A minimally invasive, field-applicable CRISPR/Cas biosensor to aid in the detection of Pseudogymnoascus destructans, the causative fungal agent of white-nose syndrome in bats.

The accessibility to CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) genetic tools has given rise to applications beyond site-directed genome editing for the detection of DNA and RNA. These tools include precise diagnostic detection of human disease pathogens, such as SARS-CoV-2 and Zika virus. Despite the technology being rapid and cost-effective, the use of CRISPR/Cas tools in the surveillance of the causative agents of wildlife diseases has not been prominent. This study presents the development of a minimally invasive, field-applicable and user-friendly CRISPR/Cas-based biosensor for the detection of Pseudogymnoascus destructans (Pd), the causative fungal agent of white-nose syndrome (WNS), an infectious disease that has killed more than five million bats in North America since its discovery in 2006. The biosensor assay combines a recombinase polymerase amplification (RPA) step followed by CRISPR/Cas12a nuclease cleavage to detect Pd DNA from bat dermal swab and guano samples. The biosensor had similar detection results when compared to quantitative PCR in distinguishing Pd-positive versus negative field samples. Although bat dermal swabs could be analysed with the biosensor without nucleic acid extraction, DNA extraction was needed when screening guano samples to overcome inhibitors. This assay can be applied to help with more rapid delineation of Pd-positive sites in the field to inform management decisions. With further optimization, this technology has broad translation potential to wildlife disease-associated pathogen detection and monitoring applications.

Click-Ready Perfluorocarbon Nanoemulsion for 19F MRI and Multimodal Cellular Detection.

We describe an in vivo imaging probe platform that is readily modifiable to accommodate binding of different molecular targeting moieties and payloads for multimodal image generation. In this work, we demonstrate the utility of perfluorocarbon (PFC) nanoemulsions incorporating dibenzocyclooctyne (DBCO) by enabling postemulsification functionalization via a click reaction with azide-containing ligands. The addition of DBCO-lipid to the surfactant in PFC nanoemulsions did not affect nanoemulsion size or nanoemulsion stability. As proof-of-concept, fluorescent dye-azides were conjugated to PFC nanoemulsions, demonstrating the feasibility of functionalization the by click reaction. Uptake of the fluorescent PFC by macrophages was demonstrated both in vitro in cultured macrophages and in situ in an acute inflammation mouse model, where fluorescence imaging and 1H/19F magnetic resonance imaging (MRI) were used for in vivo detection. Overall, these data demonstrate the potential of PFC nanoemulsions incorporating DBCO as a versatile platform for generating functionalized probes.

On the use of oxygenic photosynthesis for the sustainable production of commodity chemicals.

A sustainable society will have to largely refrain from the use of fossil carbon deposits. In such a regime, renewable electricity can be harvested as a primary source of energy. However, as for the synthesis of carbon-based materials from bulk chemicals, an alternative is required. A sustainable approach towards this is the synthesis of commodity chemicals from CO2 , water and sunlight. Multiple paths to achieve this have been designed and tested in the domains of chemistry and biology. In the latter, the use of both chemotrophic and phototrophic organisms has been advocated. 'Direct conversion' of CO2 and H2 O, catalyzed by an oxyphototroph, has excellent prospects to become the most economically competitive of these transformations, because of the relative ease of scale-up of this process. Significantly, for a wide range of energy and commodity products, a proof of principle via engineering of the corresponding production organism has been provided. In the optimization of a cyanobacterial production organism, a wide range of aspects has to be addressed. Of these, here we will put our focus on: (1) optimizing the (carbon) flux to the desired product; (2) increasing the genetic stability of the producing organism and (3) maximizing its energy conversion efficiency. Significant advances have been made on all these three aspects during the past 2 years and these will be discussed: (1) increasing the carbon partitioning to >50%; (2) aligning product formation with the growth of the cells and (3) expanding the photosynthetically active radiation region for oxygenic photosynthesis.

Septin-associated proteins Aim44 and Nis1 traffic between the bud neck and the nucleus in the yeast Saccharomyces cerevisiae.

