Collective motion and nonequilibrium cluster formation in colonies of gliding bacteria

We characterize cell motion in experiments and show that the transition to collective motion in colonies of gliding bacterial cells confined to a monolayer appears through the organization of cells into larger moving clusters. Collective motion by non-equilibrium cluster formation is detected for a critical cell packing fraction around 17%. This transition is characterized by a scale-free power-law cluster size distribution, with an exponent $0.88\pm0.07$, and the appearance of giant number fluctuations. Our findings are in quantitative agreement with simulations of self-propelled rods. This suggests that the interplay of self-propulsion of bacteria and the rod-shape of bacteria is sufficient to induce collective motion

PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus

Cell division site positioning is precisely regulated but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the similar to 15 MDa tripartite PomX/Y/Z complex associates with and translocates across the nucleoid in a PomZ ATPase-dependent manner to directly position and stimulate formation of the cytokinetic FtsZ-ring at midcell, and then undergoes fission during division. Here, we demonstrate that PomX consists of two functionally distinct domains and has three functions. The N-terminal domain stimulates ATPase activity of the ParA/MinD ATPase PomZ. The C-terminal domain interacts with PomY and forms polymers, which serve as a scaffold for PomX/Y/Z complex formation. Moreover, the PomX/PomZ interaction is important for fission of the PomX/Y/Z complex. These observations together with previous work support that the architecturally diverse ATPase activating proteins of ParA/MinD ATPases are highly modular and use the same mechanism to activate their cognate ATPase via a short positively charged N-terminal extension

Aktivierung eines kontakt-abhängigen Signalsystems durch regulierte Proteolyse in Myxococcus xanthus

Das C-Signal von Myxococcus xanthus ist ein interzelluläres Signal-Molekül, welches die Aggregation von Zellen zu Fruchtkörpern, die Sporulation und die Expression von Genen nach 6 Stunden Entwicklung induziert. Bei dem C-Signal handelt es sich um ein 17 kDa Protein (p17), welches in der äußeren Membran verankert ist und durch die proteolytische Spaltung des 25 kDa CsgA-Proteins (p25) entsteht. Es konnte gezeigt werden, dass die Protease (PopC), die in die Spaltung von p25 involviert ist, eine Serine Protease ist, dass sie während der Entwicklung hoch reguliert und voraussichtlich sekretiert wird (Lobedanz & Sogaard-Andersen, 2003). Basierend auf diesen Informationen wurde eine dreistufige Strategie zur Identifizierung von popC Kandidaten entwickelt. Als erstes wurden 146 Gene im M. xanthus Genom gefunden, die für Proteasen kodieren. 32 dieser Gene kodieren für sekretierte Serin Proteasen. Als zweites wurde auf die biochemischen Charakteristika dieser Proteasen geschaut und zu letzt die Daten von DNA-Microarrays bzw. der quantitativen real-time PCR herangezogen. Somit wurden 8 Gene gefunden, die während der Entwicklung reguliert werden. Nur die Inaktivierung von MXAN0206 führt zu einem Defekt während der Entwicklung. Die MXAN206 Mutante wird von hier an popC Mutante genannt. Die popC Mutante kann nur wenig aggregieren, aber keine Fruchtkörper ausbilden und hat einen signifikanten Defekt in der Sporulation. Zudem kann in der popC Mutante kein p17 detektiert werden. popC kodiert für eine 51 kDa Subtilisin-ähnliche Protease mit einer für Subtilisin charakteristischen katalytischen Triade. Eine „Active-Site“ Mutante von popC zeigt den gleichen Entwicklungsphänotyp wie eine popC Mutante und ist unfähig p17 zu synthetisieren. PopC kommt sowohl in vegetativen als auch in hungernden Zellen vor, aber es wird während der Entwicklung selektiv sekretiert. Weiterhin konnte in vitro gezeigt werden, dass gereinigtes PopC p25 direkt zu p17 spalten kann. Wir vermuten, dass regulierte MXAN0206 Sekretion ein Garant dafür ist, dass nur während der Entwicklung p25 und PopC im gleichen Zellkompartiment zu finden sind und somit die p17-Synthese auf hungernde Zellen beschränkt ist

