Zhao, Evan M, et al.Light-based control of metabolic flux through assembly of synthetic organelles”. Nat Chem Biol 15.6 (2019): , 15, 6, 589-597. Web.Abstract
To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
Sanfilippo, Joseph E, et al.Microfluidic-based transcriptomics reveal force-independent bacterial rheosensing”. Nat Microbiol (2019). Web.Abstract
Multiple cell types sense fluid flow as an environmental cue. Flow can exert shear force (or stress) on cells, and the prevailing model is that biological flow sensing involves the measurement of shear force. Here, we provide evidence for force-independent flow sensing in the bacterium Pseudomonas aeruginosa. A microfluidic-based transcriptomic approach enabled us to discover an operon of P. aeruginosa that is rapidly and robustly upregulated in response to flow. Using a single-cell reporter of this operon, which we name the flow-regulated operon (fro), we establish that P. aeruginosa dynamically tunes gene expression to flow intensity through a process we call rheosensing (as rheo- is Greek for flow). We further show that rheosensing occurs in multicellular biofilms, involves signalling through the alternative sigma factor FroR, and does not require known surface sensors. To directly test whether rheosensing measures force, we independently altered the two parameters that contribute to shear stress: shear rate and solution viscosity. Surprisingly, we discovered that rheosensing is sensitive to shear rate but not viscosity, indicating that rheosensing is a kinematic (force-independent) form of mechanosensing. Thus, our findings challenge the dominant belief that biological mechanosensing requires the measurement of forces.
Hsin-Jung Li, Sophia, et al.Escherichia coli translation strategies differ across carbon, nitrogen and phosphorus limitation conditions”. Nat Microbiol 38 (2018): , 3, 8, 939-947. Web.Abstract
For cells to grow faster they must increase their protein production rate. Microorganisms have traditionally been thought to accomplish this increase by producing more ribosomes to enhance protein synthesis capacity, leading to the linear relationship between ribosome level and growth rate observed under most growth conditions previously examined. Past studies have suggested that this linear relationship represents an optimal resource allocation strategy for each growth rate, independent of any specific nutrient state. Here we investigate protein production strategies in continuous cultures limited for carbon, nitrogen and phosphorus, which differentially impact substrate supply for protein versus nucleic acid metabolism. Unexpectedly, we find that at slow growth rates, Escherichia coli achieves the same protein production rate using three different strategies under the three different nutrient limitations. Under phosphorus (P) limitation, translation is slow due to a particularly low abundance of ribosomes, which are RNA-rich and thus particularly costly for phosphorous-limited cells. Under nitrogen (N) limitation, translation elongation is slowed by processes including ribosome stalling at glutamine codons. Under carbon (C) limitation, translation is slowed by accumulation of inactive ribosomes not bound to messenger RNA. These extra ribosomes enable rapid growth acceleration during nutrient upshift. Thus, bacteria tune ribosome usage across different limiting nutrients to enable balanced nutrient-limited growth while also preparing for future nutrient upshifts.
Shi, Handuo, et al.How to Build a Bacterial Cell: MreB as the Foreman of E. coli Construction”. Cell 172.6 (2018): , 172, 6, 1294-1305. Web.Abstract
Cell shape matters across the kingdoms of life, and cells have the remarkable capacity to define and maintain specific shapes and sizes. But how are the shapes of micron-sized cells determined from the coordinated activities of nanometer-sized proteins? Here, we review general principles that have surfaced through the study of rod-shaped bacterial growth. Imaging approaches have revealed that polymers of the actin homolog MreB play a central role. MreB both senses and changes cell shape, thereby generating a self-organizing feedback system for shape maintenance. At the molecular level, structural and computational studies indicate that MreB filaments exhibit tunable mechanical properties that explain their preference for certain geometries and orientations along the cylindrical cell body. We illustrate the regulatory landscape of rod-shape formation and the connectivity between cell shape, cell growth, and other aspects of cell physiology. These discoveries provide a framework for future investigations into the architecture and construction of microbes.
Trivedi, Rishi R, et al.Mechanical Genomic Studies Reveal the Role of d-Alanine Metabolism in Pseudomonas aeruginosa Cell Stiffness”. MBio 95 (2018). Web.Abstract
The stiffness of bacteria prevents cells from bursting due to the large osmotic pressure across the cell wall. Many successful antibiotic chemotherapies target elements that alter mechanical properties of bacteria, and yet a global view of the biochemistry underlying the regulation of bacterial cell stiffness is still emerging. This connection is particularly interesting in opportunistic human pathogens such as that have a large (80%) proportion of genes of unknown function and low susceptibility to different families of antibiotics, including beta-lactams, aminoglycosides, and quinolones. We used a high-throughput technique to study a library of 5,790 loss-of-function mutants covering ~80% of the nonessential genes and correlated individual genes with cell stiffness. We identified 42 genes coding for proteins with diverse functions that, when deleted individually, decreased cell stiffness by >20%. This approach enabled us to construct a "mechanical genome" for d-Alanine dehydrogenase (DadA) is an enzyme that converts d-Ala to pyruvate that was included among the hits; when DadA was deleted, cell stiffness decreased by 18% (using multiple assays to measure mechanics). An increase in the concentration of d-Ala in cells downregulated the expression of genes in peptidoglycan (PG) biosynthesis, including the peptidoglycan-cross-linking transpeptidase genes and Consistent with this observation, ultraperformance liquid chromatography-mass spectrometry analysis of murein from cells revealed that deletion mutants contained PG with reduced cross-linking and altered composition compared to wild-type cells. The mechanical properties of bacteria are important for protecting cells against physical stress. The cell wall is the best-characterized cellular element contributing to bacterial cell mechanics; however, the biochemistry underlying its regulation and assembly is still not completely understood. Using a unique high-throughput biophysical assay, we identified genes coding proteins that modulate cell stiffness in the opportunistic human pathogen This approach enabled us to discover proteins with roles in a diverse range of biochemical pathways that influence the stiffness of cells. We demonstrate that d-Ala-a component of the peptidoglycan-is tightly regulated in cells and that its accumulation reduces expression of machinery that cross-links this material and decreases cell stiffness. This research demonstrates that there is much to learn about mechanical regulation in bacteria, and these studies revealed new nonessential targets that may enhance antibacterial chemotherapies or lead to new approaches.