In budding yeast, a collar of septin filaments at the neck between a mother cell and its bud marks the incipient site for cell division and serves as a scaffold that recruits proteins required for proper spatial and temporal execution of cytokinesis. A set of interacting proteins that localize at or near the bud neck, including Aim44/Gps1, Nba1 and Nis1, also has been implicated in preventing Cdc42-dependent bud site re-establishment at the division site. We found that, at their endogenous level, Aim44 and Nis1 robustly localize sequentially at the septin collar. Strikingly, however, when overproduced, both proteins shift their subcellular distribution predominantly to the nucleus. Aim44 localizes with the inner nuclear envelope, as well as at the plasma membrane, whereas Nis1 accumulates within the nucleus, indicating that these proteins normally undergo nucleocytoplasmic shuttling. Of the 14 yeast karyopherins, Kap123/Yrb4 is the primary importin for Aim44, whereas several importins mediate Nis1 nuclear entry. Conversely, Kap124/Xpo1/Crm1 is the primary exportin for Nis1, whereas both Xpo1 and Cse1/Kap109 likely contribute to Aim44 nuclear export. Even when endogenously expressed, Nis1 accumulates in the nucleus when Nba1 is absent. When either Aim44 or Nis1 are overexpressed, Nba1 is displaced from the bud neck, further consistent with the mutual interactions of these proteins. Collectively, our results indicate that a previously unappreciated level at which localization of septin-associated proteins is controlled is via regulation of their nucleocytoplasmic shuttling, which places constraints on their availability for complex formation with other partners at the bud neck.

One Environmental Health: an emerging perspective in toxicology.

The One Environmental Health research approach, a subspecialty of the One Health initiative, focuses on toxic chemicals. Distinct disciplines work together to give a holistic perspective of a health concern through discrete disciplines, including, but not limited to, public health and the medical and veterinary sciences. In this article, we illustrate the concept of One Environmental Health with two case studies. One case study focuses on alligators and contributions to the field of endocrine disruption. The other case study focuses on whales and contributions to understanding carcinogenic metals. Both studies illustrate how the health of sentinel organisms has the potential to inform about the health of humans and the ecosystem.

Presence of a [3Fe-4S] cluster in a PsaC variant as a functional component of the photosystem I electron transfer chain in Synechococcus sp. PCC 7002.

A site-directed C14G mutation was introduced into the stromal PsaC subunit of Synechococcus sp. strain PCC 7002 in vivo in order to introduce an exchangeable coordination site into the terminal FB [4Fe-4S] cluster of Photosystem I (PSI). Using an engineered PSI-less strain (psaAB deletion), psaC was deleted and replaced with recombinant versions controlled by a strong promoter, and the psaAB deletion was complemented. Modified PSI accumulated at lower levels in this strain and supported slower photoautotrophic growth than wild type. As-isolated PSI complexes containing PsaCC14G showed resonances with g values of 2.038 and 2.007 characteristic of a [3Fe-4S]1+ cluster. When the PSI complexes were illuminated at 15 K, these resonances partially disappeared and two new sets of resonances appeared. The majority set had g values of 2.05, 1.95, and 1.85, characteristic of FA-, and the minority set had g values of 2.11, 1.90, and 1.88 from FB' in the modified site. The S = 1/2 spin state of the latter implied the presence of a thiolate as the terminal ligand. The [3Fe-4S] clusters could be partially reconstituted with iron, producing a larger population of [4Fe-4S] clusters. Rates of flavodoxin reduction were identical in PSI complexes isolated from wild type and the PsaCC14G variant strain; this implied equivalent capacity for forward electron transfer in PSI complexes that contained [3Fe-4S] and [4Fe-4S] clusters. The development of this cyanobacterial strain is a first step toward translation of in vitro PSI-based biosolar molecular wire systems in vivo and provides new insights into the formation of Fe/S clusters.

Super-Resolution Microscopy and Single-Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule.

Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. Here, we describe the use of a genetically encoded photostable fluoromodule that can be targeted to cytosolic and membrane proteins in the Gram negative bacterium Caulobacter crescentus. Additionally, we summarize methods to use this fluoromodule for single protein imaging and super-resolution microscopy using stimulated emission depletion. © 2017 by John Wiley & Sons, Inc.

A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity.

Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment.IMPORTANCE Lacking internal membrane-bound compartments, bacteria achieve subcellular organization by establishing self-assembling protein-based microdomains. The asymmetrically dividing bacterium Caulobacter crescentus uses one such microdomain to link cell cycle progression to morphogenesis, but the mechanism for the generation of this microdomain has remained unclear. Here, we demonstrate that the ordered assembly of this microdomain occurs via the polymeric network protein PopZ directly recruiting the polarity factor SpmX, which then recruits the histidine kinase DivJ to the developing cell pole. Further, we find that overexpression of the bridge protein SpmX in Caulobacter disrupts this ordered assembly, generating ectopic cell poles containing both PopZ and DivJ. Together, PopZ and SpmX assemble into a cooligomeric network that forms the basis for a polar microdomain that coordinates bacterial cell polarity.

Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor.

Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn2+-inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn2+ uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA7942) and its cognate SmtB7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA6803) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster ß-carotene 15,15'-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002. IMPORTANCE: This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn2+-inducible system, which is based on the smtA7942 operator/promoter and smtB7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae.

Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast Saccharomyces cerevisiae is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop.

Dynamic translation regulation in Caulobacter cell cycle control.