The PomXYZ cell division regulators self-organize on the nucleoid to position cell division at midcell in the rod-shaped bacterium Myxococcus xanthus

Accurate positioning of the division site is essential to produce daughter cells with the correct size, and chromosome content. Generally, the first known event of bacterial cell division is the accumulation of FtsZ at the incipient cell division site to form the circumferential, ring-like structure Z-ring. In bacteria, positioning of the division site occurs at the level of formation of the FtsZ-ring. While the cytokinesis machinery is conserved throughout bacteria, the mechanisms to position the Z-ring are diverse. The rod-shaped social bacterium Myxococcus xanthus divides precisely by binary fission at midcell but it lacks all the known systems that regulate Z-ring formation in other bacteria. Instead two novel regulators PomX and PomY together with the ParA ATPase PomZ stimulate formation and positioning of the Z-ring at midcell. PomXYZ interact and form a complex that associates with the nucleoid to translocate towards the mid-nucleoid, which coincides with midcell before nucleoids have segregated, by biased random motion. At the mid-nucleoid, at midcell, the PomXYZ complex undergoes constrained motion, not leaving midcell. Our experimental data show that cluster localization at midcell is independent of FtsZ and that clusters localize at midcell before Z-rings form. By contrast cluster localization at midcell depends on the ATPase PomZ, its associated ability to hydrolyze ATP and its ability to bind non-specifically to DNA. ATP-hydrolysis by PomZ is stimulated by PomX as well as by PomY in the presence of DNA, demonstrating that PomZ is the first ParA ATPase in which ATP-hydrolysis is stimulated by two ATPase activating proteins (AAP’s). We show that interference with ATP-hydrolysis, by mutational analysis, affects cluster translocation towards midcell. PomZ on its own interacts with the nucleoid and recruits a complex of PomX and PomY to the nucleoid. By FRAP experiments we show that PomZ is highly dynamic in the PomXYZ complex, where ATP-hydrolysis takes place and on the nucleoid. Our experimental data support a flux-based mechanism for the positioning of the PomXYZ complex by the ParA ATPase PomZ. In this model, the diffusive random PomZ dynamics on the nucleoid result in diffusive fluxes of PomZ on the nucleoid from either side into the PomXYZ cluster. These fluxes scale with the cellular asymmetry of the cluster within the cell and convert this cellular asymmetry into a PomZ concentration gradient over the PomXYZ complex. This gradient together with the PomZ-associated ATP-hydrolysis translocates the complex towards the higher concentration of PomZ to midcell. At midcell, which coincides with middle of the nucleoid, the diffusive PomZ fluxes equalize, resulting in constrained motion. To understand the molecular details of ATP-hydrolysis, a previously identified pomXK13A,R15A mutant was analyzed for its defect in PomXYZ-dependent positioning of the cell division site. Our in vivo and in vitro data show that the PomXK13A;R15A variant has a defect in stimulating PomZ ATP-hydrolysis, strongly suggesting that the N-terminal part of PomX interacts with PomZ to stimulate ATP-hydrolysis, similar to other ATPase activating proteins. In summary we identified two novel cell division regulators that work in concert with PomZ to position and promote cell division at midcell. Our data provides insights into the function of ParA-ATPases, which in this case is PomZ, and reveal that the PomXYZ system is a novel system that positions cell division at midcell most likely by recruiting the Z-ring to the incipient cell division site

