Focal research areas
The Collaborative Research Center comprises 16 groups, focusing on the following research areas:
- Cell division, growth & morphogenesis
- Chromosome organization & segregation
- Positioning of motility structures
- Dynamics of membrane protein complex assembly
Projects
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The different projects conducted in TRR 174 are listed below.
Please click on the titles to obtain more information on the aims and the people involved.
Please click on the titles to obtain more information on the aims and the people involved.
P01 Spatiotemporal regulation of cell division and motility in Myxococcus xanthus
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Lotte Søgaard-Andersen, Prof. Dr.
Department of Ecophysiology Max Planck Institute for Terrestrial Microbiology 35043 Marburg |
Summary
In the rod-shaped cells of Myxococcus xanthus cell division as well as motility depend on the dynamic localization of proteins to specific subcellular sites. We recently identified a novel type of cell division regulatory system in M. xanthus consisting of the three proteins PomXYZ. These proteins localize to the incipient division site at midcell before and independently of FtsZ and stimulate Z-ring positioning and formation. PomXYZ form a complex that transfers to midcell in a biased random walk and in a manner that depends on non-specific binding of PomZ to the nucleoid, diffusion of PomZ on the nucleoid as well as on PomZ ATPase activity.
In TRR 174, we aim to characterize the interactions between the PomXYZ proteins quantitatively in vivo as well as in vitro. Building on these findings we will seek to determine the structure of the PomXYZ complex in vivo. Furthermore, we will quantify the dynamics of the three Pom proteins in vivo to generate the data needed to refine our model for how the PomXYZ complex robustly identifies midcell. Moreover, we aim to elucidate how the PomXYZ proteins interact with FtsZ to stimulate Z-ring formation and positioning. In TRR 174, we will also collaborate closely with Ulrich Gerland to mathematically model the spatial toggle switch involved in regulation of motility in M. xanthus. Motility and its regulation in M. xanthus depends on the polar localization of motility structures and their dynamic repositioning during reversals, which are induced by the Frz chemosensory system. This spatiotemporal dynamics of motility structures is regulated by a protein module consisting of the small GTPase MglA, its cognate GTPase activating protein MglB, and the RomR response regulator. Recently we have identified three additional proteins that are also important for this process, MglC, RomX and RomY. All six proteins localize asymmetrically to the cell poles and switch polarity during reversals. Hence, between reversals these six proteins localize robustly and asymmetrically to the cell poles, and during reversals they switch poles. Together with Ulrich Gerland we will mathematically model this spatial toggle switch to understand how local interactions between the MglABC and RomRXY proteins result in their asymmetric polar localization between reversals and their dynamic relocalization during reversals. In particular, we will provide experimental data on how these six proteins interact and connect to the Frz system in vivo and in vitro.
In the rod-shaped cells of Myxococcus xanthus cell division as well as motility depend on the dynamic localization of proteins to specific subcellular sites. We recently identified a novel type of cell division regulatory system in M. xanthus consisting of the three proteins PomXYZ. These proteins localize to the incipient division site at midcell before and independently of FtsZ and stimulate Z-ring positioning and formation. PomXYZ form a complex that transfers to midcell in a biased random walk and in a manner that depends on non-specific binding of PomZ to the nucleoid, diffusion of PomZ on the nucleoid as well as on PomZ ATPase activity.
In TRR 174, we aim to characterize the interactions between the PomXYZ proteins quantitatively in vivo as well as in vitro. Building on these findings we will seek to determine the structure of the PomXYZ complex in vivo. Furthermore, we will quantify the dynamics of the three Pom proteins in vivo to generate the data needed to refine our model for how the PomXYZ complex robustly identifies midcell. Moreover, we aim to elucidate how the PomXYZ proteins interact with FtsZ to stimulate Z-ring formation and positioning. In TRR 174, we will also collaborate closely with Ulrich Gerland to mathematically model the spatial toggle switch involved in regulation of motility in M. xanthus. Motility and its regulation in M. xanthus depends on the polar localization of motility structures and their dynamic repositioning during reversals, which are induced by the Frz chemosensory system. This spatiotemporal dynamics of motility structures is regulated by a protein module consisting of the small GTPase MglA, its cognate GTPase activating protein MglB, and the RomR response regulator. Recently we have identified three additional proteins that are also important for this process, MglC, RomX and RomY. All six proteins localize asymmetrically to the cell poles and switch polarity during reversals. Hence, between reversals these six proteins localize robustly and asymmetrically to the cell poles, and during reversals they switch poles. Together with Ulrich Gerland we will mathematically model this spatial toggle switch to understand how local interactions between the MglABC and RomRXY proteins result in their asymmetric polar localization between reversals and their dynamic relocalization during reversals. In particular, we will provide experimental data on how these six proteins interact and connect to the Frz system in vivo and in vitro.
P02 In vitro reconstitution of nucleoid-guided cargo positioning by ParA ATPases
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Petra Schwille, Prof. Dr.
Department of Cellular and Molecular Biophysics Max Planck Institute of Biochemistry München |
Summary
Compared to transport processes in eukaryotic cells, we still lack a mechanistic understanding of how bacteria transport and position their cellular contents including chromosomes and protein complexes. In particular, the large-scale transformations during cell division require chromosome segregation and the definition of spatial cues for the precise positioning of the Z-ring and the divisome. We have recently focused on elucidating the self-organization processes of the MinCDE proteins that ultimately lead to Z-ring formation and positioning at midcell in E. coli. We were able to reconstitute in vitro the pole-to-pole oscillations of the MinCDE proteins leading to a well-defined zone for assembly of Z-ring protofilaments. We showed that the membrane acts as the key template for these processes by recruiting the MinD ATPase as a first step of spatiotemporal organization.
Here we focus on another class of bacterial proteins, i.e. ParA ATPases, that are close homologs of MinD, but use nucleoid DNA as a template to establish spatial patterns, position structures, generate force and transport cargo. In collaboration with partners in the Transregio-CRC, we will focus on the molecular mechanisms for force generation, cargo transport and positioning by these ATPases by focusing on (i) the PomZ ATPase that is involved in force generation and positions the PomXYZ complex to midcell to stimulate Z-ring formation in M. xanthus; and, (ii) the ParA ATPase of ParABS systems that is also involved in force generation to transport and position plasmids or chromosomal origins of replication. We propose to build a cell free minimal model system based on DNA-mimicking template structures with defined spatial proportions, analogously to the model membrane structures employed for Min/FtsZ reconstitution. In particular, we will elucidate whether force generation, cargo transport and self-organization of these systems can be reconstituted on artificial nucleoids leading to spatial patterns in vitro.
Compared to transport processes in eukaryotic cells, we still lack a mechanistic understanding of how bacteria transport and position their cellular contents including chromosomes and protein complexes. In particular, the large-scale transformations during cell division require chromosome segregation and the definition of spatial cues for the precise positioning of the Z-ring and the divisome. We have recently focused on elucidating the self-organization processes of the MinCDE proteins that ultimately lead to Z-ring formation and positioning at midcell in E. coli. We were able to reconstitute in vitro the pole-to-pole oscillations of the MinCDE proteins leading to a well-defined zone for assembly of Z-ring protofilaments. We showed that the membrane acts as the key template for these processes by recruiting the MinD ATPase as a first step of spatiotemporal organization.
Here we focus on another class of bacterial proteins, i.e. ParA ATPases, that are close homologs of MinD, but use nucleoid DNA as a template to establish spatial patterns, position structures, generate force and transport cargo. In collaboration with partners in the Transregio-CRC, we will focus on the molecular mechanisms for force generation, cargo transport and positioning by these ATPases by focusing on (i) the PomZ ATPase that is involved in force generation and positions the PomXYZ complex to midcell to stimulate Z-ring formation in M. xanthus; and, (ii) the ParA ATPase of ParABS systems that is also involved in force generation to transport and position plasmids or chromosomal origins of replication. We propose to build a cell free minimal model system based on DNA-mimicking template structures with defined spatial proportions, analogously to the model membrane structures employed for Min/FtsZ reconstitution. In particular, we will elucidate whether force generation, cargo transport and self-organization of these systems can be reconstituted on artificial nucleoids leading to spatial patterns in vitro.
P03 Generic mechanisms for spatiotemporal protein patterns in bacterial cells
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Erwin Frey, Prof. Dr.
Faculty of Physics Ludwig-Maximilians-Universität München |
Summary
The formation of protein patterns and the localization of protein clusters is a major prerequisite for many important processes in bacterial cells. Examples include Min oscillations guiding the formation of the FtsZ ring to midcell in Escherichia coli cells, the localization of chemotaxis arrays and the positioning of flagella, as well as chromosome and plasmid segregation. In all these examples, experimental evidence supports mechanisms based on reaction-diffusion dynamics. Moreover, the central elements of the biochemical reaction circuits driving these processes are P-loop NTPases. These proteins are able to switch from an NTP-bound form that preferentially binds to an intracellular interface (membrane or nucleoid) to an NDP-bound form diffusing in the cytosol.
In the planned Transregio-CRC, we will theoretically investigate mechanisms for protein localization and protein pattern formation in bacterial cells. Our focus will be on protein systems composed of one or several P-loop NTPases and associated proteins. We will develop theoretical models and investigate how the interplay between the architecture of the underlying protein circuits, the different conformational states of the proteins, the reaction rates between these states, and the diffusivities of the proteins in the respective compartments determine the broad range of observed spatio-temporal patterns. These investigations will build on and generalize our earlier work on Min oscillations in E. coli and cell polarity in budding yeast. We will closely collaborate with several experimental groups in the Transregio-CRC and explore different systems that all incorporate a ParA/MinD NTPase and illustrate the versatility of these NTPases for pattern formation. Our theoretical work will employ computational and analytical modeling that integrates and feeds back into the complementary experimental work. Upon comparing all these different bacterial systems, we ultimately aim to identify and characterize generic mechanisms of pattern-formation and self-assembly in bacterial cells.
The formation of protein patterns and the localization of protein clusters is a major prerequisite for many important processes in bacterial cells. Examples include Min oscillations guiding the formation of the FtsZ ring to midcell in Escherichia coli cells, the localization of chemotaxis arrays and the positioning of flagella, as well as chromosome and plasmid segregation. In all these examples, experimental evidence supports mechanisms based on reaction-diffusion dynamics. Moreover, the central elements of the biochemical reaction circuits driving these processes are P-loop NTPases. These proteins are able to switch from an NTP-bound form that preferentially binds to an intracellular interface (membrane or nucleoid) to an NDP-bound form diffusing in the cytosol.
In the planned Transregio-CRC, we will theoretically investigate mechanisms for protein localization and protein pattern formation in bacterial cells. Our focus will be on protein systems composed of one or several P-loop NTPases and associated proteins. We will develop theoretical models and investigate how the interplay between the architecture of the underlying protein circuits, the different conformational states of the proteins, the reaction rates between these states, and the diffusivities of the proteins in the respective compartments determine the broad range of observed spatio-temporal patterns. These investigations will build on and generalize our earlier work on Min oscillations in E. coli and cell polarity in budding yeast. We will closely collaborate with several experimental groups in the Transregio-CRC and explore different systems that all incorporate a ParA/MinD NTPase and illustrate the versatility of these NTPases for pattern formation. Our theoretical work will employ computational and analytical modeling that integrates and feeds back into the complementary experimental work. Upon comparing all these different bacterial systems, we ultimately aim to identify and characterize generic mechanisms of pattern-formation and self-assembly in bacterial cells.
P04 Budding and asymmetric cell division in Hyphomonas neptunium
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Martin Thanbichler, Prof. Dr.
Faculty of Biology Philipps-Universität Marburg |
Summary
The commonly studied model bacteria elongate symmetrically through medial and dispersed cell wall insertion and then divide by binary fission. In budding bacteria, by contrast, new offspring arises from a largely inert mother cell by deposition of cell wall material at only one of the cell poles. In a subgroup of species, the bud forms at the tip of a long stalk, with cell division occurring asymmetrically at the bud neck. However, the mechanisms that direct this unique mode of growth are still unknown.
In TRR 174, we aim to elucidate the molecular principles that underlie the spatiotemporal regulation of budding and division site placement in the stalked budding bacterium Hyphomonas neptunium. We have previously established a molecular toolbox for the genetic manipulation of this organism. Based on this work, we have started to comprehensively analyze its cell wall biosynthetic, chromosome segregation, and cell division machineries. These studies have revaled a wealth of new regulatory patterns, whose mechanistic basis will be studied in this project.
We will perform an in-depth analysis of the localization and assembly dynamics of protein complexes with a key role in growth and cell division. After characterizing their general function, we will dissect the regulatory mechanisms governing the spatiotemporal dynamics of their positioning and activity. To this end, we will use a multi-pronged approach combining the study of candidate proteins identified in our previous work with state-of-the-art global analyses and screening approaches. Regulatory factors identified will be characterized in detail to clarify the molecular mechanisms underlying their activities. Together, these studies will set the basis for understanding the spatiotemporal regulation of budding in H. neptunium and, thus, shed light on a widespread but so far poorly investigated mode of growth in bacteria.
The commonly studied model bacteria elongate symmetrically through medial and dispersed cell wall insertion and then divide by binary fission. In budding bacteria, by contrast, new offspring arises from a largely inert mother cell by deposition of cell wall material at only one of the cell poles. In a subgroup of species, the bud forms at the tip of a long stalk, with cell division occurring asymmetrically at the bud neck. However, the mechanisms that direct this unique mode of growth are still unknown.
In TRR 174, we aim to elucidate the molecular principles that underlie the spatiotemporal regulation of budding and division site placement in the stalked budding bacterium Hyphomonas neptunium. We have previously established a molecular toolbox for the genetic manipulation of this organism. Based on this work, we have started to comprehensively analyze its cell wall biosynthetic, chromosome segregation, and cell division machineries. These studies have revaled a wealth of new regulatory patterns, whose mechanistic basis will be studied in this project.
We will perform an in-depth analysis of the localization and assembly dynamics of protein complexes with a key role in growth and cell division. After characterizing their general function, we will dissect the regulatory mechanisms governing the spatiotemporal dynamics of their positioning and activity. To this end, we will use a multi-pronged approach combining the study of candidate proteins identified in our previous work with state-of-the-art global analyses and screening approaches. Regulatory factors identified will be characterized in detail to clarify the molecular mechanisms underlying their activities. Together, these studies will set the basis for understanding the spatiotemporal regulation of budding in H. neptunium and, thus, shed light on a widespread but so far poorly investigated mode of growth in bacteria.
P05 Apical cell growth and generation of asymmetry in corynebacteria
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Marc Bramkamp, Prof. Dr.
Biologiezentrum Christian-Albrechts-Universität Kiel |
Summary
Actinobacteria grow apically by polar cell wall synthesis. In Corynebacterium glutamicum, a DivIVA-like protein is the topological marker that restricts elongation growth to the cell poles by recruiting the cell wall synthetic machinery to this position. We have shown recently that DivIVA additionally serves in chromosome tethering to the cell poles through direct interaction with ParB, a protein binding to specific sites (parS) in the chromosomal replication origin region. We found that this mechanism is conserved among actinobacteria. It is therefore an attractive hypothesis to assume that chromosome segregation and cell elongation are functionally coupled at the DivIVA interaction hub.
We will test this hypothesis by generating mutants in DivIVA, ParB and the ParB-interacting ATPase ParA and analyzing their phenotypes on the single-cell level using ready-made microfluidic growth chambers. Furthermore, we will identify new protein components of the apical growth machinery and determine how the septal wall synthesis complex is replaced by the elongation machinery at the newly generated poles after cytokinesis. We have shown that FtsZ and DivIVA are post-translationally modified by serine/threonine kinases (PknAB). Therefore, we will analyze the consequences of their phosphorylation on cell division and elongation using biochemical, genetic, and microscopic analyses. To shed light on the interplay between chromosome segregation and cell growth, in vitro reconstruction of a minimal segrosome composed of DivIVA, ParAB, and plasmid DNA carrying parS sites will be performed in membrane-clad soft-polymer compartments. Protein dynamics will be analyzed microscopically, and the data obtained will be used to model ParAB-driven DNA segregation. A long-term goal is to understand spatiotemporal assembly and control of apical growth in bacteria and its coupling to other cellular processes such as chromosome organisation.
Actinobacteria grow apically by polar cell wall synthesis. In Corynebacterium glutamicum, a DivIVA-like protein is the topological marker that restricts elongation growth to the cell poles by recruiting the cell wall synthetic machinery to this position. We have shown recently that DivIVA additionally serves in chromosome tethering to the cell poles through direct interaction with ParB, a protein binding to specific sites (parS) in the chromosomal replication origin region. We found that this mechanism is conserved among actinobacteria. It is therefore an attractive hypothesis to assume that chromosome segregation and cell elongation are functionally coupled at the DivIVA interaction hub.
We will test this hypothesis by generating mutants in DivIVA, ParB and the ParB-interacting ATPase ParA and analyzing their phenotypes on the single-cell level using ready-made microfluidic growth chambers. Furthermore, we will identify new protein components of the apical growth machinery and determine how the septal wall synthesis complex is replaced by the elongation machinery at the newly generated poles after cytokinesis. We have shown that FtsZ and DivIVA are post-translationally modified by serine/threonine kinases (PknAB). Therefore, we will analyze the consequences of their phosphorylation on cell division and elongation using biochemical, genetic, and microscopic analyses. To shed light on the interplay between chromosome segregation and cell growth, in vitro reconstruction of a minimal segrosome composed of DivIVA, ParAB, and plasmid DNA carrying parS sites will be performed in membrane-clad soft-polymer compartments. Protein dynamics will be analyzed microscopically, and the data obtained will be used to model ParAB-driven DNA segregation. A long-term goal is to understand spatiotemporal assembly and control of apical growth in bacteria and its coupling to other cellular processes such as chromosome organisation.
P06 Physical principles of chromosome organization and segregation by ParABS and SMC
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Chase Broedersz, Prof. Dr.
Department of Biology I Ludwig-Maximilians-Universität München |
Summary
Chromosomal organization and segregation presents a major challenge in all organisms. In many bacteria, the chromosome is arranged longitudinally with a striking juxtaposition between loci on the left and right arms. This organization can be attributed in part to a variety of DNA binding proteins. For instance, in B. subtilis the 3D chromosome organization with interarm interaction requires both the widely conserved SMC condensin complexes and the ParABS segregation machinery. It remains unclear how the intricate organization of the bacterial chromosome emerges from interactions between SMC, ParB, and the DNA. Our goal is to theoretically investigate the physical principles of how SMC and the ParABS system act together to spatially organize the chromosome, resolve replicating origins, and contribute to DNA segregation.
Recently, we introduced a lattice polymer model to investigate the 3D spatial organization of interacting DNA-binding proteins such as ParB. We will now extend this model to include two replicating origins, ParB, and SMC. This polymer-based lattice Monte Carlo model will be geared to investigate the organizational behavior of the chromosome, including its 3D folding statistics as determined by chromosome capture experiments, in the presence of SMC and ParB proteins and parS. This approach will be complemented with Brownian dynamics simulations of DNA with interacting proteins such as ParA and ParB, allowing us to develop a quantitative model for the dynamics of the origin separation process and chromosome segregation.
The polymer-based approaches in this project will allow us to address a fundamental question: Is the dynamics of the bacterial chromosome primarily driven by thermal diffusive processes or rather by active energy-consuming processes? All current methods to address such a question require mechanical or chemical perturbations and are thus inherently invasive. We will develop and apply a novel non-invasive statistical method based on the principle of detailed balance to study the physical origins of chromosomal dynamics prior to segregation. We will collaborate with several members in this Transregio-CRC to obtain microscopy data on the steady-state dynamics of two or more chromosomal loci in living bacteria, which will allow us to elucidate the role of active processes in chromosome dynamics.
Chromosomal organization and segregation presents a major challenge in all organisms. In many bacteria, the chromosome is arranged longitudinally with a striking juxtaposition between loci on the left and right arms. This organization can be attributed in part to a variety of DNA binding proteins. For instance, in B. subtilis the 3D chromosome organization with interarm interaction requires both the widely conserved SMC condensin complexes and the ParABS segregation machinery. It remains unclear how the intricate organization of the bacterial chromosome emerges from interactions between SMC, ParB, and the DNA. Our goal is to theoretically investigate the physical principles of how SMC and the ParABS system act together to spatially organize the chromosome, resolve replicating origins, and contribute to DNA segregation.
Recently, we introduced a lattice polymer model to investigate the 3D spatial organization of interacting DNA-binding proteins such as ParB. We will now extend this model to include two replicating origins, ParB, and SMC. This polymer-based lattice Monte Carlo model will be geared to investigate the organizational behavior of the chromosome, including its 3D folding statistics as determined by chromosome capture experiments, in the presence of SMC and ParB proteins and parS. This approach will be complemented with Brownian dynamics simulations of DNA with interacting proteins such as ParA and ParB, allowing us to develop a quantitative model for the dynamics of the origin separation process and chromosome segregation.
