ParB-type DNA Segregation Proteins Are CTP-Dependent Molecular Switches.

During cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions.

The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes.

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

General protein diffusion barriers create compartments within bacterial cells.

In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are also widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell-cycle-dependent manner. Their presence is critical for cellular fitness because they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins.

MipZ caps the plus-end of FtsZ polymers to promote their rapid disassembly.

The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ-FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell.

Closing the ring: a new twist to bacterial chromosome condensation.

The mechanisms underlying chromosome organization in bacteria are still shrouded in mystery. Sullivan et al. (2009) and Gruber and Errington (2009) now report that the DNA-binding protein ParB ensures proper arrangement and partitioning of chromosomal DNA in Bacillus subtilis by recruiting the condensin SMC to the replication origin region.

Functional Degeneracy in Paracoccus denitrificans Pd1222 Is Coordinated via RamB, Which Links Expression of the Glyoxylate Cycle to Activity of the Ethylmalonyl-CoA Pathway.

Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.

Integrative and quantitative view of the CtrA regulatory network in a stalked budding bacterium.

The Alphaproteobacteria show a remarkable diversity of cell cycle-dependent developmental patterns, which are governed by the conserved CtrA pathway. Its central component CtrA is a DNA-binding response regulator that is controlled by a complex two-component signaling network, mediating distinct transcriptional programs in the two offspring. The CtrA pathway has been studied intensively and was shown to consist of an upstream part that reads out the developmental state of the cell and a downstream part that integrates the upstream signals and mediates CtrA phosphorylation. However, the role of this circuitry in bacterial diversification remains incompletely understood. We have therefore investigated CtrA regulation in the morphologically complex stalked budding alphaproteobacterium Hyphomonas neptunium. Compared to relatives dividing by binary fission, H. neptunium shows distinct changes in the role and regulation of various pathway components. Most notably, the response regulator DivK, which normally links the upstream and downstream parts of the CtrA pathway, is dispensable, while downstream components such as the pseudokinase DivL, the histidine kinase CckA, the phosphotransferase ChpT and CtrA are essential. Moreover, CckA is compartmentalized to the nascent bud without forming distinct polar complexes and CtrA is not regulated at the level of protein abundance. We show that the downstream pathway controls critical functions such as replication initiation, cell division and motility. Quantification of the signal flow through different nodes of the regulatory cascade revealed that the CtrA pathway is a leaky pipeline and must involve thus-far unidentified factors. Collectively, the quantitative system-level analysis of CtrA regulation in H. neptunium points to a considerable evolutionary plasticity of cell cycle regulation in alphaproteobacteria and leads to hypotheses that may also hold in well-established model organisms such as Caulobacter crescentus.

Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus.

The spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus is mediated by two different P-loop ATPases, ParA and MipZ. Both of these proteins form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization depends on their nucleotide-dependent cycling between a monomeric and a dimeric state and on the ability of the dimeric species to associate with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to comprehensively map the residues mediating the interactions of MipZ and ParA with DNA. We show that MipZ has non-specific DNA-binding activity that relies on an array of positively charged and hydrophobic residues lining both sides of the dimer interface. Extending our analysis to ParA, we find that the MipZ and ParA DNA-binding sites differ markedly in composition, although their relative positions on the dimer surface and their mode of DNA binding are conserved. In line with previous experimental work, bioinformatic analysis suggests that the same principles may apply to other members of the P-loop ATPase family. P-loop ATPases thus share common mechanistic features, although their functions have diverged considerably during the course of evolution.

A specialized MreB-dependent cell wall biosynthetic complex mediates the formation of stalk-specific peptidoglycan in Caulobacter crescentus.

Many bacteria have complex cell shapes, but the mechanisms producing their distinctive morphologies are still poorly understood. Caulobacter crescentus, for instance, exhibits a stalk-like extension that carries an adhesive holdfast mediating surface attachment. This structure forms through zonal peptidoglycan biosynthesis at the old cell pole and elongates extensively under phosphate-limiting conditions. We analyzed the composition of cell body and stalk peptidoglycan and identified significant differences in the nature and proportion of peptide crosslinks, indicating that the stalk represents a distinct subcellular domain with specific mechanical properties. To identify factors that participate in stalk formation, we systematically inactivated and localized predicted components of the cell wall biosynthetic machinery of C. crescentus. Our results show that the biosynthesis of stalk peptidoglycan involves a dedicated peptidoglycan biosynthetic complex that combines specific components of the divisome and elongasome, suggesting that the repurposing of preexisting machinery provides a straightforward means to evolve new morphological traits.