Progression of the Caulobacter cell cycle requires temporal and spatial control of gene expression, culminating in an asymmetric cell division yielding distinct daughter cells. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. Translational efficiency (TE) was used as a metric for the relative rate of protein production from each mRNA. TE profiles with similar cell cycle patterns were found across multiple clusters of genes, including those in operons or in subsets of operons. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. The cell cycle-regulatory pathways that produce specific temporal TE patterns are separate from-but highly coordinated with-the transcriptional cell cycle circuitry, suggesting that the scheduling of translational regulation is organized by the same cyclical regulatory circuit that directs the transcriptional control of the Caulobacter cell cycle.

Super-resolution Imaging of Live Bacteria Cells Using a Genetically Directed, Highly Photostable Fluoromodule.

The rapid development in fluorescence microscopy and imaging techniques has greatly benefited our understanding of the mechanisms governing cellular processes at the molecular level. In particular, super-resolution microscopy methods overcome the diffraction limit to observe nanoscale cellular structures with unprecedented detail, and single-molecule tracking provides precise dynamic information about the motions of labeled proteins and oligonucleotides. Enhanced photostability of fluorescent labels (i.e., maximum emitted photons before photobleaching) is a critical requirement for achieving the ultimate spatio-temporal resolution with either method. While super-resolution imaging has greatly benefited from highly photostable fluorophores, a shortage of photostable fluorescent labels for bacteria has limited its use in these small but relevant organisms. In this study, we report the use of a highly photostable fluoromodule, dL5, to genetically label proteins in the Gram-negative bacterium Caulobacter crescentus, enabling long-time-scale protein tracking and super-resolution microscopy. dL5 imaging relies on the activation of the fluorogen Malachite Green (MG) and can be used to label proteins sparsely, enabling single-protein detection in live bacteria without initial bleaching steps. dL5-MG complexes emit 2-fold more photons before photobleaching compared to organic dyes such as Cy5 and Alexa 647 in vitro, and 5-fold more photons compared to eYFP in vivo. We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion microscopy, yielding a 4-fold resolution enhancement compared to diffraction-limited imaging. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. To the best of our knowledge, this is the first demonstration of the use of fluorogen activating proteins for super-resolution imaging in live bacterial cells.

Complementation of Cobalamin Auxotrophy in Synechococcus sp. Strain PCC 7002 and Validation of a Putative Cobalamin Riboswitch In Vivo.

UNLABELLED: The euryhaline cyanobacterium Synechococcus sp. strain PCC 7002 has an obligate requirement for exogenous vitamin B12 (cobalamin), but little is known about the roles of this compound in cyanobacteria. Bioinformatic analyses suggest that only the terminal enzyme in methionine biosynthesis, methionine synthase, requires cobalamin as a coenzyme in Synechococcus sp. strain PCC 7002. Methionine synthase (MetH) catalyzes the transfer of a methyl group from N(5)-methyl-5,6,7,8-tetrahydrofolate to l-homocysteine during l-methionine synthesis and uses methylcobalamin as an intermediate methyl donor. Numerous bacteria and plants alternatively employ a cobalamin-independent methionine synthase isozyme, MetE, that catalyzes the same methyl transfer reaction as MetH but uses N(5)-methyl-5,6,7,8-tetrahydrofolate directly as the methyl donor. The cobalamin auxotrophy of Synechococcus sp. strain PCC 7002 was complemented by using the metE gene from the closely related cyanobacterium Synechococcus sp. strain PCC 73109, which possesses genes for both methionine synthases. This result suggests that methionine biosynthesis is probably the sole use of cobalamin in Synechococcus sp. strain PCC 7002. Furthermore, a cobalamin-repressible gene expression system was developed in Synechococcus sp. strain PCC 7002 that was used to validate the presence of a cobalamin riboswitch in the promoter region of metE from Synechococcus sp. strain PCC 73109. This riboswitch acts as a cobalamin-dependent transcriptional attenuator for metE in that organism. IMPORTANCE: Synechococcus sp. strain PCC 7002 is a cobalamin auxotroph because, like eukaryotic marine algae, it uses a cobalamin-dependent methionine synthase (MetH) for the final step of l-methionine biosynthesis but cannot synthesize cobalamin de novo Heterologous expression of metE, encoding cobalamin-independent methionine synthase, from Synechococcus sp. strain PCC 73109, relieved this auxotrophy and enabled the construction of a truly autotrophic Synechococcus sp. strain PCC 7002 more suitable for large-scale industrial applications. Characterization of a cobalamin riboswitch expands the genetic toolbox for Synechococcus sp. strain PCC 7002 by providing a cobalamin-repressible expression system.

Identification and Regulation of Genes for Cobalamin Transport in the Cyanobacterium Synechococcus sp. Strain PCC 7002.

