Loss of PopZ At activity in Agrobacterium tumefaciens by Deletion or Depletion Leads to Multiple Growth Poles, Minicells, and Growth Defects.

Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ At ), we created deletions of the five PopZ At domains and assayed their localization. In addition, we created a popZAt deletion strain (ΔpopZAt ) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ At strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAt cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ At , to stabilize growth pole accumulation of PopZ AtIMPORTANCEAgrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZ At , a homologue of Caulobacter crescentus PopZ (PopZ Cc ), a protein essential in many aspects of pole identity in C. crescentus We report that the loss of PopZ At leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process in A. tumefaciens may lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies of A. tumefaciens will provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division

The material properties of a bacterial-derived biomolecular condensate tune biological function in natural and synthetic systems

Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells

A Self-Associating Protein Critical for Chromosome Attachment, Division, and Polar Organization in Caulobacter

Cell polarization is an integral part of many unrelated bacterial processes. How intrinsic cell polarization is achieved is poorly understood. Here, we provide evidence that Caulobacter crescentus uses a multimeric pole-organizing factor (PopZ) that serves as a hub to concurrently achieve several polarizing functions. During chromosome segregation, polar PopZ captures the ParB•ori complex and thereby anchors sister chromosomes at opposite poles. This step is essential for stabilizing bipolar gradients of a cell division inhibitor and setting up division near midcell. PopZ also affects polar stalk morphogenesis and mediates the polar localization of the morphogenetic and cell cycle signaling proteins CckA and DivJ. Polar accumulation of PopZ, which is central to its polarizing activity, can be achieved independently of division and does not appear to be dictated by the pole curvature. Instead, evidence suggests that localization of PopZ largely relies on PopZ multimerization in chromosome-free regions, consistent with a self-organizing mechanism

PopZ and FtsZ coordinate polar growth termination and cell division in Agrobacterium tumefaciens

Understanding how bacterial cells expand their cell walls is an important question with relevance to development of antibiotics. While many studies have focused on the regulation of bacterial elongation utilizing lateral cell wall biogenesis, polar growth in bacteria is less well understood. Yet, polar growth has been observed across taxonomically diverse bacteria like Actinobacteria and the alphaproteobacterial clade Rhizobiales (Howell and Brown, 2016). Interestingly, polar-growing bacteria within Rhizobiales lack canonical scaffolding proteins for spatial and temporal regulation of peptidoglycan synthesis during elongation. Here, we dissect the role of two candidate scaffolding proteins in directing cell wall synthesis in the bacterial plant pathogen, Agrobacterium tumefaciens. Since cell wall (peptidoglycan) biosynthesis during elongation and cell division is vital for bacterial survival, we expected many key proteins involved in these processes to be essential for cell survival. Thus, we developed a depletion system for A. tumefaciens (Figureroa-Cuilan et al. 2016). We further optimized a suite of target-specific fluorescent labeling techniques which allow us to visualize morphological changes during essential cell processes (Howell, Daniel, and Brown, 2017). We use these techniques to dissect the contributions of PopZ and FtsZ to polar growth and cell division. Although PopZ is not required for polar growth, it is required for proper coordination of polar growth, chromosome segregation, and cell division. This PopZ-mediated coordination ensures that daughter cells are the proper size and contain a complete complement of genetic material (Howell et al 2017). Next, we find that FtsZ is required for both termination of polar growth and cell division. This finding suggests that FtsZ has at least two important functions in regulation of cell wall biogenesis. First, FtsZ enables cell wall biogenesis machinery to be released or inactivated from the growth pole. Second, FtsZ must recruit additional proteins to mid cell to assemble the divisome, enabling activation of cell wall biogenesis to promote septum formation and cell separation. While further research is needed to understand how growth is targeted to the pole during elongation, our work provides mechanistic insights about the coordination of polar growth termination, chromosome segregation, and cell division. We hypothesize that our findings will be applicable to other closely related polar growing Rhizobiales, including plant, animal, and human pathogens.Includes bibliographical reference

Chromosome arrangement and dynamics in the budding bacterium Hyphomonas neptunium

Faithful chromosome replication and segregation are essential for every living cell and must be tightly coordinated with other cell cycle events such as cell division. Our knowledge about prokaryotic chromosome dynamics is based on studies of only a few model organisms that divide by binary fission and are mostly characterized by a rod-like morphology. To broaden our insight into bacterial chromosome segregation, our lab has recently started to analyze chromosome dynamics in the marine alphaproteobacterium Hyphomonas neptunium, which divides by budding at the tip of the stalk and uses its stalk as a reproductive structure. This mode of reproduction distinguishes H. neptunium from so far studied model organisms and renders it an exciting candidate for the study of chromosome dynamics, since the duplicated chromosome must transit the stalk to reach the newly generated daughter cell. Recent work has revealed that the H. neptunium chromosome is segregated in a unique two-step process. At first, one of the duplicated origins is segregated within the mother cell, possibly in a ParABS-dependent manner, and remains at the stalked mother cell pole until a visible bud has formed at the tip of the stalk. In a second step, it is then segregated through the stalk into the bud. Several lines of evidence suggest that the transport through the stalk is mediated by a novel, yet unidentified, segregation mechanism. Commonly, chromosome replication and segregation occur concomitantly in bacteria. However, this two-step segregation mechanism implies a temporal uncoupling of chromosome replication and segregation through the stalk, reminiscent of eukaryotic mitosis. In this work, we analyzed the role of the ParABS system in chromosome segregation of H. neptunium. The ParABS system was shown to be essential for cell viability and chromosome segregation. Impairment of ParA functioning leads to morphological alterations and incomplete origin segregation within the mother cell and, consequently, hampers chromosome segregation through the stalk. This shows that the ParABS system mediates origin segregation within the mother cell. It also implies that chromosome segregation within the mother cell and through the stalk are sequential processes. Furthermore, we analyzed the role of PopZ and SMC in H. neptunium, since these proteins were shown to be involved in chromosome segregation in other bacteria. PopZ localizes to the pole opposite the stalk in the newly generated bud and appears to play only a minor role in the positioning of the ParABS partitioning machinery. SMC seems to be essential in H. neptunium and shows a similar localization pattern as ParB. Determination of the location of seven genomic loci in new-born cells revealed that the chromosome shows a longitudinal arrangement with the origin located at the flagellated pole and the terminus at the opposite cell pole. The other loci are arranged between both cell poles in a linear order that correlates with their position on the genomic map. Moreover, analysis of chromosome dynamics indicates that the ParB/parS complex is the region to be segregated first within the mother cell and also through the stalk, emphasizing its central role in the segregation process. As mentioned above, the observed two-step chromosome segregation mechanism suggested a temporal uncoupling of chromosome replication and its segregation through the stalk. To investigate the coordination between these two processes in more detail, we followed replisome dynamics by fluorescence labeling of different replisome components. The replication machinery shows a dynamic localization within the mother cell: in cells that are most likely at the swarmer-to-stalked cell transition as well as in stalked cells, it assembles at the pole opposite the (future) stalk and moves, via midcell, close to the stalked cell pole, where it disassembles again. This localization pattern is consistent with the observed location of the origin and terminus region. Furthermore, the replisomes appear to track independently along the two chromosome arms. Co-localization of ParB (origin) and DnaN (replisome) revealed that a large part of the chromosome is replicated before its segregation through the stalk commences, indicating that these processes are partially temporally uncoupled. Collectively, these observations expand our insight into chromosome dynamics in H. neptunium and suggest that it combines previously described segregation mechanisms, such as the ParABS system, with a novel segregation mechanism that awaits discovery

Biochemistry of the key spatial regulators MipZ and PopZ in Caulobacter crescentus

Bacteria are known to tightly control the spatial distribution of certain proteins by positioning them to distinct regions of the cell, particularly the cell poles. These regions represent important organizing platforms for several processes essential for bacterial survival and reproduction. The proteins localized at the cell poles are recruited to these positions by interaction with other polar proteins or protein complexes. The α-proteobacterium Caulobacter crescentus possesses a self-organizing polymeric polar matrix constituted of the scaffolding protein PopZ. PopZ recruits to the cell poles several proteins involved in various essential processes such as chromosome segregation and the regulation of cell division. This latter process is controlled by the spatial regulator MipZ, which coordinates chromosome segregation with cell division. The two essential proteins PopZ and MipZ both physically interact with the centromere binding protein ParB, an essential element of the chrosomome segregation system of Caulobacter crescentus. The main function of the ATPase MipZ is to position the cell division apparatus by spatially restricting the localization of the key cell division protein FtsZ to midcell. MipZ accomplishes this function by interacting with chromosomal DNA and forming a shallow gradient, with a high concentration at the cell poles and a low concentration near the midcell, therefore permitting FtsZ polymerization solely at midcell. The formation of the MipZ bipolar gradient is intimately linked to the establishment of the multimeric matrix PopZ at the cell poles, which insures the anchorage of ParB-parS complexes at the cell poles. In this study, we have uncovered the inhibitory mode of action of the polar element MipZ on FtsZ polymerization and identified the interaction regions of MipZ with its three interaction partners, ParB, FtsZ and the chromosomal DNA. We found that similarly to the FtsZ assembly inhibitor from Escherichia coli MinC, MipZ is capable of inhibiting FtsZ polymerization as well as shortening FtsZ polymers into smaller oligomers. Our results show also that the inhibitory effect of MipZ on FtsZ polymerization is independent of its ability to stimulate the FtsZ GTPase activity. Mapping of the binding interfaces of MipZ revealed that the DNA- and ParB-binding regions are overlapping and mainly constituted of positively charged residues, whereas two distinct regions appear to be involved in FtsZ-binding. We also purified the polar factor PopZ from soluble fractions and provided relevant data related to its secondary structure composition and its assembly into higher-order structures. Our in vitro analysis on PopZ, revealed among others that it is mainly composed of α-helices and unstructured regions and forms relatively straight filament-like structures differing from what was previously reported. Altogether, the data obtained in this work bring more knowledge about two key elements of C. crescentus

Regulatory Mechanisms of a Bacterial Multi-Kinase Network

Cells sense and respond to their environment through signaling pathways which often require processing several signals prior to implementing a biological response. Bacterial signaling pathways are responsible for processes such as virulence, biofilm formation, survival, and symbiosis that are of research interest for medical, environmental, and industrial advancements. Emerging discoveries suggest that the systems that control these responses are more complex and intertwined than the previously understood two-component systems. Proteins such as scaffolds and pseudokinases regulate the localization, activity, and timing of the phosphotransfer reactions that dictate cellular decisions. This dissertation describes regulatory mechanisms of a multi-kinase network that controls asymmetric division in the model bacterium C. crescentus. It has been proposed that the novel pseudokinase DivL reverses signal flow by exploiting conserved kinase conformational changes and protein-protein interactions. Chapter 2 describes the development and characterization of a series of DivL-based modulators to synthetically stimulate reverse signaling of the network in vivo. I propose that synthetic stimulation and sensor disruption provide strategies to define signaling circuit organization principles for the rational design and validation of synthetic pathways. In Chapter 3, I further dissect the roles of each DivL domain on subcellular localization and downstream activity. While not catalytically active, pseudokinases have been repurposed to serve functions including complex signal recognition, integration, competition, and intermolecular allostery. I provide a refined model detailing how DivL plays each of these parts within its broader network. The work in Chapter 3 also revealed multiple scaffolding interactions that orchestrate the multi-kinase network in time and space. In Chapter 4 I identify factors that lead to the accumulation of two biochemically distinct signaling hubs at opposite cell poles to provide the foundation for asymmetry. I also provide evidence that a scaffold not only recruits a key signaling protein to the correct location but mediates its switch between kinase and phosphatase activities that drives the cell cycle. In each chapter, I discuss questions that remain and suggest future directions for study. Overall, this dissertation contributes strategies that can be used to interrogate other relevant multi-kinase networks in bacteria