An analysis of two-component regulatory systems in Myxococcus xanthus

Proteins of two-component regulatory systems (TCS) have essential functions in the sensing of external and self-generated signals in bacteria as well as in the generation of appropriate output responses. Accordingly, in Myxococcus xanthus TCS are important for fruiting body formation and sporulation as well as normal motility. In this study, I analyzed the M. xanthus genome for the presence and genetic organization of genes encoding TCS. 272 genes that encode TCS proteins were identified including 21 genes in eight loci, which encode TCS proteins that are part of chemotaxis-like systems. Sebsequent analyses focused on 251 TCS proteins (non chemotaxis-like) consisting of 118 histidine protein kinases (HPKs), 119 response regulators (RRs) and 14 HPK-like genes. 71% of the TCS genes are organized in unusual manners as orphan genes or in complex gene clusters whereas the remaining 29% display the standard paired gene organization. Bioinformatics analyses suggest that TCS proteins encoded by orphan genes and complex gene clusters are functionally distinct from TCS proteins encoded by paired genes. Experimentally, microarray data and quantitative real-time PCR suggest that orphan TCS genes are overrepresented among TCS genes that display altered transcription during fruiting body formation. The genetic analysis of 25 orphan HPKs, which are transcriptionally up-regulated during development, led to the identification of two HPKs that are likely essential for viability and seven HPKs including four novel HPKs that have important function in fruiting body formation or spore germination. As an attempt to identify functional partners of orphan TCS proteins in M. xanthus, I focused on the RR FruA, which has a key role in the C-signal transduction pathway. To identify the FruA kinase, two candidate approaches were used. The first candidate approach is based on the hypothesis that a FruA kinase gene shares characteristics with the fruA gene, i.e. it is orphan, developmentally up-regulated at the transcriptional level and a null mutant is deficient in development. Yeast two-hybrid analysis was used to investigate potential interactions between FruA and developmentally regulated orphan HPKs. Three best FruA kinase candidates (SdeK, Hpk8 and Hpk12) and four potentially redundant candidates (Hpk9, Hpk11, Hpk13 and Hpk29) were identified. In vivo analyses of the three best FruA kinase candidates support a model in which SdeK is the main FruA kinase, Hpk12 is a minor FruA kinase and Hpk8 is a phosphatase of FruA~P. Furthermore, SdeK may have other downstream targets in addition to FruA and there may be other HPKs that phosphorylate or cross talk to FruA. To obtain direct evidence for an interaction between FruA and the FruA kinase candidates in vitro, the relevant proteins have been purified. To date, the Hpk8 and Hpk12 proteins have been shown to autophosphorylate in vitro. Intriguingly, Hpk8 does not appear to be phosphorylated on the conserved His residue but is likely phosphorylated on a Tyr residue. Preliminary phosphotransfer assay suggests that Hpk8 engages in phosphotransfer to or phosphorylation of FruA. A possible interaction in vitro between SdeK and Hpk12 with FruA still remains to be shown. Hpk37 belongs to the group of orphan HPKs that are transcriptionally up-regulated during development and essential for development. However, the yeast two-hybrid analyses to determine a possible direct interaction with FruA were inconclusive. In vivo analyses demonstrated that Hpk37 is likely involved in the production or response to (p)ppGpp or the A-signal suggesting that Hpk37 is not a FruA kinase. Domain analyses of Hpk37 and analyses of the genetic organization of the hpk37 locus suggest that regulation of Hpk37 activity could involve a unique methylation/demethylation mechanism similar to that resulting in adaptation in chemosensory pathways. In a second candidate approach to identify a FruA kinase, candidates were predicted using an in silico method (White et al., 2007). In vivo analyses of mutants carrying mutations in the genes encoding the six best candidates strongly suggest that these HPK are not FruA kinases