UNLABELLED: The cyanobacterium Synechococcus sp. strain PCC 7002 is a cobalamin auxotroph and utilizes this coenzyme solely for the synthesis of l-methionine by methionine synthase (MetH). Synechococcus sp. strain PCC 7002 is unable to synthesize cobalamin de novo, and because of the large size of this tetrapyrrole, an active-transport system must exist for cobalamin uptake. Surprisingly, no cobalamin transport system was identified in the initial annotation of the genome of this organism. With more sophisticated in silico prediction tools, a btuB-cpdA-btuC-btuF operon encoding components putatively required for a B12 uptake (btu) system was identified. The expression of these genes was predicted to be controlled by a cobalamin riboswitch. Global transcriptional profiling by high-throughput RNA sequencing of a cobalamin-independent form of Synechococcus sp. strain PCC 7002 grown in the absence or presence of cobalamin confirmed regulation of the btu operon by cobalamin. Pérez et al. (A. A. Pérez, Z. Liu, D. A. Rodionov, Z. Li, and D. A. Bryant, J Bacteriol 198:2743-2752, 2016, http://dx.doi.org/10.1128/JB.00475-16) developed a cobalamin-dependent yellow fluorescent protein reporter system in a Synechococcus sp. strain PCC 7002 variant that had been genetically modified to allow cobalamin-independent growth. This reporter system was exploited to validate components of the btu uptake system by assessing the ability of targeted mutants to transport cobalamin. The btuB promoter and a variant counterpart mutated in an essential element of the predicted cobalamin riboswitch were fused to a yfp reporter. The combined data indicate that the btuB-cpdA-btuF-btuC operon in this cyanobacterium is transcriptionally regulated by a cobalamin riboswitch. IMPORTANCE: With a cobalamin-regulated reporter system for expression of yellow fluorescent protein, genes previously misidentified as encoding subunits of a siderophore transporter were shown to encode components of cobalamin uptake in the cyanobacterium Synechococcus sp. strain PCC 7002. This study demonstrates the importance of experimental validation of in silico predictions and provides a general scheme for in vivo verification of similar cobalamin transport systems. A putative cobalamin riboswitch was identified in Synechococcus sp. strain PCC 7002. This riboswitch acts as a potential transcriptional attenuator of the btu operon that encodes the components of the cobalamin active-transport system.

Electron transfer from the A1A and A1B sites to a tethered Pt nanoparticle requires the FeS clusters for suppression of the recombination channel.

In this work, a previously described model of electron withdrawal from the A1A/A1B sites of Photosystem I (PS I) was tested using a dihydrogen-producing PS I-NQ(CH2)15S-Pt nanoconstruct. According to this model, the rate of electron transfer from A1A/A1B to a tethered Pt nanoparticle is kinetically unfavorable relative to the rate of forward electron transfer to the FeS clusters. Dihydrogen is produced only when an external donor rapidly reduces P700(+), thereby suppressing the recombination channel and allowing the electron in the FeS clusters to proceed via uphill electron transfer through the A1A/A1B quinones to the Pt nanoparticle. We tested this model by sequentially removing the FeS clusters, FB, FA, and FX, and determining the concentration of cytochrome c6 (Cyt c6) at which the backreaction was outcompeted and dihydrogen production was observed. P700-FA cores were generated in a menB insertionally inactivated strain by removing FB with HgCl2; P700-FX cores were generated in a menB psaC insertionally inactivated strain that lacks FA and FB, and P700-A1 cores were generated in a menB rubA insertionally inactivated strain that lacks FX, FA and FB. Quinone incorporation was measured using transient electron paramagnetic resonance spectroscopy and time resolved optical spectroscopy. Cyt c6 was titrated into each of these PS I preparations and the kinetics of P700(+) reduction were measured. A similar experiment was carried out on PS I-NQ(CH2)15S-Pt nanoconstructs assembled from these PS I preparations. This study showed that the concentration of Cyt c6 needed to produce dihydrogen was comparable to that needed to suppress the backreaction. We conclude that the FeS clusters serve to 'park' the electron and thereby extend the duration of the charge-separated state; however, in doing so, the redox advantage of removing the electron at A1A/A1B is lost.

Bacterial scaffold directs pole-specific centromere segregation.

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.

Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold.

In Caulobacter crescentus, the PopZ polar scaffold protein supports asymmetric cell division by recruiting distinct sets of binding partners to opposite cell poles. To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub-cellular localization and recruitment activity. We identified a domain within the C-terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N-terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. Mutations that inhibited dynamic PopZ localization inhibited the recruitment of other factors to cell poles. Mutations in the C-terminal domain also blocked discrete steps in the assembly of higher-order structures. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into filaments, and these structures are important for sub-cellular localization. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter.