What are we doing?

 

Shewanella

 

Our group is mainly using species of the genus Shewanella as model organisms to study adaptation processes in two very different aspects of the bacterial life style: the formation of biofilms and flagella-mediated motility. Shewanella sp. are versatile facultatively anaerobic gammaproteobacteria that can thrive in a wide range of environmental conditions and are capable of using an impressive array of alternative terminal electron acceptors, including numerous metal oxides. Shewanella species have potential to be used in different bioremediation processes and applications such as microbial fuel cells, but have also been identified as commensal pathogens. The numerous sequenced species that were isolated from various environments make Shewanella a well-suited model for long-term environmental adaptation. To study our fields of interest, we are using a wide range of experimental approaches. Almost all genetic tools are available for members of this genus.


 

Flagella

 

Flagella are long helical proteinaceous filaments extending from the cell’s surface that are rotated at the filaments base by a membrane-embedded motor. The flagellar motor is a highly intricate nanomachine which is powered by transmembrane ion gradients. Most bacterial motors interact with one or more corresponding chemotaxis systems to convert the perception of environmental gradients of attractants or repellents into directed movement. Production and placement of the huge flagellar and chemotaxis machinery requires tight spatiotemporal control and thus serve as great models to study the cellular organization of bacterial cells.


provided by M. Beeby
provided by M. Beeby

Adaptation of motor function and cellular navigation

 

The flagellar as well as chemotaxis systems within the genus Shewanella are remarkably heterogeneous. All species are motile by a single polar flagellum rotated by a sodium ion-dependent motor. In addition, a number or species possess a complete individual secondary flagellar system. One of our model species, S. oneidensis MR-1, harbors two distinct separate sets of important motor components, the so-called stators which are attached to the cell wall and surround the MS-ring, a part of the rotor, in a ring-like fashion. This stator ring is highly dynamic and the units are constantly exchanged with a pool of membrane-diffusing stator units which are activated upon incorporation into the motor. In contrast to the primary set of stator units, the second stator of the S. oneidensis MR-1 proton-dependent. We found that stators are both produced and are synchronously present in most of the cells. We could show that composition of the stator ring and turnover of the units depends on the environmental concentration of sodium ions.

The model strongly suggests that S. oneidensis MR-1 possesses a hybrid motor whose composition is directly mediated by environmental conditions. As many bacterial species possess a similar motor set-up with more than a single stator to drive flagellar rotation, we think that stator swapping represents a widespread means of functional adaptation of the flagellar motor. We could further show how the second proton-dependent stator improves its function upon simple spontaneous mutations, demonstrating how these important motor proteins can functionally adapt, e.g. after being recruited by horizontal gene transfer.

 

Our second Shewanella model species, S. putrefaciens CN-32 possesses two complete flagellar systems which are synchronously assembled in a subpopulation of the cells. The primary system forms the sodium ion-dependent single polar flagellum typical for Shewanella sp., the second system produces one or more lateral flagellar filaments. The subpopulation with two active flagellar systems shows improved spreading through complex environments, which we could attribute to an efficient lowering of the cell turning angles. Current projects are also dealing with more novel factor we identified to impact flagellar rotation.

 


Cell polarity

 

Spatiotemporal organization is crucial for a number of fundamental cell processes such as cell division or motility. In S. putrefaciens, the primary flagellar system and the associated flagellar systems are localized to the cell pole, while the secondary flagellar system is not. Recruitment of one, but not the other complex flagellar machinery to a specific location within the cell, as in S. putrefaciens CN-32, provides an excellent system to study the underlying mechanisms of regulation and flagellar placement which is conducted in close collaboration with the lab of Gert Bange in Marburg.

 

In many bacterial species, number and position of flagella depend on two proteins, the SRP-type GTPase FlhF and the MinD-like ATPase FlhG (also named YlxH, MinD2, FleN or MotR). In S. putrefaciens CN-32, loss of one or both proteins results in an aberrant flagellation pattern of the primary, but not the secondary flagellar system. We could show that for proper function, FlhG needs to interact with building blocks of the flagellar basal body, namely FliM and N. The corresponding binding motif is absent in the secondary flagellar system which is therefore not addressed by FlhG. Thus, FlhG links regulation and assembly of a multiprotein complex. We are currently further investigating the function and mechanism by which the two highly similar proteins mediate a wide range of different flagellation patterns in various bacterial species (see also here).

 

In addition, we have identified the polar marker protein HubP in Shewanella. HubP and the homologous FimV are present in various gammaproteobacteria where they mediate a range of different aspects related to flagella- or pili-mediated motility, chromosome segregation, and more. In Shewanella, HubP recruits the chemotaxis system to the flagellated cell pole and ensures proper segregation of the chromosome origin. HubP is not required for polar recruitment of the flagellum but interacts with both FlhF and FlhG and positively affects the function of the flagellar motor. Current studies address the role and mechanism of HubP-mediated cellular polarity.

 



eDNA in biofilms of Shewanella oneidensis
CLSM projection of an S. oneidensis MR-1 biofilm (cyan) with extracellular DNA (yellow)

Microbial biofilm formation

 

To study bacterial life on surfaces, we have established and adapted systems that allow us to monitor bacterial biofilm formation by means of confocal laser scanning and fluorescent microscopy. Although sometimes resembling developmental cycles, biofilm formation of many bacterial species is a dynamic process strongly depending on environmental cues and lacking a specific genetic program. We (mainly) use Shewanella oneidensis MR-1 as model organism for our biofilm studies. As many other bacterial species, S. oneidensis requires type IV pili and flagella for attachment and forms a matrix composed of exopolysaccharides, proteins, nucleic acids (see image) and other compounds. Intriguingly, normal biofilm formation also requires the activity of phages which are located within the chromosome. In our lab, we study the different aspects of Shewanella biofilm formation, in particular with respect to phage-host interactions, and the role eDNA matrix formation and turnover.

 

 


supported by the

AG Thormann

Department for Microbiology and Molecular Biology

Justus-Liebig-Universität Gießen

Heinrich-Buff-Ring 26-32 | 35392 Gießen | Germany

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the dual flagellar system of Shewanella putrefaciens