BacStalk: A comprehensive and interactive image analysis software tool for bacterial cell biology.

Prokaryotic cells display a striking subcellular organization. Studies of the underlying mechanisms in different species have greatly enhanced our understanding of the morphological and physiological adaptation of bacteria to different environmental niches. The image analysis software tool BacStalk is designed to extract comprehensive quantitative information from the images of morphologically complex bacteria with stalks, flagella, or other appendages. The resulting data can be visualized in interactive demographs, kymographs, cell lineage plots, and scatter plots to enable fast and thorough data analysis and representation. Notably, BacStalk can generate demographs and kymographs that display fluorescence signals within the two-dimensional cellular outlines, to accurately represent their subcellular location. Beyond organisms with visible appendages, BacStalk is also suitable for established, non-stalked model organisms with common or uncommon cell shapes. BacStalk, therefore, contributes to the advancement of prokaryotic cell biology and physiology, as it widens the spectrum of easily accessible model organisms and enables highly intuitive and interactive data analysis and visualization.

A gradient-forming MipZ protein mediating the control of cell division in the magnetotactic bacterium Magnetospirillum gryphiswaldense.

Cell division needs to be tightly regulated and closely coordinated with other cellular processes to ensure the generation of fully viable offspring. Here, we investigate division site placement by the cell division regulator MipZ in the alphaproteobacterium Magnetospirillum gryphiswaldense, a species that forms linear chains of magnetosomes to navigate within the geomagnetic field. We show that M. gryphiswaldense contains two MipZ homologs, termed MipZ1 and MipZ2. MipZ2 localizes to the division site, but its absence does not cause any obvious phenotype. MipZ1, by contrast, forms a dynamic bipolar gradient, and its deletion or overproduction cause cell filamentation, suggesting an important role in cell division. The monomeric form of MipZ1 interacts with the chromosome partitioning protein ParB, whereas its ATP-dependent dimeric form shows non-specific DNA-binding activity. Notably, both the dimeric and, to a lesser extent, the monomeric form inhibit FtsZ polymerization in vitro. MipZ1 thus represents a canonical gradient-forming MipZ homolog that critically contributes to the spatiotemporal control of FtsZ ring formation. Collectively, our findings add to the view that the regulatory role of MipZ proteins in cell division is conserved among many alphaproteobacteria. However, their number and biochemical properties may have adapted to the specific needs of the host organism.

Generating asymmetry in a changing environment: cell cycle regulation in dimorphic alphaproteobacteria.

While many bacteria divide by symmetric binary fission, some alphaproteobacteria have strikingly asymmetric cell cycles, producing offspring that differs significantly in their morphology and reproductive state. To establish this asymmetry, these species employ a complex cell cycle regulatory pathway based on two-component signaling cascades. At the center of this network is the essential DNA-binding response regulator CtrA, which acts as a transcription factor controlling numerous genes with cell cycle-relevant functions as well as a regulator of chromosome replication. The DNA-binding activity of CtrA is controlled at the level of both protein phosphorylation and stability, dependent on an intricate network of regulatory proteins, whose function is tightly coordinated in time and space. CtrA is differentially activated in the two (developing) offspring, thereby establishing distinct transcriptional programs that ultimately determine their distinct cell fates. Phase-separated polar microdomains of changing composition sequester proteins involved in the (in-)activation and degradation of CtrA specifically at each pole. In this review, we summarize the current knowledge of the CtrA pathway and discuss how it has evolved to regulate the cell cycle of morphologically distinct alphaproteobacteria.

Localized dimerization and nucleoid binding drive gradient formation by the bacterial cell division inhibitor MipZ.

Protein gradients play a central role in the spatial organization of cells, but the mechanisms of their formation are incompletely understood. This study analyzes the determinants responsible for establishing bipolar gradients of the ATPase MipZ, a key regulator of division site placement in Caulobacter crescentus. We have solved the crystal structure of MipZ in different nucleotide states, dissected its ATPase cycle, and investigated its interaction with FtsZ, ParB, and the nucleoid. Our results suggest that the polar ParB complexes locally stimulate the formation of ATP-bound MipZ dimers, which are then retained near the cell poles through association with chromosomal DNA. Due to their intrinsic ATPase activity, dimers eventually dissociate into freely diffusible monomers that undergo spontaneous nucleotide exchange and are recaptured by ParB. These findings clarify the molecular function of a conserved gradient-forming system and reveal mechanistic principles that might be commonly used to sustain protein gradients within cells.

LytM factors affect the recruitment of autolysins to the cell division site in Caulobacter crescentus.

Most bacteria possess a peptidoglycan cell wall that determines their morphology and provides mechanical robustness during osmotic challenges. The biosynthesis of this structure is achieved by a large set of synthetic and lytic enzymes with varying substrate specificities. Although the biochemical functions of these proteins are conserved and well-investigated, the precise roles of individual factors and the regulatory mechanisms coordinating their activities in time and space remain incompletely understood. Here, we comprehensively analyze the autolytic machinery of the alphaproteobacterial model organism Caulobacter crescentus, with a specific focus on LytM-like endopeptidases, soluble lytic transglycosylases and amidases. Our data reveal a high degree of redundancy within each protein family but also specialized functions for individual family members under stress conditions. In addition, we identify two lytic transglycosylases and an amidase as new divisome components that are recruited to midcell at distinct stages of the cell cycle. The midcell localization of these proteins is affected by two LytM factors with degenerate catalytic domains, DipM and LdpF, which may serve as regulatory hubs coordinating the activities of multiple autolytic enzymes during cell constriction and fission respectively. These findings set the stage for in-depth studies of the molecular mechanisms that control peptidoglycan remodeling in C. crescentus.

Dynamics of the peptidoglycan biosynthetic machinery in the stalked budding bacterium Hyphomonas neptunium.

Most commonly studied bacteria grow symmetrically and divide by binary fission, generating two siblings of equal morphology. An exception to this rule are budding bacteria, in which new offspring emerges de novo from a morphologically invariant mother cell. Although this mode of proliferation is widespread in diverse bacterial lineages, the underlying mechanisms are still incompletely understood. Here, we report the first molecular-level analysis of growth and morphogenesis in the stalked budding alphaproteobacterium Hyphomonas neptunium. Peptidoglycan labeling shows that, in this species, buds originate from a stalk-like extension of the mother cell whose terminal segment is gradually remodeled into a new cell compartment. As a first step toward identifying the machinery mediating the budding process, we performed comprehensive mutational and localization studies of predicted peptidoglycan biosynthetic proteins in H. neptunium. These analyses identify factors that localize to distinct zones of dispersed and zonal growth, and they suggest a critical role of the MreB-controlled elongasome in cell morphogenesis. Collectively, our work shows that the mechanism of growth in H. neptunium is distinct from that in related, polarly growing members of the order Rhizobiales, setting the stage for in-depth analyses of the molecular principles regulating the fascinating developmental cycle of this species.

ß-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR.

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly ß-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified ß-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of ß-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a ß-helical architecture, in which 18 ß-strand segments are arranged in six consecutive windings of a ß-helix.

Mutations targeting the plug-domain of the Shewanella oneidensis proton-driven stator allow swimming at increased viscosity and under anaerobic conditions.

Shewanella oneidensis MR-1 possesses two different stator units to drive flagellar rotation, the Na+ -dependent PomAB stator and the H+ -driven MotAB stator, the latter possibly acquired by lateral gene transfer. Although either stator can independently drive swimming through liquid, MotAB-driven motors cannot support efficient motility in structured environments or swimming under anaerobic conditions. Using ΔpomAB cells we isolated spontaneous mutants able to move through soft agar. We show that a mutation that alters the structure of the plug domain in MotB affects motor functions and allows cells to swim through media of increased viscosity and under anaerobic conditions. The number and exchange rates of the mutant stator around the rotor were not significantly different from wild-type stators, suggesting that the number of stators engaged is not the cause of increased swimming efficiency. The swimming speeds of planktonic mutant MotAB-driven cells was reduced, and overexpression of some of these stators caused reduced growth rates, implying that mutant stators not engaged with the rotor allow some proton leakage. The results suggest that the mutations in the MotB plug domain alter the proton interactions with the stator ion channel in a way that both increases torque output and allows swimming at decreased pmf values.

Molecular toolbox for genetic manipulation of the stalked budding bacterium Hyphomonas neptunium.

The alphaproteobacterium Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions in H. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species.

Function and localization dynamics of bifunctional penicillin-binding proteins in Caulobacter crescentus.

The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are cross-linked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide cross-links. The stalked model bacterium Caulobacter crescentus possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY, and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of C. crescentus against the noncanonical amino acid d-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of C. crescentus are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis, and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.

Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus.

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.