Regulierung der Polarität des A-Bewegungssystems in Myxococcus xanthus

Die Zellen des stäbchenförmigen Bakteriums Myxococcus xanthus bewegen sich mit einer gleitenden Bewegung vorwärts. Hierfür verwenden die Zellen zwei verschiedene Fortbewegungssysteme. Die S-Bewegung („social“) und die A-Bewegung („adventurous“). Die A-Bewegung befähigt die Zellen eine individuelle und von anderen Zellen unabhängige Vorwärtsbewegung auszuführen. M. xanthus Zellen wechseln regelmäßig die Richtung ihrer Bewegung, wobei der alte vordere Pol zum neuen hinteren Zellpol wird. Während eines Richtungswechsels müssen die beiden Bewegungsmaschinerien synchron ihre Polarität innerhalb der Zelle ändern, um eine erneute Vorwärtsbewegung in die entgegengesetzte Richtung zu garantieren. Um den molekularen Mechanismus, welcher den Polaritätswechsel der Bewegungsmaschinerien vermittelt, zu erforschen wurden das RomR Protein und das MglA Protein genauer analysiert. Der Response Regulator RomR besteht aus einer N-terminalen Receiver Domäne und einer C-terminalen Prolinreichen Output Domäne. Das Protein ist essentiell für die A-Bewegung der Zellen. Diese Arbeit demonstriert, dass das RomR-GFP Fusionsprotein in einer asymmetrischen Verteilung an beiden Zellpolen lokalisiert. Wobei eine verstärkte Ansammlung des RomR-GFP Proteins am hinteren Zellpol lokalisiert, wenn die Zelle sich vorwärts bewegt. Parallel mit einem Richtungswechsel in der Bewegung wechselt die verstärkte Ansammlung von RomR-GFP von dem alten hinteren Pol zum neuen hinteren Zellpol. Dies deutet an, dass RomR am hinteren Zellpol stimulierend auf eine Komponente der A-Bewegungsmaschinerie einwirkt. Das MglA Protein weist in der Primärstruktur Ähnlichkeiten zu eukaryontischen kleinen GTPasen der Ras/Rac/Rho Superfamilie auf und wird für die A-Bewegung benötigt. Eine inaktive, im GDP gebundene MglA Form (MglAlof) ist nicht fähig, die A-Bewegung zu stimulieren, während eine konstitutiv aktive MglA Form, gebundenen im GTP Zustand (MglAgof), die A-Bewegung der Zellen anregt und die Durchführung häufiger Richtungswechsel stimuliert. Das native YFP-MglA Protein lokalisiert am vorderen Zellpol und wechselt zwischen den beiden Polen, während die Zelle einen Richtungswechsel vollzieht. Das YFP-MglAgof Protein hingegen oszilliert ständig zwischen den Zellpolen, während sich das YFP-MglAlof Protein homogen in der gesamten Zelle verteilt. Aufgrund dieser Beobachtungen, nehmen wir an, dass eine mittlere Menge an MglA im GTP gebundenen Zustand am vorderen Zellpol lokalisiert und die A-Bewegung stimuliert. Wird ein erhöhter Level an MglA im GTP gebundenen Zustand erreicht, so wird das MglA Protein an dem vorderen Zellpol freigesetzt und gelangt von dort zum hinteren Zellpol. Sobald die erhöhte Menge an MglA im GTP gebundenen Zustand den hinteren Zellpol erreicht, wechselt die Zelle ihre Bewegungsrichtung. Dieser Zusammenhang weißt daraufhin, dass der Transfer eines erhöhten Levels an MglA in der GTP gebundenen Form, gefolgt von der vollständigen Lokalisierung am hinteren Zellpol einen Richtungswechsel in der A-Bewegung induziert. Auf genetischer Ebene wurden Hinweise gefunden, dass MglA stromabwärts des Frz-Sytems in demselben Signalweg agiert, um Richtungswechsel in dem A-Bewegungssystem einzuleiten. Weiterhin scheint RomR stromabwärts von MglA zu agieren, bezüglich der Richtungswechsel, jedoch gibt es ebenfalls eine Rückkopplung zwischen diesen beiden Proteinen, da beide die jeweilige Lokalisierung des anderen Proteins beeinflussen. Basierend auf den Ergebnissen dieser Arbeit nehmen wir an, dass das Frz-System ein Signal wahrnimmt und daraufhin eine GTP Bindung an MglA stimuliert. Die vermehrte GTP Bindung führt zu einem Transfer des MglA-GTP Proteins vom vorderen Pol zum hinteren Zellpol. Sobald die erhöhte Menge an MglA im GTP gebundenen Zustand den hinteren Zellpol erreicht, interagiert MglA-GTP mit dem unphosphoprylierten RomR Protein. Gleichzeitig wird RomR phosphoryliert und die GTP Hydrolyse von MglA wird stimuliert. Das phosphorylierte RomR wechselt zum neuen hinteren Zellpol, durch die GTP Hydrolyse wird erneut eine mittlere Menge an MglA im GTP gebundenen Zustand erreicht, welche an dem neuen vorderen Pol der Zelle lokalisiert.Folglich reguliert die prokaryontische kleine GTPase MglA der Ras/Rac/Rho Familie die Zellpolarität über die Festlegung der RomR Lokalisierung in der Zelle

Identification and characterization of RomX and RomY, two novel motility regulators in Myxoccoxus xanthus

Eine klar definierte Polarität des vorderen und hinteren Zellpols ist für eine gerichtete Zellbewegung essentiell. Die stäbchenförmigen Zellen von Myxococcus xanthus benötigen für ihre Bewegung zwei Motilitätssysteme und eine klar definierte Polarität der Zellpole. Dabei sind beide Motilitätssysteme polarisiert: die Typ IV Pili sind am vorderen Pol angeordnet, dagegen werden die Komplexe, die für die Gleitbewewegung benötigt werden, zwar am vorderen Pol zusammengebaut, wandern aber im Zuge der Gleitbewegung zum hinteren Zellpol, wo sie anschließend abgebaut werden. M. xanthus Zellen wechseln regelmäßig die Richtung ihrer Bewegung, wobei der alte vordere Pol zum neuen hinteren Zellpol wird. Während eines Richtungswechsels müssen die beiden Motilitätssysteme synchron ihre Polarität innerhalb der Zelle ändern, um eine erneute Vorwärtsbewegung in die entgegengesetzte Richtung zu garantieren. Die Ras-ähnliche GTPase MglA bildet zusammen mit MglB, dem verwandten MglA GTPase aktivierenden Protein (GAP) und dem RomR Response-Regulator ein Modul, das die Polarität des vorderen und hinteren Zellpols bestimmt. Die polare Lokalisation von MglA-GTP und MglB definiert den vorderen und hinteren Zellpol und ist abhängig von dem polar lokalisierten RomR. Während des durch das Frz-System induzierten Richtungswechsels wechseln MglA-, MglB- und RomR von dem einen zum anderen Pol. In Rahmen einer großen vergleichenden Genomanalyse konnten wir RomX und RomY als weitere integrale Komponenten dieses Polaritätsmoduls identifizieren. RomX lokalisiert asymmetrisch an den Polen mit einem großen Cluster am hinteren Pol. In-vivo- und in-vitro-Experimente zeigten, dass das polare RomX zwischen seinem polaren Rekrutierungsfaktor RomR und MglA-GTP liegt. Der RomR / RomX / MglA-GTP-Komplex stimuliert den Aufbau von Gleitmotilitätskomplexen am vorderen Pol und wird dabei selbst Teil des Komplexes. Überraschenderweise sind RomX und RomR nur dann für die Gleitbewegung notwendig, wenn MglB abwesend ist. In Abwesenheit von MglB translozieren die Gleitmotilitätskomplexe weniger gerichtet zum hinteren Zellpol und die von den Zellen zurückgelegte Nettodistanz ist stark reduziert. Unsere Daten legen übereinstimmend nahe, dass ein RomX / RomR Komplex als Guanin-Nukleotid-Faktor (GEF) auf MglA-GDP wirkt und somit die gerichtete Zellbewegung reguliert. Am vorderen Zellpol bindet der RomX / RomR Komplex MglA-GTP und stimuliert dadurch den Aufbau der Gleitmotilitätskomplexe. Am hinteren Pol stimuliert der RomX / RomR Komplex dagegen den Abbau der Gleitmotilitätskomplexe.RomY lokalisiert unipolar mit einem Cluster am hinteren Zellpol. In vivo Experimente zeigten, dass RomY Richtungswechsel und Zellpolarität ähnlich wie MglB reguliert. Darüber hinaus hängt die RomY-Lokalisierung von MglB ab, was eine funktionelle Verbindung zwischen den Proteinen vermuten läßt. Proteininteraktionsstudien haben gezeigt, dass RomY direkt mit MglA und RomX interagiert. Bemerkenswerterweise sind RomX und RomR in Abwesenheit von RomY für die Gleitmotilität entbehrlich. Basierend auf diesen Daten schlagen wir vor, dass RomY die MglB GAP oder MglA GTPase Aktivität reguliert

The small G-protein MglA connects the motility machinery to the bacterial actin cytoskeleton

Motility of Myxococcus xanthus cells is powered by two distinct engines: S-motility allows grouped cells movement and is driven by type IV pili (T4P) at the leading cell pole that use ATP for their function and pull the cell forward upon their retraction. Single cell movement is called gliding or A-motility and its AglQ/R/S engine is powered by proton-motive force and is incorporated at focal adhesion complexes in the cell. The control of motility and its direction is accomplished by cells rapidly switching their leading into lagging cell pole (cellular reversal), a process regulated by the small Ras-like G-protein MglA and its cognate GTPase activating protein (GAP) MglB. Using fluorescence microscopy it was previously shown that MglA localizes at the leading cell pole and MglB at the lagging cell pole and both proteins dynamically switch polarity during cellular reversal. Further, recent experiments showed that an A-motility protein AglZ, and A-motility engine AglQ/R/S localize at clusters distributed along the cell body that stay fixed relative to the substratum as the cell moves forming focal adhesion complexes (FACs). Based on the in vivo experiments it has been proposed that gliding motility machinery assembles at the leading cell pole and that it is guided by the cytoskeletal element to the lagging cell pole, where it disassembles. In this work we investigated the function of MglA during gliding motility. First, we demonstrate that MglA in its active state forms a focal adhesion cluster, which co-localizes with AglZ and AglQ, thus showing that active MglA is a component of the FACs. We show that MglA is essential for incorporation of AlgQ in the FACs, and that MglA GTPase cycle regulates the number of AglQ clusters. Further, we provide evidence that the GTPase negative MglA variant MglAQ82A leads to regularly reversing cells after movement of only one cell length, and that MglA GTPase cycle regulates the disassembly of the FACs at the lagging cell pole. Fluorescent YFP-MglAQ82A forms a focal adhesion cluster which appears to regularly oscillate between the poles, and causes the cell to move in a pendulum-like manner. Unlike wildtype MglA, MglAQ82A is insensitive to the GAP activity of MglB, and upon reaching the lagging cell pole where MglB localizes, it causes a cellular reversal by starting to oscillate in the opposite direction. The co-localizing YFP-MglAQ82A/AglZ-mCherry and YFP-MglAQ82A/AglQ-mCherry FAC also appear to continuously oscillate between the poles suggesting that the gliding motility machinery coupled to active MglA needs to be disassembled at the lagging cell pole by MglB GAP, and in this way allow uni-directional motility for distances longer than one cell length. Furthermore, in this work we demonstrate that active wt MglA and MglAQ82L variant interact directly with filament forming MreB actin homolog. Additionally, our results show that the formation and localization of FACs depend on intact MreB, thus indicating that MreB acts as a scaffold for the assembly of gliding motility machinery. The addition of antibiotics which inhibit peptidoglycan (PG) synthesis and reduce the dynamics of MreB in other bacteria did not inhibit single cell motility and did not cause mislocalization of MglA and AglQ. This strongly suggests that the major proposed function of MreB as a scaffold for PG elongation machinery is not coupled to its essential role during gliding motility in M. xanthus. Thus, we demonstrate that MreB is required for MglA, AglZ and AglQ localization at FACs during gliding, and this function of MreB is separable from its major proposed function in PG synthesis

Deciphering the assembly pathway of type IV pili in Myxococcus xanthus

Type IV pili (T4P) are hairlike surface structures, present on a variety of different bacteria. They are polymers involved in diverse functions such as motility, adherence, protein secretion, DNA uptake and in many pathogens they are found to be the primary colonization factor. Especially their role in virulence makes T4P particularly relevant for studying pilus function and assembly. The T4P machinery consists of 12 conserved proteins building an envelope-spanning macromolecular machinery, which localizes polarly in Myxococcus xanthus. Although most of the proteins have been known and studied for a long time, the precise mechanism of how and in which order the individual components are assembled to generate a macromolecular machinery remain largely unknown. Here we uncovered a sequential, outside-in assembly pathway starting with the outer membrane (OM) PilQ secretin, and proceeding inwards over the periplasm and inner membrane (IM) to the cytoplasm. Specifically, by taking advantage of the cell biology tools for studying T4P in M. xanthus, we carried out one of the largest screens comprising 11 of the 12 proteins of the T4P machinery by systematically profiling the stability and localization of T4P proteins in the absence of each individual other T4P protein in combination with mapping direct protein-protein interactions. Using these approaches, we show that assembly of the T4P machinery initiates with the formation of the PilQ secretin ring, assisted by its pilotin Tgl, in the OM. Oligomeric PilQ serves as an assembly platform for further T4P components. PilQ recruits TsaP, a peptidoglycan binding protein, as well as PilP by direct interactions with PilP. PilP, in turn, recruits the IM proteins PilN and PilO. PilP/PilO/PilN likely make up a complex aligning IM and OM components of the T4P machinery. The PilP/PilO/PilN complex recruits cytoplasmic PilM by direct interaction between PilN and PilM and recruits PilC, presumably by direct interaction between PilC and PilO. Finally, the ATPases PilB and PilT that power extension and retraction of T4P, localize independently of other T4P machinery proteins. In this study, we elucidate the assembly process and functional interactions between T4P proteins. This work lays the basis for further understanding of these functionally highly versatile surface structures. Interestingly, the assembly of the type II and III secretion systems also initiates from the OM secretin and proceeds inwards. Thus, an outside-in assembly pathway is emerging as a conserved feature in secretin-containing trans-envelope export machines

The ParA-like protein AgmE positively regulates cell division in Myxococcus xanthus

Correct positioning of the division plane is a prerequisite for the generation of daughter cells with a normal chromosome complement and with a correct size. So far, all bacterial systems, which contribute to correctly place the division plane, regulate formation of the FtsZ cytokinetic ring at mid-cell. Myxococcus xanthus belongs to the δ-proteobacteria and divides by binary fission. However, the mechanisms that ensure proper cell division are not known. In contrast, M. xanthus has been studied in detail because of its complex life cycle and for its gliding motility. M. xanthus possesses two motility systems referred to as the social (S) and the adventurous (A)- motility systems. While analyzing the adventurous gliding motility E protein (AgmE), which is a member of the ParA/Soj family of ATPases, we discovered that an in-frame deletion of the agmE gene results in the formation of filamentous cells and chromosome-free mini-cells. We determined that an ΔagmE mutant is neither impaired in chromosome replication nor in chromosome segregation. Moreover, an ΔagmE mutant cells displayed fewer division sites and frequently these sites were not located to mid-cell. These data strongly suggest that AgmE has a central function in cell division and in placing the division plane at mid-cell. Consequently, FtsZ localization was investigated. In WT, FtsZ localizes in a speckled-pattern in small-sized cells, i.e. early in the cell cycle, whereas in larger cells, i.e. later in the cell cycle, FtsZ localizes at mid-cell. On the contrary, in an ΔagmE mutant FtsZ localizes in a speckled-pattern independently of cell length and cell cycle. We hypothesized that AgmE is required to properly localize FtsZ and could act either positively by directing FtsZ to mid-cell or by stabilizing the FtsZ cytokinetic ring or negatively by inhibiting FtsZ cytokinetic ring formation at the poles.To distinguish between these hypotheses, AgmE localization was studied. AgmE was found to localize in three distinct patterns, which correlates with cell length, i.e. the cell cycle. In small-sized cells, AgmE localizes in a patchy pattern, as cell size increases AgmE localizes to a single focus slightly off mid-cell and when the cells have reached a specific cell length AgmE localizes at mid-cell. Genetic data suggest that the cell cycle dependent localization of AgmE depends on ATPase activity. The localization of AgmE at mid-cell implies that AgmE acts positively on FtsZ localization or stabilizes the FtsZ cytokinetic ring. To distinguish between these models, co-localization studies were carried out. These analyses demonstrated that FtsZ and AgmE co-localize at mid-cell. Intriguingly, some cells displayed mid-cell localization of AgmE in the absence of mid-cell localization of FtsZ. Moreover, in vitro analyses showed that AgmE interact directly with FtsZ. On the basis of the localization of AgmE to mid-cell and the observation that AgmE localize to mid-cell before FtsZ, we propose that AgmE is a novel cell division regulator that acts positively to direct FtsZ to mid-cell. A systematic analysis of the M. xanthus proteome revealed a unique combination of cell division regulators, i.e. M. xanthus encodes an orthologue of DivIVA and lacks orthologues of MinCDE, MipZ, SlmA and Noc. These observations suggest that cell division regulators are yet to be discovered in M. xanthus. In this study, we have shown that M. xanthus possesses the orphan ParA-like protein AgmE, which is involved in cell division. Importantly, AgmE is the first example of a protein shown to positively regulate FtsZ localization. Interestingly, an in-frame deletion of divIVA has no obvious effect on cell division and a DivIVA-mCherry protein localizes in a speckled pattern. Moreover, MXAN0636, the downstream gene of agmE, seems to be an additional component for the regulation of cell division in M. xanthus