The polymer-based approaches in this project will allow us to address a fundamental question: Is the dynamics of the bacterial chromosome primarily driven by thermal diffusive processes or rather by active energy-consuming processes? All current methods to address such a question require mechanical or chemical perturbations and are thus inherently invasive. We will develop and apply a novel non-invasive statistical method based on the principle of detailed balance to study the physical origins of chromosomal dynamics prior to segregation. We will collaborate with several members in this Transregio-CRC to obtain microscopy data on the steady-state dynamics of two or more chromosomal loci in living bacteria, which will allow us to elucidate the role of active processes in chromosome dynamics.
P07 Spatial organization and temporal dynamics of multi-replicon genomes in alpha-rhizobia
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Anke Becker, Prof. Dr.
Faculty of Biology Philipps-Universität Marburg |
Summary
A considerable share of bacterial species maintains multipartite genomes. The precise spatiotemporal coordination of genome replication and segregation with cell growth and division is vital for proliferation of these bacteria. Multipartite genomes are particularly prevalent in plant-symbiotic alpha-rhizobia. In this group, they usually comprise one chromosome and two to six RepABC-family plasmids, which are characterized by the combined replication and partitioning repABC locus. While RepC most likely acts as the replication initiator protein at the origin of replication, RepA, RepB and the partitioning sites are required for vertical transmission of the megaplasmids to the daughter cells. Sinorhizobium meliloti possesses a tripartite genome composed of one main chromosome (3.65 Mb) and the RepABC-type megaplasmids pSymA (1.35 Mb) and pSymB (1.68 Mb). Duplication of the chromosomal and megaplasmid origins of replication is spatially and temporally uncoupled and occurs only once per cell cycle. Origin partitioning follows a strict temporal order, commencing with the chromosome and followed by pSymA and then by pSymB.
We aim at elucidating the molecular principles underlying the spatial organization, replication and partitioning of bacterial multipartite genomes employing the α-proteobacterium S. meliloti as a model organism. We will investigate the 3D-architecture of the tripartite genome and how it is affected by genome reduction or re-organization. Furthermore, we will study the regulatory mechanisms underlying the spatiotemporal coordination of replication and segregation of the main chromosome and both megaplasmids. The results are expected to point out characteristic features in the 3D-configuration as well as in the motion and positioning of specifc genomic loci, arising from the division of the genome into a main chromosome and additional chromosome-like replicons. This study will contribute to a better understanding of the benefits, structural properties and challenges associated with the maintenance of bacterial multipartite genomes.
A considerable share of bacterial species maintains multipartite genomes. The precise spatiotemporal coordination of genome replication and segregation with cell growth and division is vital for proliferation of these bacteria. Multipartite genomes are particularly prevalent in plant-symbiotic alpha-rhizobia. In this group, they usually comprise one chromosome and two to six RepABC-family plasmids, which are characterized by the combined replication and partitioning repABC locus. While RepC most likely acts as the replication initiator protein at the origin of replication, RepA, RepB and the partitioning sites are required for vertical transmission of the megaplasmids to the daughter cells. Sinorhizobium meliloti possesses a tripartite genome composed of one main chromosome (3.65 Mb) and the RepABC-type megaplasmids pSymA (1.35 Mb) and pSymB (1.68 Mb). Duplication of the chromosomal and megaplasmid origins of replication is spatially and temporally uncoupled and occurs only once per cell cycle. Origin partitioning follows a strict temporal order, commencing with the chromosome and followed by pSymA and then by pSymB.
We aim at elucidating the molecular principles underlying the spatial organization, replication and partitioning of bacterial multipartite genomes employing the α-proteobacterium S. meliloti as a model organism. We will investigate the 3D-architecture of the tripartite genome and how it is affected by genome reduction or re-organization. Furthermore, we will study the regulatory mechanisms underlying the spatiotemporal coordination of replication and segregation of the main chromosome and both megaplasmids. The results are expected to point out characteristic features in the 3D-configuration as well as in the motion and positioning of specifc genomic loci, arising from the division of the genome into a main chromosome and additional chromosome-like replicons. This study will contribute to a better understanding of the benefits, structural properties and challenges associated with the maintenance of bacterial multipartite genomes.
P08 Modeling the spatial organization of bacterial chromosomes
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Peter Lenz, Prof. Dr.
Faculty of Physics Philipps-Universität Marburg |
Summary
Bacterial chromosomes are orders of magnitude longer than the spatial extension of the cells they reside in. Bacteria therefore have to massively compact DNA and organize it in a way that is compatible with DNA replication, segregation, and transcription/translation. Recent advances in cell-imaging techniques have revealed that the bacterial chromosome is highly organized and reliably oriented within the cell.
This project focuses on the modeling of the spatial organization of chromosomes in bacteria with a single chromosome (Bacillus subtilis, Caulobacter crescentus and Escherichia coli) and with a main chromosome and additional megaplasmids (Sinorhizobium meliloti). It aims to enhance our understanding of how the spatial arrangement of the chromosome affects gene expression and motion inside cells. We will investigate how membrane-chromosome interactions affect the spatial arrangement of the chromosome. Such interactions are, e.g., mediated by expression of membrane proteins that, upon induction, shift their position towards the membrane. We will also study the opposite effect, i.e., if and how the spatial arrangement of the chromosome can regulate expression from such loci. Furthermore, in bacteria with multipartite genomes, we will analyze the geometric properties of the spatial arrangement of the several replicons and study how they affect long-range genomic interactions and the positioning and movement of particular chromosomal loci.
Basis of our approach is a theoretical model that we have recently developed and used to explain the experimentally observed spatial ordering of the chromosome of C. crescentus. We will systematically extend this model to describe the above scenarios. To do so we will closely collaborate with several experimental groups in the Transregio-CRC who will test our theoretical predictions and whose experimental data will be integrated into our model.
Bacterial chromosomes are orders of magnitude longer than the spatial extension of the cells they reside in. Bacteria therefore have to massively compact DNA and organize it in a way that is compatible with DNA replication, segregation, and transcription/translation. Recent advances in cell-imaging techniques have revealed that the bacterial chromosome is highly organized and reliably oriented within the cell.
This project focuses on the modeling of the spatial organization of chromosomes in bacteria with a single chromosome (Bacillus subtilis, Caulobacter crescentus and Escherichia coli) and with a main chromosome and additional megaplasmids (Sinorhizobium meliloti). It aims to enhance our understanding of how the spatial arrangement of the chromosome affects gene expression and motion inside cells. We will investigate how membrane-chromosome interactions affect the spatial arrangement of the chromosome. Such interactions are, e.g., mediated by expression of membrane proteins that, upon induction, shift their position towards the membrane. We will also study the opposite effect, i.e., if and how the spatial arrangement of the chromosome can regulate expression from such loci. Furthermore, in bacteria with multipartite genomes, we will analyze the geometric properties of the spatial arrangement of the several replicons and study how they affect long-range genomic interactions and the positioning and movement of particular chromosomal loci.
Basis of our approach is a theoretical model that we have recently developed and used to explain the experimentally observed spatial ordering of the chromosome of C. crescentus. We will systematically extend this model to describe the above scenarios. To do so we will closely collaborate with several experimental groups in the Transregio-CRC who will test our theoretical predictions and whose experimental data will be integrated into our model.
P09 Spatiotemporal dynamics of low-copy number receptors
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Kirsten Jung, Prof. Dr.
Department of Biology I Ludwig-Maximilians-Universität München |
Summary
Bacteria constantly respond to environmental alterations, a process mediated mostly by membrane-integrated receptors and their associated signal transduction proteins. The signalling network as a whole links the perception of environmental stimuli to various cellular responses, such as transcriptional regulation, regulation of motility or regulation of enzyme activity. However, little is known about how the spatiotemporal dynamics of particularly low-copy number membrane-integrated receptors correlates with their function and the output response in individual cells within a population.
We aim to unravel the localisation and the spatiotemporal dynamics of different types of polyproline (polyPro) containing low-copy number receptors in interplay with their co-sensors in E. coli and V. cholerae. In particular, some of these receptors, called one-component systems, combine two functions in a single polypeptide, the periplasmic sensory and the cytoplasmic DNA-binding activity. The contribution of these membrane-integrated transcriptional regulators to chromosome-membrane interactions and chromosome organization will be investigated. Finally, we will study the role of the translation elongation factor P (EF-P) in these processes, a factor that alleviates ribosome stalling at polyPro motifs. This work will contribute to a better understanding of the cellular organization and functioning of the signalling network that allows the sophisticated information flow in bacteria.
In the first funding period we will analyse the localisation and the relative abundance of the fluorophore-tagged pH-sensor and transcriptional activator CadC and its lysine co-sensor LysP as well as the labelled CadC-DNA-binding site under non-stress and acid stress conditions in E. coli. In E. coli the copy number of CadC and hence the signalling output of the Cad-system is tightly controlled by EF-P dependent translation. However, Vibrionaceae, e.g. Vibrio cholerae, naturally encode an EF-P independent CadC and lack the co-sensor LysP. Therefore, we will also investigate the spatiotemporal dynamics and functioning of CadC in this species. The contribution of CadC in E. coli and V. cholerae on chromosome organization will be studied. Finally, we will extend our studies on the role of ribosome stalling and EF‑P stall release to three other polyPro-dependent receptors: the osmosensor EnvZ, the acid/drug sensor EvgS, and the phosphate sensor PhoR. Altogether, we expect new insights into the dynamics of low-copy number receptors at the cellular level as well as a detailed understanding of the molecular mechanism in the context of chromosome-membrane interactions and translation elongation factor P in bacteria.
Bacteria constantly respond to environmental alterations, a process mediated mostly by membrane-integrated receptors and their associated signal transduction proteins. The signalling network as a whole links the perception of environmental stimuli to various cellular responses, such as transcriptional regulation, regulation of motility or regulation of enzyme activity. However, little is known about how the spatiotemporal dynamics of particularly low-copy number membrane-integrated receptors correlates with their function and the output response in individual cells within a population.
We aim to unravel the localisation and the spatiotemporal dynamics of different types of polyproline (polyPro) containing low-copy number receptors in interplay with their co-sensors in E. coli and V. cholerae. In particular, some of these receptors, called one-component systems, combine two functions in a single polypeptide, the periplasmic sensory and the cytoplasmic DNA-binding activity. The contribution of these membrane-integrated transcriptional regulators to chromosome-membrane interactions and chromosome organization will be investigated. Finally, we will study the role of the translation elongation factor P (EF-P) in these processes, a factor that alleviates ribosome stalling at polyPro motifs. This work will contribute to a better understanding of the cellular organization and functioning of the signalling network that allows the sophisticated information flow in bacteria.
In the first funding period we will analyse the localisation and the relative abundance of the fluorophore-tagged pH-sensor and transcriptional activator CadC and its lysine co-sensor LysP as well as the labelled CadC-DNA-binding site under non-stress and acid stress conditions in E. coli. In E. coli the copy number of CadC and hence the signalling output of the Cad-system is tightly controlled by EF-P dependent translation. However, Vibrionaceae, e.g. Vibrio cholerae, naturally encode an EF-P independent CadC and lack the co-sensor LysP. Therefore, we will also investigate the spatiotemporal dynamics and functioning of CadC in this species. The contribution of CadC in E. coli and V. cholerae on chromosome organization will be studied. Finally, we will extend our studies on the role of ribosome stalling and EF‑P stall release to three other polyPro-dependent receptors: the osmosensor EnvZ, the acid/drug sensor EvgS, and the phosphate sensor PhoR. Altogether, we expect new insights into the dynamics of low-copy number receptors at the cellular level as well as a detailed understanding of the molecular mechanism in the context of chromosome-membrane interactions and translation elongation factor P in bacteria.
P10 Quantitative analysis of a spatial toggle switch and one-component signaling in bacteria
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Ulrich Gerland, Prof. Dr.
Faculty of Physics Technische Universität München |
Summary
The cellular behavior of bacteria is orchestrated by signaling pathways, which sense environmental cues and control the downstream adaptive responses. This project focuses on two spatio-temporal phenomena in bacterial signaling, (i) the regulation of Myxococcus xanthus motility, which involves the dynamics of controlled cell polarity switching, and (ii) the signal transduction from a membrane-localized signaling system to its target gene.
Exquisite control of cell motility is crucial for the efficient feeding and the social behaviors of bacterial cells. The control of gliding motility in the rod-shaped bacterium M. xanthus involves an intricate spatiotemporal dynamics, based on the establishment of a cell polarity axis and induced reversals of this axis. Key components of the system have been identified, including MglA, which accumulates at the leading cell pole and MglB, which localizes to the lagging cell pole. The correct localization of the Mgl proteins is also mutually dependent on other adaptor proteins, including RomR. Together, these proteins form an intriguing “spatial toggle switch”. The reversal of this switch, and thus of the cell polarity axis, is known to be controlled by the Frz pathway, which has homologies to the chemotaxis pathway of E. coli. While the key components of this system have been identified, its working principle remains unclear. This project first seeks a biophysical understanding of the Mgl-Rom-based polarity mechanism using quantitative modeling in close collaboration with the experimental investigation of this system in the Søgaard-Andersen lab. The approach will be based on a coarse-grained modeling approach that distinguishes three spatial localizations: localized at the leading cell pole, at the lagging pole, and delocalized in the cytosol. These three pools of molecules can also be clearly separated experimentally using fluorescence microscopy, yielding time-dependent information about their relative abundance. Next, we will study the mechanism underlying polarity switching. Using the coarse-grained modeling approach, we will identify different categories of possible switching mechanisms. Based on the experimental evidence, we will then seek to discriminate between the different possibilities. Furthermore, we will identify experimental tests that can feed back into the complementary experimental work to clarify the switching mechanism.
The second focus of this project is on the stochastic dynamics of “one-component” signaling in bacteria, in the context of the E. coli CadC-LysP system that is experimentally studied by Kirsten Jung within TRR 174. One-component systems consist of only a single protein that implements both functions of a signaling system, sensing and response regulation. CadC is one of several known membrane-integrated one-component systems that directly bind to the genomic DNA to regulate transcription. This pH-stress response system also receives signaling input from the lysine-specific permease LysP. The proper functioning of the CadC-LysP system relies on a target search process whereby CadC locates its cognate binding site on the DNA. To understand the biophysics of one-component signal transduction, and to describe experiments probing the CadC-LysP system, we will develop a stochastic modeling framework. The framework will enable us to characterize the dependence of the target search process on several system parameters and to test working hypotheses extracted from the experiments of the Jung Lab. Taken together, this project leverages biophysical modeling to elucidate two spatiotemporal regulation phenomena that are studied experimentally within the proposed Transregio-CRC.
The cellular behavior of bacteria is orchestrated by signaling pathways, which sense environmental cues and control the downstream adaptive responses. This project focuses on two spatio-temporal phenomena in bacterial signaling, (i) the regulation of Myxococcus xanthus motility, which involves the dynamics of controlled cell polarity switching, and (ii) the signal transduction from a membrane-localized signaling system to its target gene.
Exquisite control of cell motility is crucial for the efficient feeding and the social behaviors of bacterial cells. The control of gliding motility in the rod-shaped bacterium M. xanthus involves an intricate spatiotemporal dynamics, based on the establishment of a cell polarity axis and induced reversals of this axis. Key components of the system have been identified, including MglA, which accumulates at the leading cell pole and MglB, which localizes to the lagging cell pole. The correct localization of the Mgl proteins is also mutually dependent on other adaptor proteins, including RomR. Together, these proteins form an intriguing “spatial toggle switch”. The reversal of this switch, and thus of the cell polarity axis, is known to be controlled by the Frz pathway, which has homologies to the chemotaxis pathway of E. coli. While the key components of this system have been identified, its working principle remains unclear. This project first seeks a biophysical understanding of the Mgl-Rom-based polarity mechanism using quantitative modeling in close collaboration with the experimental investigation of this system in the Søgaard-Andersen lab. The approach will be based on a coarse-grained modeling approach that distinguishes three spatial localizations: localized at the leading cell pole, at the lagging pole, and delocalized in the cytosol. These three pools of molecules can also be clearly separated experimentally using fluorescence microscopy, yielding time-dependent information about their relative abundance. Next, we will study the mechanism underlying polarity switching. Using the coarse-grained modeling approach, we will identify different categories of possible switching mechanisms. Based on the experimental evidence, we will then seek to discriminate between the different possibilities. Furthermore, we will identify experimental tests that can feed back into the complementary experimental work to clarify the switching mechanism.
The second focus of this project is on the stochastic dynamics of “one-component” signaling in bacteria, in the context of the E. coli CadC-LysP system that is experimentally studied by Kirsten Jung within TRR 174. One-component systems consist of only a single protein that implements both functions of a signaling system, sensing and response regulation. CadC is one of several known membrane-integrated one-component systems that directly bind to the genomic DNA to regulate transcription. This pH-stress response system also receives signaling input from the lysine-specific permease LysP. The proper functioning of the CadC-LysP system relies on a target search process whereby CadC locates its cognate binding site on the DNA. To understand the biophysics of one-component signal transduction, and to describe experiments probing the CadC-LysP system, we will develop a stochastic modeling framework. The framework will enable us to characterize the dependence of the target search process on several system parameters and to test working hypotheses extracted from the experiments of the Jung Lab. Taken together, this project leverages biophysical modeling to elucidate two spatiotemporal regulation phenomena that are studied experimentally within the proposed Transregio-CRC.
P11 Spatiotemporal regulation of peritrichous flagellation in Bacillus subtilis
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Gert Bange, Prof. Dr.
Faculty of Chemistry Philipps-Universität Marburg |
Summary
Flagella are the organelles of motility in many bacteria, and show an asymmetric, but distinct distribution (pattern). The Gram-positive bacterium Bacillus subtilis exhibits approximately 25 regularly spaced flagella that are found at the lateral sides but not at the cell pole. The nucleotide-binding proteins FlhF and FlhG are essential for establishing the correct peritrichous flagellation pattern in B. subtilis. This proposal aims at unraveling the molecular mechanism by which these proteins control the spatiotemporal formation of the peritrichous flagellation pattern in B. subtilis.
Flagella are the organelles of motility in many bacteria, and show an asymmetric, but distinct distribution (pattern). The Gram-positive bacterium Bacillus subtilis exhibits approximately 25 regularly spaced flagella that are found at the lateral sides but not at the cell pole. The nucleotide-binding proteins FlhF and FlhG are essential for establishing the correct peritrichous flagellation pattern in B. subtilis. This proposal aims at unraveling the molecular mechanism by which these proteins control the spatiotemporal formation of the peritrichous flagellation pattern in B. subtilis.
P12 Molecular and mechanistic basis for spatiotemporal organization of polar and lateral flagella
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Kai Thormann, Prof. Dr.
Department of Microbiology and Molecular Biology Justus-Liebig-Universität Gießen |
Summary
Bacterial flagella are intricate multiprotein complexes that need to be specifically positioned for proper function. Several diverse flagellation patterns exist among bacterial species, with the major difference that one or more of these molecular machines are either targeted to the cell pole, or they occur in lateral positions. For numerous bacterial species, FlhF and FlhG are mediating spatiotemporal control of diverse flagellar patterns and flagellar number. The molecular principle by which these two proteins establish such a variety of flagellation patterns is mostly unknown. In this planned Transregio-CRC, we aim to explore the molecular mechanism by which FlhF and FlhG orchestrate positioning, number, and function of a polar flagellar system in our model organism Shewanella putrefaciens CN-32. Our preliminary data suggests that FlhF mediates polar targeting of the first flagellar building blocks via the signal recognition pathway (SRP). We could also show that the MinD-like ATPase FlhG functions through direct interaction with the building blocks of the flagellar basal body.
In this project we will, together with our collaborating partners, further analyse localization and activity of FlhF and FlhG and their interaction partners. One part of the project will concentrate on the role of FlhF in polar recruitment of flagellar components via the SRP pathway. In a second part, we will aim at establishing how FlhG executes its function at the flagellar basal body to restrict the number of polar flagella to one, and how FlhF/FlhG may affect transcriptional control of flagellar production. By comparison of the role of FlhF/FlhF in polarly flagellated S. putrefaciens CN-32 and the lateral flagellation pattern of B. subtilis, we will aim at getting further insights into the evolution of flagellar pattern formation. Taken together, the proposed studies are expected to give fundamentally novel insights into the molecular mechanism underlying spatiotemporal control of multiprotein complex assembly in bacteria.
Bacterial flagella are intricate multiprotein complexes that need to be specifically positioned for proper function. Several diverse flagellation patterns exist among bacterial species, with the major difference that one or more of these molecular machines are either targeted to the cell pole, or they occur in lateral positions. For numerous bacterial species, FlhF and FlhG are mediating spatiotemporal control of diverse flagellar patterns and flagellar number. The molecular principle by which these two proteins establish such a variety of flagellation patterns is mostly unknown. In this planned Transregio-CRC, we aim to explore the molecular mechanism by which FlhF and FlhG orchestrate positioning, number, and function of a polar flagellar system in our model organism Shewanella putrefaciens CN-32. Our preliminary data suggests that FlhF mediates polar targeting of the first flagellar building blocks via the signal recognition pathway (SRP). We could also show that the MinD-like ATPase FlhG functions through direct interaction with the building blocks of the flagellar basal body.
In this project we will, together with our collaborating partners, further analyse localization and activity of FlhF and FlhG and their interaction partners. One part of the project will concentrate on the role of FlhF in polar recruitment of flagellar components via the SRP pathway. In a second part, we will aim at establishing how FlhG executes its function at the flagellar basal body to restrict the number of polar flagella to one, and how FlhF/FlhG may affect transcriptional control of flagellar production. By comparison of the role of FlhF/FlhF in polarly flagellated S. putrefaciens CN-32 and the lateral flagellation pattern of B. subtilis, we will aim at getting further insights into the evolution of flagellar pattern formation. Taken together, the proposed studies are expected to give fundamentally novel insights into the molecular mechanism underlying spatiotemporal control of multiprotein complex assembly in bacteria.
P13 Co-translational membrane targeting by SRP and FlhF
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Roland Beckmann, Prof. Dr.
Department of Biochemistry Ludwig-Maximilians-Universität München |
Summary
Co-translational targeting of integral membrane proteins to the plasma membrane is an essential part of the spatiotemporal organization in bacteria. The universally conserved signal recognition particle (SRP) and its cognate receptor (SR) play a crucial role in this process by recognizing and guiding the translating ribosome to the site of co-translational membrane insertion, usually via the SecYEG protein-conducting channel. Both, the signal sequence recognizing subunit of SRP (Srp54 or Ffh in bacteria) and SR (FtsY in bacteria) are G proteins that can form heterodimers in a GTP-dependent manner in order to coordinate the translating ribosome with the appropriate translocation/insertion machinery at the membrane. Interestingly, FlhF, a third Srp54-like GTPase, has been discovered in bacteria and has been shown to be involved in the biogenesis and correct pattern formation of flagella. Recent data of the Bange lab and the Thormann lab point towards an intricate connection between the FlhF system and the SRP system in Bacillus subtilis and Shewanella putrefaciens. However, it is completely unclear how this interplay eventually allows for proper regulation of flagellar biogenesis. This proposal aims at unraveling the structural basis and molecular mechanisms by which these proteins contribute to the correct spatiotemporal coordination of flagella biogenesis in B. subtilis and S. putrefaciens.
Co-translational targeting of integral membrane proteins to the plasma membrane is an essential part of the spatiotemporal organization in bacteria. The universally conserved signal recognition particle (SRP) and its cognate receptor (SR) play a crucial role in this process by recognizing and guiding the translating ribosome to the site of co-translational membrane insertion, usually via the SecYEG protein-conducting channel. Both, the signal sequence recognizing subunit of SRP (Srp54 or Ffh in bacteria) and SR (FtsY in bacteria) are G proteins that can form heterodimers in a GTP-dependent manner in order to coordinate the translating ribosome with the appropriate translocation/insertion machinery at the membrane. Interestingly, FlhF, a third Srp54-like GTPase, has been discovered in bacteria and has been shown to be involved in the biogenesis and correct pattern formation of flagella. Recent data of the Bange lab and the Thormann lab point towards an intricate connection between the FlhF system and the SRP system in Bacillus subtilis and Shewanella putrefaciens. However, it is completely unclear how this interplay eventually allows for proper regulation of flagellar biogenesis. This proposal aims at unraveling the structural basis and molecular mechanisms by which these proteins contribute to the correct spatiotemporal coordination of flagella biogenesis in B. subtilis and S. putrefaciens.
P14 Chromosome and protein dynamics during the insertion of membrane proteins into the cell membrane
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Peter Graumann, Prof. Dr.
Faculty of Chemistry Philipps-Universität Marburg |
Summary
It is still unclear how the synthesis of membrane proteins is spatiotemporally organized within bacterial cells. Signal recognition particle (SRP) binds to N-terminal signal sequences of actively translated proteins, reduces translation rate, and together with the SRP receptor, FtsY, mediates the transfer of the ribosome/nascent chain complex (RNC) to the membrane-embedded SecEYG translocon. We will use single molecule microscopy and automated particle tracking (SMT) to investigate if SRP binds to RNC complexes in the cytosol, or whether RNCs are targeted to the membrane, where they interact with SRP/FtsY. We will localize mRNA encoding for soluble and for membrane proteins and determine their subcellular localization and mobility. Bacterial chromosomes have a preferred arrangement and are roughly folded according to their physical organization, such that neighbouring genes are also closely positioned within the cell, at a relatively defined location. In most bacterial species, chromosomes do not fill the entire cells but are compacted into a structure called the nucleoid. Ribosomes are present at the nucleoid-free space within the cell, and it is still unclear if newly synthesized RNA leaves the nucleoid, or if actively transcribing genes move towards the periphery of the nucleoid, due to the coupling of transcription and translation in bacteria. We will investigate the 3D organization of the Bacillus subtilis chromosome using structured illumination super-resolution fluorescence microscopy.
It is still unclear how the synthesis of membrane proteins is spatiotemporally organized within bacterial cells. Signal recognition particle (SRP) binds to N-terminal signal sequences of actively translated proteins, reduces translation rate, and together with the SRP receptor, FtsY, mediates the transfer of the ribosome/nascent chain complex (RNC) to the membrane-embedded SecEYG translocon. We will use single molecule microscopy and automated particle tracking (SMT) to investigate if SRP binds to RNC complexes in the cytosol, or whether RNCs are targeted to the membrane, where they interact with SRP/FtsY. We will localize mRNA encoding for soluble and for membrane proteins and determine their subcellular localization and mobility. Bacterial chromosomes have a preferred arrangement and are roughly folded according to their physical organization, such that neighbouring genes are also closely positioned within the cell, at a relatively defined location. In most bacterial species, chromosomes do not fill the entire cells but are compacted into a structure called the nucleoid. Ribosomes are present at the nucleoid-free space within the cell, and it is still unclear if newly synthesized RNA leaves the nucleoid, or if actively transcribing genes move towards the periphery of the nucleoid, due to the coupling of transcription and translation in bacteria. We will investigate the 3D organization of the Bacillus subtilis chromosome using structured illumination super-resolution fluorescence microscopy.
P15 Spatiotemporal dynamics of a membrane-associated RNA-binding protein and its cargo in Vibrio cholerae
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Kai Papenfort, Prof. Dr.
Institute of Microbiology Friedrich-Schiller-Universität Jena |
Summary
The localization of RNA to subcellular compartments provides a mechanism for regulating gene expression with exquisite temporal and spatial control. In bacteria, many proteins localize to distinct subcellular domains regulating the spatial deployment of other proteins, DNA or lipids. Similarly, bacterial RNAs can be targeted to specific cellular locations affecting stability and translation efficiency of these molecules. In this project, we will characterize a novel RNA-binding protein encoded on the chromosome of the major human pathogen, Vibrio cholerae. This protein, named MbrA (membrane-bound RNA-binding protein A), contains two trans-membrane domains at the N-terminus and a conserved RRM-type RNA-binding domain located towards the C-terminus. Our preliminary data show that MbrA localizes to the cell envelope, and that mutants lacking the mbrA gene display altered expression of key genes controlling quorum-sensing (QS) and virulence of V. cholerae.
To understand the contribution of MbrA to RNA localization in V. cholerae globally and how it affects QS specifically, we will determine the RNA-binding profile of MbrA in V. cholerae and explore the parameters driving RNA localization in the cell. Targeted mutagenesis of the membrane- and RNA-binding elements of MbrA will pinpoint the roles of the individual domains in RNA stability, translation efficiency, localization and ultimately complex behaviors, such as QS. How RNA-molecules are targeted to specific domains in bacteria has not been understood. Membrane-associated RNA-binding proteins, such as MbrA, could provide a solution to this problem by guiding RNAs to discrete cellular positions, modulating their accessibility to and interaction with other cellular components.
The localization of RNA to subcellular compartments provides a mechanism for regulating gene expression with exquisite temporal and spatial control. In bacteria, many proteins localize to distinct subcellular domains regulating the spatial deployment of other proteins, DNA or lipids. Similarly, bacterial RNAs can be targeted to specific cellular locations affecting stability and translation efficiency of these molecules. In this project, we will characterize a novel RNA-binding protein encoded on the chromosome of the major human pathogen, Vibrio cholerae. This protein, named MbrA (membrane-bound RNA-binding protein A), contains two trans-membrane domains at the N-terminus and a conserved RRM-type RNA-binding domain located towards the C-terminus. Our preliminary data show that MbrA localizes to the cell envelope, and that mutants lacking the mbrA gene display altered expression of key genes controlling quorum-sensing (QS) and virulence of V. cholerae.
To understand the contribution of MbrA to RNA localization in V. cholerae globally and how it affects QS specifically, we will determine the RNA-binding profile of MbrA in V. cholerae and explore the parameters driving RNA localization in the cell. Targeted mutagenesis of the membrane- and RNA-binding elements of MbrA will pinpoint the roles of the individual domains in RNA stability, translation efficiency, localization and ultimately complex behaviors, such as QS. How RNA-molecules are targeted to specific domains in bacteria has not been understood. Membrane-associated RNA-binding proteins, such as MbrA, could provide a solution to this problem by guiding RNAs to discrete cellular positions, modulating their accessibility to and interaction with other cellular components.
P16 Diffusional properties of proteins in a bacterial cell
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Victor Sourjik, Prof. Dr.
Department of Systems and Synthetic Biology Max Planck Institute for Terrestrial Microbiology Marburg |
Summary
Protein diffusion is one of the key factors in many biochemical reactions as well as the assembly and dynamic reorganization of multiprotein complexes. Consequently, diffusion has to be taken into account by any quantitative, mathematical description of spatiotemporal dynamics of cellular processes. Although the theory of diffusion in dilute solutions is well established, the crowded and non-homogeneous nature of the cellular environment makes protein movement within cells very different. Several recent studies suggest that diffusion of proteins is strongly obstructed in the cell, but the details of this obstruction in different sub-compartments and its dependence on various cellular structures are only poorly understood. Moreover, the degree to which protein mobility depends on the shape and charge of a protein, the degree of molecular crowding within the cell, and on the cellular state remains largely unknown. Finally, it is unclear how changes in protein mobility, e.g. due to changes in molecular crowding, affect cellular processes.
The aim of this project is to systematically characterize protein diffusion within all four sub-cellular compartments of Gram-negative bacteria (cytoplasm, periplasm, and inner and outer membranes) using Escherichia coli as a model. Extending our previous work on protein diffusion in E. coli, we will use a set of fluorescently tagged model proteins to investigate protein mobility within each of these four sub-cellular compartments. We will apply complementary techniques – FCS, FRAP and SPT – that enable analysis of diffusion on different spatial and temporal scales. We will analyze how the diffusion of individual proteins and protein complexes depends on their molecular mass and shape, on cellular structures and on crowding within a cell. We will further investigate how protein diffusion depends on the growth phase, the growth rate, the energy state of the cell, osmolarity and temperature. Finally, using the well-studied Min system and the chemotaxis system as models, we will quantitatively explore the effects of changing protein mobility and molecular crowding on the dynamics of cellular processes as well as on assembly and stability of multiprotein complexes.
We expect this analysis to provide information about the nanoscale structure of bacterial cells and how it depends on the environment and cellular state, thus enabling a more precise quantitative description of the spatiotemporal dynamics of cellular processes.
Protein diffusion is one of the key factors in many biochemical reactions as well as the assembly and dynamic reorganization of multiprotein complexes. Consequently, diffusion has to be taken into account by any quantitative, mathematical description of spatiotemporal dynamics of cellular processes. Although the theory of diffusion in dilute solutions is well established, the crowded and non-homogeneous nature of the cellular environment makes protein movement within cells very different. Several recent studies suggest that diffusion of proteins is strongly obstructed in the cell, but the details of this obstruction in different sub-compartments and its dependence on various cellular structures are only poorly understood. Moreover, the degree to which protein mobility depends on the shape and charge of a protein, the degree of molecular crowding within the cell, and on the cellular state remains largely unknown. Finally, it is unclear how changes in protein mobility, e.g. due to changes in molecular crowding, affect cellular processes.
The aim of this project is to systematically characterize protein diffusion within all four sub-cellular compartments of Gram-negative bacteria (cytoplasm, periplasm, and inner and outer membranes) using Escherichia coli as a model. Extending our previous work on protein diffusion in E. coli, we will use a set of fluorescently tagged model proteins to investigate protein mobility within each of these four sub-cellular compartments. We will apply complementary techniques – FCS, FRAP and SPT – that enable analysis of diffusion on different spatial and temporal scales. We will analyze how the diffusion of individual proteins and protein complexes depends on their molecular mass and shape, on cellular structures and on crowding within a cell. We will further investigate how protein diffusion depends on the growth phase, the growth rate, the energy state of the cell, osmolarity and temperature. Finally, using the well-studied Min system and the chemotaxis system as models, we will quantitatively explore the effects of changing protein mobility and molecular crowding on the dynamics of cellular processes as well as on assembly and stability of multiprotein complexes.
We expect this analysis to provide information about the nanoscale structure of bacterial cells and how it depends on the environment and cellular state, thus enabling a more precise quantitative description of the spatiotemporal dynamics of cellular processes.