bacteria. Left cell, regulators (red) inhibit Z-ring (green) formation at the cell poles and over the nucleoid (light blue) leaving only midcell free for Z-ring formation; right cell, regulators (red) directly stimulate Z-ring formation at midcell. [less]

Fruiting body formation and spore formation are closely coupled to cell cycle progression. While rod-shaped vegetative cells contain one chromosome after cell division, mature spores contain two chromosomes suggesting that at some point during the sporulation process cell division is inhibited. To understand the coupling between cell cycle regulation and development, we are focusing on understanding regulation of cell division and chromosome replication and segregation.

The rod-shaped M. xanthus cells divide by binary fission. We have shown that the PomZ protein, which is a member of the ParA subfamily of P-loop ATPases, regulates positioning of the cell division site in M. xanthus. In particular, we were able to show that lack of PomZ results in a severe reduction in Z-ring formation, and abnormal positioning of the few Z-rings formed. PomZ localization changes with cell cycle progression and culminates in the localization to the incipient division site at mid-cell before and in the absence of FtsZ. Based on these observations we have proposed that PomZ is a novel spatial regulator of cell division that provides positional information for Z-ring formation in that way positively regulating positioning of the cell division site. In addition, our data suggest that PomZ stabilizes the Z-ring. The finding that PomZ may positively regulate cell division site positioning illustrates that bacteria may also employ positively acting systems to specify the site of cell division.

Lately, we have determined the spatial organization of the 9.1 Mb M. xanthus chromosome as well as chromosome dynamics during replication and segregation in vegetative cells. For chromosome segregation, M. xanthus uses a ParABS system. The genome is organized about a longitudinal axis with ori in a subpolar region and ter in the opposite subpolar region. Upon replication, one ori remains at the original subpolar region while the second copy in a directed and ParABS-dependent manner segregates to the opposite subpolar region followed by the rest of the chromosome. In parallel, ter relocates from a subpolar region to midcell. Replication involves replisomes that track independently of each other from the ori-containing subpolar region towards ter. In M. xanthus the dynamics of chromosome replication and segregation combine features from previously described systems leading to a novel spatiotemporal organization pattern.

<strong>Chromosome organization and dynamics in M. xanthus.</strong> Left panel, the diagram illustrates a cell through seven stages of chromosome replication, chromosome segregation and division during a cell cycle. The two chromosomes with their associated ori (blue dot) and ter (green dot) are shown in black and grey, respectively. The replisomes are shown as yellow dots. ParA is shown in light and dark red. Right panel, ParA-mCherry (red) and ParB-YFP (green) localize at the edge of chromosomes (blue). Zoom Image
Chromosome organization and dynamics in M. xanthus. Left panel, the diagram illustrates a cell through seven stages of chromosome replication, chromosome segregation and division during a cell cycle. The two chromosomes with their associated ori (blue dot) and ter (green dot) are shown in black and grey, respectively. The replisomes are shown as yellow dots. ParA is shown in light and dark red. Right panel, ParA-mCherry (red) and ParB-YFP (green) localize at the edge of chromosomes (blue). [less]

In our current research, we focus on understanding how PomZ identifies midcell. Also, we are aiming to understand how cell division and chromosome dynamics are regulated during the sporulation process.


Some of our recent publications on regulation of chromosome replication & cell division:


Treuner-Lange, A., Aguiluz, K., van der Does, C., Gómez-Santos, N., Lenz, P., Harms, A., Schumacher, D., Hoppert, M., Kahnt, J., Muñoz-Dorado, J. & Søgaard-Andersen, L. (2013). PomZ, a ParA-like protein, regulates Z-ring formation and cell division in Myxococcus xanthus. Mol. Microbiol. 87, 235-253.

Harms, A., Treuner-Lange, A., Schumacher, D. & Søgaard-Andersen, L. (2013) Tracking of chromosome and replisome dynamics in Myxococcus xanthus reveals a novel chromosome arrangement. PLoS Genet. 9, e1003802.

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

Bacterial adaption and differentiation

Intercellular communication

Self-organization and pattern formation

Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Motility

Regulation of motility & cell polarity

Cell cycle regulation with an emphasis on chromosome replication & cell division

Synthetic (micro)biology & cell polarity modularity

The genetic basis underlying differences in fruiting body morphology


Synthetic (micro)biology & cell polarity modularity

All cells are polarized and contain proteins that localize asymmetrically to specific subcellular regions. In bacteria as well as in eukaryotic cells, polarized proteins are important for fundamental cellular processes such as cell division, growth, motility and differentiation. We have as a working hypothesis that streamlined natural cells as well as synthetic cells depend on some level of subcellular organization for optimal function. Therefore, the overall goal of our work in synthetic microbiology is to generate a module for regulating dynamic cell polarity in streamlined natural cells as well as in synthetic cells.

For most dynamically localized proteins, the localization pattern changes in a cell cycle dependent manner. An exception to this general rule are motility proteins in M. xanthus that localize dynamically to the cell poles in a cell cycle-independent manner, i.e. during a cellular reversal motility proteins localizing to the lagging cell pole switch to the new lagging cell pole and proteins and the leading cell pole switch to the new leading cell pole. The specific goal of our synthetic microbiology is to identify and characterize the components of the regulatory system that underlies the dynamic polarity of motility proteins in M. xanthus. On the basis of this system, we aim to define a minimal module for regulation of dynamic cell polarity in bacteria and to establish this system in other microorganisms as well as in synthetic cells. As part of this research, we are addressing a more fundamental question, i.e. are cell polarity systems modular? In other words, can these systems be transferred between organisms and still function? Or are they so tightly integrated with host cell physiology that function is restricted to the original host?

The regulatory system that controls polarity of motility proteins in M. xanthus is built around the MglA, MglB and RomR proteins, which interact to define the leading/lagging polarity axis. MglA is a small Ras-like GTPase that functions as a nucleotide-dependent molecular switch to regulate motility in M. xanthus. The MglB protein functions as a GTPase activating protein (GAP) and converts active MglA-GTP to the inactive MglA-GDP. Between reversals MglA-GTP localizes to the leading cell pole together with RomR while MglB localizes to the lagging cell pole also together with RomR. MglA-GTP between reversals sets up the correct polarity of dynamically motility proteins. In response to signaling activity of the Frz chemosensory system MglA, MglB and RomR are released from the poles and then relocate to the respective opposite poles. In total, this results in an inversion of the leading/lagging polarity axis and the relocation of dynamic motility proteins.

Currently, we focus on defining the parts of this cell polarity system and on elucidating how the various proteins interact. In parallel, we are attempting to establish the system in heterologous hosts.

Bacterial adaption and differentiation

Intercellular communication

Self-organization and pattern formation

Signal transduction by two component systems & the second messengers (p)ppGpp and c-di-GMP

Motility

Regulation of motility & cell polarity

Cell cycle regulation with an emphasis on chromosome replication & cell division

Synthetic (micro)biology & cell polarity modularity

The genetic basis underlying differences in fruiting body morphology


The genetic basis underlying differences in fruiting body morphology

<strong>Phylogeny within the myxobacteria.</strong> The species included in our comparative analyses are indicated by the asterisks (*).Bootstrap values (percentages), shown at nodes, are based on 1000 replications. The vertical bar indicates the three suborders within the myxobacteria: light gray, Cystobacterineae; dark gray, Nannocystineae; and black, Sorangineae. Zoom Image
Phylogeny within the myxobacteria. The species included in our comparative analyses are indicated by the asterisks (*).Bootstrap values (percentages), shown at nodes, are based on 1000 replications. The vertical bar indicates the three suborders within the myxobacteria: light gray, Cystobacterineae; dark gray, Nannocystineae; and black, Sorangineae. [less]

M. xanthus has emerged as the model organism to understand the molecular mechanisms underlying fruiting body formation in Myxobacteria. All Myxobacteria tested - with the exception of one - initiate fruiting body formation in response to starvation suggesting that the last common ancestor of the Myxobacteria harboured a genetic program for fruiting body formation and that fruiting Myxobacteria would share in common a genetic program underlying fruiting body formation.

We used comparative and functional genomics on four complete genomes of fruiting myxobacteria (M. xanthus, Stigmatella aurantiaca, Sorangium cellulosum and Haliangium ochraceum) and one genome of the only known non-fruiting myxobacterium (Anaeromyxobacter dehalogenans) to test this hypothesis. Surprisingly, these comparative analyses strongly indicate that the genetic programs for fruiting body formation in M. xanthus and S. aurantiaca are very similar but significantly different from the genetic program directing fruiting body formation in S. cellulosum and H. ochraceum. In other words, our analyses reveal an unexpected level of plasticity in the genetic programs for fruiting body formation in the Myxobacteria and suggest that the genetic program underlying fruiting body formation in different Myxobacteria is not conserved. The differences in these genetic programs may either reflect convergent evolution, i.e. fruiting bodies are not homologous structures, or divergent evolution, i.e. fruiting bodies are homologous structures generated from non-homologous proteins.

To follow up on these surprising findings, we are currently generating high quality, completed genome sequences of selected Myxobacteria. Moreover, we conduct comparative transcriptomics analyses to map similarities and differences between transcriptional programs in different Myxobacteria during fruiting body formation.


Some of our recent publications on fruiting body morphology & evolution:


Kadam, S.V., Wegener-Feldbrügge, S., Søgaard-Andersen, L., & Velicer, G.J. (2008) Novel transcriptome patterns accompany evolutionary restoration of defective social development in the bacterium Myxococcus xanthus. Mol. Biol. Evol. 25, 1274-1281.

Huntley, S., Hamann, N., Wegener-Feldbrügge, S., Treuner-Lange, A., Kube, M., Reinhardt, R., Klages, S., Müller, R., Ronning, C.M., Nierman, W.C. & Søgaard-Andersen, L. (2011) Comparative genomic analysis of fruiting body formation in Myxococcales. Mol. Biol. Evol. 28, 1083-1097.

Huntley, S., Zhang, Y., Treuner-Lange, A., Kneip, S., Sensen, C.W. & Søgaard-Andersen, L. (2012) Complete genome sequence of the fruiting myxobacterium Corallococcus coralloides DSM 2259. J. Bacteriol. 194, 3012-3013.

Huntley, S., Wuichet, K. & Søgaard-Andersen, L. (2013) Genome evolution and content in the myxobacteria. In "Myxobacteria: Genomics, Cellular and Molecular Biology" ed. Higgs, P.I. & Yang, Z. Horizon Press. pp. 31-50.

Huntley, S., Kneip, S., Treuner-Lange, A. & Søgaard-Andersen, L. (2013) Complete genome sequence of Myxococcus stipitatus strain DSM 14675, a fruiting myxobacterium. Genome Announc. 1, e00100-13.

Claessen, D., Rozen, D.E., Kuipers, O.P., Søgaard-Andersen, L. & van Wezel, G.P (2014) Bacterial solutions to multicellularity: A tale of biofilms, filaments and fruiting bodies. Nat. Rev. Microbiol. 2, 115-124.

 
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