Bratton, Benjamin P, et al.MreB polymers and curvature localization are enhanced by RodZ and predict E. coli's cylindrical uniformity”. Nat Commun 91 (2018): , 9, 1, 2797. Web.Abstract
The actin-like protein MreB has been proposed to coordinate the synthesis of the cell wall to determine cell shape in bacteria. MreB is preferentially localized to areas of the cell with specific curved geometries, avoiding the cell poles. It remains unclear whether MreB's curvature preference is regulated by additional factors, and which specific features of MreB promote specific features of rod shape growth. Here, we show that the transmembrane protein RodZ modulates MreB curvature preference and polymer number in E. coli, properties which are regulated independently. An unbiased machine learning analysis shows that MreB polymer number, the total length of MreB polymers, and MreB curvature preference are key correlates of cylindrical uniformity, the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r = 0.93). Our data thus suggest RodZ promotes the assembly of geometrically-localized MreB polymers that lead to the growth of uniform cylinders.
Fröhlich, Kathrin S, Konrad U Förstner, and Zemer Gitai. “Post-transcriptional gene regulation by an Hfq-independent small RNA in Caulobacter crescentus”. Nucleic Acids Res 46.20 (2018): , 46, 20, 10969-10982. Web.Abstract
Bacterial small RNAs (sRNAs) are a heterogeneous group of post-transcriptional regulators that often act at the heart of large networks. Hundreds of sRNAs have been discovered by genome-wide screens and most of these sRNAs exert their functions by base-pairing with target mRNAs. However, studies addressing the molecular roles of sRNAs have been largely confined to gamma-proteobacteria, such as Escherichia coli. Here we identify and characterize a novel sRNA, ChvR, from the alpha-proteobacterium Caulobacter crescentus. Transcription of chvR is controlled by the conserved two-component system ChvI-ChvG and it is expressed in response to DNA damage, low pH, and growth in minimal medium. Transient over-expression of ChvR in combination with genome-wide transcriptome profiling identified the mRNA of the TonB-dependent receptor ChvT as the sole target of ChvR. Genetic and biochemical analyses showed that ChvR represses ChvT at the post-transcriptional level through direct base-pairing. Fine-mapping of the ChvR-chvT interaction revealed the requirement of two distinct base-pairing sites for full target regulation. Finally, we show that ChvR-controlled repression of chvT is independent of the ubiquitous RNA-chaperone Hfq, and therefore distinct from previously reported mechanisms employed by prototypical bacterial sRNAs. These findings have implications for the mechanism and evolution of sRNA function across bacterial species.
Liu, George S, et al.The effect of antibiotics on protein diffusion in the Escherichia coli cytoplasmic membrane”. PLoS One 12.10 (2017): , 12, 10, e0185810. Web.Abstract
Accumulating evidence suggests that molecular motors contribute to the apparent diffusion of molecules in cells. However, current literature lacks evidence for an active process that drives diffusive-like motion in the bacterial membrane. One possible mechanism is cell wall synthesis, which involves the movement of protein complexes in the cell membrane circumferentially around the cell envelope and may generate currents in the lipid bilayer that advectively transport other transmembrane proteins. We test this hypothesis in Escherichia coli using drug treatments that slow cell wall synthesis and measure their effect on the diffusion of the transmembrane protein mannitol permease using fluorescence recovery after photobleaching. We found no clear decrease in diffusion in response to vancomycin and no decrease in response to mecillinam treatment. These results suggest that cell wall synthesis is not an active contributor to mobility in the cytoplasmic membrane.
Lynch, Eric M, et al.Human CTP synthase filament structure reveals the active enzyme conformation”. Nat Struct Mol Biol 24.6 (2017): , 24, 6, 507-514. Web.Abstract
The universally conserved enzyme CTP synthase (CTPS) forms filaments in bacteria and eukaryotes. In bacteria, polymerization inhibits CTPS activity and is required for nucleotide homeostasis. Here we show that for human CTPS, polymerization increases catalytic activity. The cryo-EM structures of bacterial and human CTPS filaments differ considerably in overall architecture and in the conformation of the CTPS protomer, explaining the divergent consequences of polymerization on activity. The structure of human CTPS filament, the first structure of the full-length human enzyme, reveals a novel active conformation. The filament structures elucidate allosteric mechanisms of assembly and regulation that rely on a conserved conformational equilibrium. The findings may provide a mechanism for increasing human CTPS activity in response to metabolic state and challenge the assumption that metabolic filaments are generally storage forms of inactive enzymes. Allosteric regulation of CTPS polymerization by ligands likely represents a fundamental mechanism underlying assembly of other metabolic filaments.
Bartlett, Thomas M, et al.A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis.”. Cell 168.1-2 (2017): , 168, 1-2, 172-185.e15. Web.Abstract
Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.
Habrian, Chris, et al.Inhibition of Escherichia coli CTP Synthetase by NADH and Other Nicotinamides and Their Mutual Interactions with CTP and GTP.”. Biochemistry 55.39 (2016): , 55, 39, 5554-5565. Web.Abstract
CTP synthetases catalyze the last step of pyrimidine biosynthesis and provide the sole de novo source of cytosine-containing nucleotides. As a central regulatory hub, they are regulated by ribonucleotide and enzyme concentration through ATP and UTP substrate availability, CTP product inhibition, GTP allosteric modification, and quaternary structural changes including the formation of CTP-inhibited linear polymers (filaments). Here, we demonstrate that nicotinamide redox cofactors are moderate inhibitors of Escherichia coli CTP synthetase (EcCTPS). NADH and NADPH are the most potent, and the primary inhibitory determinant is the reduced nicotinamide ring. Although nicotinamide inhibition is noncompetitive with substrates, it apparently enhances CTP product feedback inhibition and GTP allosteric regulation. Further, CTP and GTP also enhance each other's effects, consistent with the idea that NADH, CTP, and GTP interact with a common intermediate enzyme state. A filament-blocking mutation that reduces CTP inhibitory effects also reduced inhibition by GTP but not NADH. Protein-concentration effects on GTP inhibition suggest that, like CTP, GTP preferentially binds to the filament. All three compounds display nearly linear dose-dependent inhibition, indicating a complex pattern of cooperative interactions between binding sites. The apparent synergy between inhibitors, in consideration with physiological nucleotide concentrations, points to metabolically relevant inhibition by nicotinamides, and implicates cellular redox state as a regulator of pyrimidine biosynthesis.
Wilson, Maxwell Z, et al.Mode of action and resistance studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl cycle as a proton sink.”. Proc Natl Acad Sci U S A 113.6 (2016): , 113, 6, 1630-5. Web.Abstract
While we have come to appreciate the architectural complexity of microbially synthesized secondary metabolites, far less attention has been paid to linking their structural features with possible modes of action. This is certainly the case with tropodithietic acid (TDA), a broad-spectrum antibiotic generated by marine bacteria that engage in dynamic symbioses with microscopic algae. TDA promotes algal health by killing unwanted marine pathogens; however, its mode of action (MoA) and significance for the survival of an algal-bacterial miniecosystem remains unknown. Using cytological profiling, we herein determine the MoA of TDA and surprisingly find that it acts by a mechanism similar to polyether antibiotics, which are structurally highly divergent. We show that like polyether drugs, TDA collapses the proton motive force by a proton antiport mechanism, in which extracellular protons are exchanged for cytoplasmic cations. The α-carboxy-tropone substructure is ideal for this purpose as the proton can be carried on the carboxyl group, whereas the basicity of the tropylium ion facilitates cation export. Based on similarities to polyether anticancer agents we have further examined TDA's cytotoxicity and find it to exhibit potent, broad-spectrum anticancer activities. These results highlight the power of MoA-profiling technologies in repurposing old drugs for new targets. In addition, we identify an operon that confers TDA resistance to the producing marine bacteria. Bioinformatic and biochemical analyses of these genes lead to a previously unknown metabolic link between TDA/acid resistance and the γ-glutamyl cycle. The implications of this resistance mechanism in the context of the algal-bacterial symbiosis are discussed.
Ouzounov, Nikolay, et al.MreB Orientation Correlates with Cell Diameter in Escherichia coli.”. Biophys J 111.5 (2016): , 111, 5, 1035-43. Web.Abstract
Bacteria have remarkably robust cell shape control mechanisms. For example, cell diameter only varies by a few percent across a given population. The bacterial actin homolog, MreB, is necessary for establishment and maintenance of rod shape although the detailed properties of MreB that are important for shape control remained unknown. In this study, we perturb MreB in two ways: by treating cells with the polymerization-inhibiting drug A22 and by creating point mutants in mreB. These perturbations modify the steady-state diameter of cells over a wide range, from 790 ± 30 nm to 1700 ± 20 nm. To determine which properties of MreB are important for diameter control, we correlated structural characteristics of fluorescently tagged MreB polymers with cell diameter by simultaneously analyzing three-dimensional images of MreB and cell shape. Our results indicate that the helical pitch angle of MreB inversely correlates with the cell diameter of Escherichia coli. Other correlations between MreB and cell diameter are not found to be significant. These results demonstrate that the physical properties of MreB filaments are important for shape control and support a model in which MreB organizes the cell wall growth machinery to produce a chiral cell wall structure and dictate cell diameter.
Inclan, Yuki F, et al.A scaffold protein connects type IV pili with the Chp chemosensory system to mediate activation of virulence signaling in Pseudomonas aeruginosa.”. Mol Microbiol 101.4 (2016): , 101, 4, 590-605. Web.Abstract
Type IV pili (TFP) function as mechanosensors to trigger acute virulence programs in Pseudomonas aeruginosa. On surface contact, TFP retraction activates the Chp chemosensory system phosphorelay to upregulate 3', 5'-cyclic monophosphate (cAMP) production and transcription of virulence-associated genes. To dissect the specific interactions mediating the mechanochemical relay, we used affinity purification/mass spectrometry, directed co-immunoprecipitations in P. aeruginosa, single cell analysis of contact-dependent transcriptional reporters, subcellular localization and bacterial two hybrid assays. We demonstrate that FimL, a Chp chemosensory system accessory protein of unknown function, directly links the integral component of the TFP structural complex FimV, a peptidoglycan binding protein, with one of the Chp system output response regulators PilG. FimL and PilG colocalize at cell poles in a FimV-dependent manner. While PilG phosphorylation is required for TFP function and mechanochemical signaling, it is not required for polar localization or binding to FimL. Phylogenetic analysis reveals other bacterial species simultaneously encode TFP, the Chp system, FimL, FimV and adenylate cyclase homologs, suggesting that surface sensing may be widespread among TFP-expressing bacteria. We propose that FimL acts as a scaffold enabling spatial colocalization of TFP and Chp system components to coordinate signaling leading to cAMP-dependent upregulation of virulence genes on surface contact.
Siryaporn, Albert, et al.Colonization, competition, and dispersal of pathogens in fluid flow networks.”. Curr Biol 25.9 (2015): , 25, 9, 1201-7. Web.Abstract
The colonization of bacteria in complex fluid flow networks, such as those found in host vasculature, remains poorly understood. Recently, it was reported that many bacteria, including Bacillus subtilis [1], Escherichia coli [2], and Pseudomonas aeruginosa [3, 4], can move in the opposite direction of fluid flow. Upstream movement results from the interplay between fluid shear stress and bacterial motility structures, and such rheotactic-like behavior is predicted to occur for a wide range of conditions [1]. Given the potential ubiquity of upstream movement, its impact on population-level behaviors within hosts could be significant. Here, we find that P. aeruginosa communities use a diverse set of motility strategies, including a novel surface-motility mechanism characterized by counter-advection and transverse diffusion, to rapidly disperse throughout vasculature-like flow networks. These motility modalities give P. aeruginosa a selective growth advantage, enabling it to self-segregate from other human pathogens such as Proteus mirabilis and Staphylococcus aureus that outcompete P. aeruginosa in well-mixed non-flow environments. We develop a quantitative model of bacterial colonization in flow networks, confirm our model in vivo in plant vasculature, and validate a key prediction that colonization and dispersal can be inhibited by modifying surface chemistry. Our results show that the interaction between flow mechanics and motility structures shapes the formation of mixed-species communities and suggest a general mechanism by which bacteria could colonize hosts. Furthermore, our results suggest novel strategies for tuning the composition of multi-species bacterial communities in hosts, preventing inappropriate colonization in medical devices, and combatting bacterial infections.
Persat, Alexandre, et al.The mechanical world of bacteria.”. Cell 161.5 (2015): , 161, 5, 988-97. Web.Abstract
In the wild, bacteria are predominantly associated with surfaces as opposed to existing as free-swimming, isolated organisms. They are thus subject to surface-specific mechanics, including hydrodynamic forces, adhesive forces, the rheology of their surroundings, and transport rules that define their encounters with nutrients and signaling molecules. Here, we highlight the effects of mechanics on bacterial behaviors on surfaces at multiple length scales, from single bacteria to the development of multicellular bacterial communities such as biofilms.
Morgenstein, Randy M, et al.RodZ links MreB to cell wall synthesis to mediate MreB rotation and robust morphogenesis.”. Proc Natl Acad Sci U S A 112.40 (2015): , 112, 40, 12510-5. Web.Abstract
The rod shape of most bacteria requires the actin homolog, MreB. Whereas MreB was initially thought to statically define rod shape, recent studies found that MreB dynamically rotates around the cell circumference dependent on cell wall synthesis. However, the mechanism by which cytoplasmic MreB is linked to extracytoplasmic cell wall synthesis and the function of this linkage for morphogenesis has remained unclear. Here we demonstrate that the transmembrane protein RodZ mediates MreB rotation by directly or indirectly coupling MreB to cell wall synthesis enzymes. Furthermore, we map the RodZ domains that link MreB to cell wall synthesis and identify mreB mutants that suppress the shape defect of ΔrodZ without restoring rotation, uncoupling rotation from rod-like growth. Surprisingly, MreB rotation is dispensable for rod-like shape determination under standard laboratory conditions but is required for the robustness of rod shape and growth under conditions of cell wall stress.
Persat, Alexandre, et al.Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa.”. Proc Natl Acad Sci U S A 112.24 (2015): , 112, 24, 7563-8. Web.Abstract
Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.
Persat, Alexandre, and Zemer Gitai. “Bacterial evolution: rewiring modules to get in shape.”. Curr Biol 24.11 (2014): , 24, 11, R522-4. Web.Abstract
Bacterial species take on a wide variety of shapes, but the mechanisms by which specific shapes evolve have remained poorly understood. A recent study demonstrates that two Asticcacaulis species repurposed an ancestral regulatory protein to rewire the modules of stalk regulation, localization, and synthesis, thereby generating new shapes.
Persat, Alexandre, Howard A Stone, and Zemer Gitai. “The curved shape of Caulobacter crescentus enhances surface colonization in flow.”. Nat Commun 5 (2014): , 5, 3824. Web.Abstract
Each bacterial species has a characteristic shape, but the benefits of specific morphologies remain largely unknown. To understand potential functions for cell shape, we focused on the curved bacterium Caulobacter crescentus. Paradoxically, C. crescentus curvature is robustly maintained in the wild but straight mutants have no known disadvantage in standard laboratory conditions. Here we demonstrate that cell curvature enhances C. crescentus surface colonization in flow. Imaging the formation of microcolonies at high spatial and temporal resolution indicates that flow causes curved cells to orient such that they arc over the surface, thereby decreasing the distance between the surface and polar adhesive pili, and orienting pili to face the surface. C. crescentus thus repurposes pilus retraction, typically used for surface motility, for surface attachment. The benefit provided by curvature is eliminated at high flow intensity, raising the possibility that diversity in curvature adapts related species for life in different flow environments.
Castellana, Michele, et al.Enzyme clustering accelerates processing of intermediates through metabolic channeling.”. Nat Biotechnol 32.10 (2014): , 32, 10, 1011-8. Web.Abstract
We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation.
Barry, Rachael M, et al.Large-scale filament formation inhibits the activity of CTP synthetase.”. Elife 3 (2014): , 3, e03638. Web.Abstract
CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.
Siryaporn, Albert, et al.Surface attachment induces Pseudomonas aeruginosa virulence.”. Proc Natl Acad Sci U S A 111.47 (2014): , 111, 47, 16860-5. Web.Abstract
Pseudomonas aeruginosa infects every type of host that has been examined by deploying multiple virulence factors. Previous studies of virulence regulation have largely focused on chemical cues, but P. aeruginosa may also respond to mechanical cues. Using a rapid imaging-based virulence assay, we demonstrate that P. aeruginosa activates virulence in response to attachment to a range of chemically distinct surfaces, suggesting that this bacterial species responds to mechanical properties of its substrates. Surface-activated virulence requires quorum sensing, but activating quorum sensing does not induce virulence without surface attachment. The activation of virulence by surfaces also requires the surface-exposed protein PilY1, which has a domain homologous to a eukaryotic mechanosensor. Specific mutation of the putative PilY1 mechanosensory domain is sufficient to induce virulence in non-surface-attached cells, suggesting that PilY1 mediates surface mechanotransduction. Triggering virulence only when cells are both at high density and attached to a surface—two host-nonspecific cues—explains how P. aeruginosa precisely regulates virulence while maintaining broad host specificity.
Wilson, Maxwell Z, and Zemer Gitai. “Beyond the cytoskeleton: mesoscale assemblies and their function in spatial organization.”. Curr Opin Microbiol 16.2 (2013): , 16, 2, 177-83. Web.Abstract
Recent studies have identified a growing number of mesoscale protein assemblies in both bacterial and eukaryotic cells. Traditionally, these polymeric assemblies are thought to provide structural support for the cell and thus have been classified as the cytoskeleton. However a new class of macromolecular structure is emerging as an organizer of cellular processes that occur on scales hundreds of times larger than a single protein. We propose two types of self-assembling structures, dynamic globules and crystalline scaffolds, and suggest they provide a means to achieve cell-scale order. We discuss general mechanisms for assembly and regulation. Finally, we discuss assemblies that are found to organize metabolism and what possible mechanisms may serve these metabolic enzyme complexes.
Yakhnina, Anastasiya A, and Zemer Gitai. “Diverse functions for six glycosyltransferases in Caulobacter crescentus cell wall assembly.”. J Bacteriol 195.19 (2013): , 195, 19, 4527-35. Web.Abstract
The essential process of peptidoglycan synthesis requires two enzymatic activities, transpeptidation and transglycosylation. While the PBP2 and PBP3 transpeptidases perform highly specialized functions that are widely conserved, the specific roles of different glycosyltransferases are poorly understood. For example, Caulobacter crescentus encodes six glycosyltransferase paralogs of largely unknown function. Using genetic analyses, we found that Caulobacter glycosyltransferases are primarily redundant but that PbpX is responsible for most of the essential glycosyltransferase activity. Cells containing PbpX as their sole glycosyltransferase are viable, and the loss of pbpX leads to a general defect in the integrity of the cell wall structure even in the presence of the other five glycosyltransferases. However, neither PbpX nor any of its paralogs is required for the specific processes of cell elongation or division, while the cell wall synthesis required for stalk biogenesis is only partially disrupted in several of the glycosyltransferase mutants. Despite their genetic redundancy, Caulobacter glycosyltransferases exhibit different subcellular localizations. We suggest that these enzymes have specialized roles and normally function in distinct subcomplexes but retain the ability to substitute for one another so as to ensure the robustness of the peptidoglycan synthesis process.
Klein, Eric A, and Zemer Gitai. “Draft Genome Sequence of Uropathogenic Escherichia coli Strain J96.”. Genome Announc 11 (2013). Web.Abstract
Escherichia coli J96 (O4:K6) was isolated from a human pyelonephritis patient. Here, we report the draft genome sequence of E. coli J96, which contains virulence genes, including adhesion factors, alpha-hemolysins, and cytotoxic necrotizing factor. J96 infects the kidney and bladder, making it an important tool for studying E. coli pathogenesis.
Klein, Eric A, et al.Physiological role of stalk lengthening in Caulobacter crescentus.”. Commun Integr Biol 64 (2013): , 6, 4, e24561. Web.Abstract
The Gram-negative bacterium Caulobacter crescentus forms a thin polar stalk, which mediates its attachment to solid surfaces. Whereas stalks remain short (1 µm) in nutrient-rich conditions, they lengthen dramatically (up to 30 µm) upon phosphate starvation. A long-standing hypothesis is that the Caulobacter stalk functions as a nutrient scavenging "antenna" that facilitates phosphate uptake and transport to the cell body. The mechanistic details of this model must be revisited, given our recent identification of a protein-mediated diffusion barrier, which prevents the exchange of both membrane and soluble proteins between the stalk extension and the cell body. In this report, we discuss the potential of stalks to facilitate nutrient uptake and propose additional physiological roles for stalk elongation in Caulobacter cells.
Cowles, Kimberly N, et al.The putative Poc complex controls two distinct Pseudomonas aeruginosa polar motility mechanisms.”. Mol Microbiol 90.5 (2013): , 90, 5, 923-38. Web.Abstract
Each Pseudomonas aeruginosa cell localizes two types of motility structures, a single flagellum and one or two clusters of type IV pili, to the cell poles. Previous studies suggested that these motility structures arrive at the pole through distinct mechanisms. Here we performed a swimming motility screen to identify polar flagellum localization factors and discovered three genes homologous to the TonB/ExbB/ExbD complex that have defects in both flagella-mediated swimming and pilus-mediated twitching motility. We found that deletion of tonB3, PA2983 or PA2982 led to non-polar localization of the flagellum and FlhF, which was thought to sit at the top of the flagellar localization hierarchy. Surprisingly, these mutants also exhibited pronounced changes in pilus formation or localization, indicating that these proteins may co-ordinate both the pilus and flagellum motility systems. Thus, we have renamed PA2983 and PA2982, pocA and pocB, respectively, for polar organelle co-ordinator to reflect this function. Our results suggest that TonB3, PocA and PocB may form a membrane-associated complex, which we term the Poc complex. These proteins do not exhibit polar localization themselves, but are required for increased expression of pilus genes upon surface association, indicating that they regulate motility structures through either localization or transcriptional mechanisms.
Bos, Julia, Anastasiya A Yakhnina, and Zemer Gitai. “BapE DNA endonuclease induces an apoptotic-like response to DNA damage in Caulobacter.”. Proc Natl Acad Sci U S A 109.44 (2012): , 109, 44, 18096-101. Web.Abstract
In the presence of extensive DNA damage, eukaryotes activate endonucleases to fragment their chromosomes and induce apoptotic cell death. Apoptotic-like responses have recently been described in bacteria, but primarily in specialized mutant backgrounds, and the factors responsible for DNA damage-induced chromosome fragmentation and death have not been identified. Here we find that wild-type Caulobacter cells induce apoptotic-like cell death in response to extensive DNA damage. The bacterial apoptosis endonuclease (BapE) protein is induced by damage but not involved in DNA repair itself, and mediates this cell fate decision. BapE fragments chromosomes by cleaving supercoiled DNA in a sequence-nonspecific manner, thereby perturbing chromosome integrity both in vivo and in vitro. This damage-induced chromosome fragmentation pathway resembles that of eukaryotic apoptosis. We propose that damage-induced programmed cell death can be a primary stress response for some bacterial species, providing isogenic bacterial communities with advantages similar to those that apoptosis provides to multicellular organisms.
Shen, Yi, et al.Flow directs surface-attached bacteria to twitch upstream.”. Biophys J 103.1 (2012): , 103, 1, 146-51. Web.Abstract
Bacteria inhabit a wide variety of environments in which fluid flow is present, including healthcare and food processing settings and the vasculature of animals and plants. The motility of bacteria on surfaces in the presence of flow has not been well characterized. Here we focus on Pseudomonas aeruginosa, an opportunistic human pathogen that thrives in flow conditions such as in catheters and respiratory tracts. We investigate the effects of flow on P. aeruginosa cells and describe a mechanism in which surface shear stress orients surface-attached P. aeruginosa cells along the flow direction, causing cells to migrate against the flow direction while pivoting in a zig-zag motion. This upstream movement is due to the retraction of type IV pili by the ATPase motors PilT and PilU and results from the effects of flow on the polar localization of type IV pili. This directed upstream motility could be beneficial in environments where flow is present, allowing bacteria to colonize environments that cannot be reached by other surface-attached bacteria.
Ingerson-Mahar, Michael, and Zemer Gitai. “A growing family: the expanding universe of the bacterial cytoskeleton.”. FEMS Microbiol Rev 36.1 (2012): , 36, 1, 256-66. Web.Abstract
Cytoskeletal proteins are important mediators of cellular organization in both eukaryotes and bacteria. In the past, cytoskeletal studies have largely focused on three major cytoskeletal families, namely the eukaryotic actin, tubulin, and intermediate filament (IF) proteins and their bacterial homologs MreB, FtsZ, and crescentin. However, mounting evidence suggests that these proteins represent only the tip of the iceberg, as the cellular cytoskeletal network is far more complex. In bacteria, each of MreB, FtsZ, and crescentin represents only one member of large families of diverse homologs. There are also newly identified bacterial cytoskeletal proteins with no eukaryotic homologs, such as WACA proteins and bactofilins. Furthermore, there are universally conserved proteins, such as the metabolic enzyme CtpS, that assemble into filamentous structures that can be repurposed for structural cytoskeletal functions. Recent studies have also identified an increasing number of eukaryotic cytoskeletal proteins that are unrelated to actin, tubulin, and IFs, such that expanding our understanding of cytoskeletal proteins is advancing the understanding of the cell biology of all organisms. Here, we summarize the recent explosion in the identification of new members of the bacterial cytoskeleton and describe a hypothesis for the evolution of the cytoskeleton from self-assembling enzymes.
van Teeffelen, Sven, Joshua W Shaevitz, and Zemer Gitai. “Image analysis in fluorescence microscopy: bacterial dynamics as a case study.”. Bioessays 34.5 (2012): , 34, 5, 427-36. Web.Abstract
Fluorescence microscopy is the primary tool for studying complex processes inside individual living cells. Technical advances in both molecular biology and microscopy have made it possible to image cells from many genetic and environmental backgrounds. These images contain a vast amount of information, which is often hidden behind various sources of noise, convoluted with other information and stochastic in nature. Accessing the desired biological information therefore requires new tools of computational image analysis and modeling. Here, we review some of the recent advances in computational analysis of images obtained from fluorescence microscopy, focusing on bacterial systems. We emphasize techniques that are readily available to molecular and cell biologists but also point out examples where problem-specific image analyses are necessary. Thus, image analysis is not only a toolkit to be applied to new images but also an integral part of the design and implementation of a microscopy experiment.
Yakhnina, Anastasiya A, and Zemer Gitai. “The small protein MbiA interacts with MreB and modulates cell shape in Caulobacter crescentus.”. Mol Microbiol 85.6 (2012): , 85, 6, 1090-104. Web.Abstract
In Caulobacter crescentus, the actin homologue MreB is critical for cell shape maintenance. Despite the central importance of MreB for cell morphology and viability, very little is known about MreB-interacting factors. Here, we use an overexpression approach to identify a novel MreB interactor, MbiA. MbiA interacts with MreB in both biochemical and genetic assays, colocalizes with MreB throughout the cell cycle, and relies on MreB for its localization. MbiA overexpression mimics the loss of MreB function, severely perturbing cell morphology, inhibiting growth and inducing cell lysis. Additionally, mbiA deletion shows a synthetic growth phenotype with a hypomorphic allele of the MreB interactor RodZ, suggesting that these two MreB-interacting proteins either have partially redundant functions or participate in the same functional complex. Our work thus establishes MbiA as a novel cell shape regulator that appears to function through regulating MreB, and opens avenues for discovery of more MreB-regulating factors by showing that overexpression screens are a valuable tool for uncovering potentially redundant cell shape effectors.
van Teeffelen, Sven, et al.The bacterial actin MreB rotates, and rotation depends on cell-wall assembly.”. Proc Natl Acad Sci U S A 108.38 (2011): , 108, 38, 15822-7. Web.Abstract
Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis.
van Teeffelen, Sven, and Zemer Gitai. “Rotate into shape: MreB and bacterial morphogenesis.”. EMBO J 30.24 (2011): , 30, 24, 4856-7. Web.
Barry, Rachael M, and Zemer Gitai. “Self-assembling enzymes and the origins of the cytoskeleton.”. Curr Opin Microbiol 14.6 (2011): , 14, 6, 704-11. Web.Abstract
The bacterial cytoskeleton is composed of a complex and diverse group of proteins that self-assemble into linear filaments. These filaments support and organize cellular architecture and provide a dynamic network controlling transport and localization within the cell. Here, we review recent discoveries related to a newly appreciated class of self-assembling proteins that expand our view of the bacterial cytoskeleton and provide potential explanations for its evolutionary origins. Specifically, several types of metabolic enzymes can form structures similar to established cytoskeletal filaments and, in some cases, these structures have been repurposed for structural uses independent of their normal roles. The behaviors of these enzymes suggest that some modern cytoskeletal proteins may have evolved from dual-role proteins with catalytic and structural functions.
Michaelis, Allison M, and Zemer Gitai. “Dynamic polar sequestration of excess MurG may regulate enzymatic function.”. J Bacteriol 192.18 (2010): , 192, 18, 4597-605. Web.Abstract
Advances in bacterial cell biology have demonstrated the importance of protein localization for protein function. In general, proteins are thought to localize to the sites where they are active. Here we demonstrate that in Escherichia coli, MurG, the enzyme that mediates the last step in peptidoglycan subunit biosynthesis, becomes polarly localized when expressed at high cellular concentrations. MurG only becomes polarly localized at levels that saturate MurG's cellular requirement for growth, and E. coli cells do not insert peptidoglycan at the cell poles, indicating that the polar MurG is not active. Fluorescence recovery after photobleaching (FRAP) and single-cell biochemistry experiments demonstrate that polar MurG is dynamic. Polar MurG foci are distinct from inclusion body aggregates, and polar MurG can be remobilized when MurG levels drop. These results suggest that polar MurG represents a temporary storage mechanism for excess protein that can later be remobilized into the active pool. We investigated and ruled out several candidate pathways for polar MurG localization, including peptidoglycan biosynthesis, the MreB cytoskeleton, and polar cardiolipin, as well as MurG enzymatic activity and lipid binding, suggesting that polar MurG is localized by a novel mechanism. Together, our results imply that inactive MurG is dynamically sequestered at the cell poles and that prokaryotes can thus utilize subcellular localization as a mechanism for negatively regulating enzymatic activity.
Ingerson-Mahar, Michael, et al.The metabolic enzyme CTP synthase forms cytoskeletal filaments.”. Nat Cell Biol 12.8 (2010): , 12, 8, 739-46. Web.Abstract
Filament-forming cytoskeletal proteins are essential for the structure and organization of all cells. Bacterial homologues of the major eukaryotic cytoskeletal families have now been discovered, but studies suggest that yet more remain to be identified. We demonstrate that the metabolic enzyme CTP synthase (CtpS) forms filaments in Caulobacter crescentus. CtpS is bifunctional, as the filaments it forms regulate the curvature of C. crescentus cells independently of its catalytic function. The morphogenic role of CtpS requires its functional interaction with the intermediate filament, crescentin (CreS). Interestingly, the Escherichia coli CtpS homologue also forms filaments both in vivo and in vitro, suggesting that CtpS polymerization may be widely conserved. E. coli CtpS can replace the enzymatic and morphogenic functions of C. crescentus CtpS, indicating that C. crescentus has adapted a conserved filament-forming protein for a secondary role. These results implicate CtpS as a novel bifunctional member of the bacterial cytoskeleton and suggest that localization and polymerization may be important properties of metabolic enzymes.
Shaevitz, Joshua W, and Zemer Gitai. “The structure and function of bacterial actin homologs.”. Cold Spring Harb Perspect Biol 29 (2010): , 2, 9, a000364. Web.Abstract
During the past decade, the appreciation and understanding of how bacterial cells can be organized in both space and time have been revolutionized by the identification and characterization of multiple bacterial homologs of the eukaryotic actin cytoskeleton. Some of these bacterial actins, such as the plasmid-borne ParM protein, have highly specialized functions, whereas other bacterial actins, such as the chromosomally encoded MreB protein, have been implicated in a wide array of cellular activities. In this review we cover our current understanding of the structure, assembly, function, and regulation of bacterial actins. We focus on ParM as a well-understood reductionist model and on MreB as a central organizer of multiple aspects of bacterial cell biology. We also discuss the outstanding puzzles in the field and possible directions where this fast-developing area may progress in the future.
Cowles, Kimberly N, and Zemer Gitai. “Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa.”. Mol Microbiol 76.6 (2010): , 76, 6, 1411-26. Web.Abstract
Spatial organization of bacterial proteins influences many cellular processes, including division, chromosome segregation and motility. Virulence-associated proteins also localize to specific destinations within bacterial cells. However, the functions and mechanisms of virulence factor localization remain largely unknown. In this work, we demonstrate that polar assembly of the Pseudomonas aeruginosa PAO1 type IV pilus is regulated by surface association in a manner that affects gene transcription, protein levels and protein localization. We also uncover one mechanism for this regulation that acts through the actin homologue MreB. Inactivation of MreB leads to mislocalization of the pilus retraction ATPase PilT, mislocalization of the pili themselves and a reduction in motility. Furthermore, the role of MreB in polar localization of PilT is modulated by surface association, corroborating our results that environmental factors influence the regulation of pilus production. Specifically, MreB mediates both the initiation and maintenance of PilT localization when cells are grown in suspension but only affects the initiation of localization when cells are grown on a surface. Together, these results suggest that the bacterial cytoskeleton provides a mechanism for the polar localization of P. aeruginosa pili and demonstrate that protein localization may represent an important aspect of virulence factor regulation in bacterial pathogens.
Shebelut, Conrad W, et al.Caulobacter chromosome segregation is an ordered multistep process.”. Proc Natl Acad Sci U S A 107.32 (2010): , 107, 32, 14194-8. Web.Abstract
Despite its fundamental nature, bacterial chromosome segregation remains poorly understood. Viewing segregation as a single process caused multiple proposed mechanisms to appear in conflict and failed to explain how asymmetrically dividing bacteria break symmetry to move only one of their chromosomes. Here, we demonstrate that the ParA ATPase extends from one cell pole and pulls the chromosome by retracting upon association with the ParB DNA-binding protein. Surprisingly, ParA disruption has a specific effect on chromosome segregation that only perturbs the latter stages of this process. Using quantitative high-resolution imaging, we demonstrate that this specificity results from the multistep nature of chromosome translocation. We propose that Caulobacter chromosome segregation follows an ordered pathway of events with distinct functions and mechanisms. Initiation releases polar tethering of the origin of replication, distinction spatially differentiates the two chromosomes, and commitment irreversibly translocates the distal centromeric locus. Thus, much as eukaryotic mitosis involves a sequence of distinct subprocesses, Caulobacter cells also segregate their chromosomes through an orchestrated series of steps. We discuss how the multistep view of bacterial chromosome segregation can help to explain and reconcile outstanding puzzles and frame future investigation.
Werner, John N, and Zemer Gitai. “High-throughput screening of bacterial protein localization.”. Methods Enzymol 471 (2010): , 471, 185-204. Web.Abstract
The ever-increasing number of sequenced genomes and subsequent sequence-based analysis has provided tremendous insight into cellular processes; however, the ability to experimentally manipulate this genomic information in the laboratory requires the development of new high-throughput methods. To translate this genomic information into information on protein function, molecular and cell biological techniques are required. One strategy to gain insight into protein function is to observe where each specific protein is subcellularly localized. We have developed a pipeline of methods that allows rapid, efficient, and scalable gene cloning, imaging, and image analysis. This work focuses on a high-throughput screen of the Caulobacter crescentus proteome to identify proteins with unique subcellular localization patterns. The cloning, imaging, and image analysis techniques described here are applicable to any organism of interest.
Shebelut, Conrad W, Rasmus B Jensen, and Zemer Gitai. “Growth conditions regulate the requirements for Caulobacter chromosome segregation.”. J Bacteriol 191.3 (2009): , 191, 3, 1097-100. Web.Abstract
Growth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect on Caulobacter chromosome segregation of a small-molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation determinants, suggesting that multiple distinct mechanisms can mediate Caulobacter chromosome segregation and that their relative contributions can be environmentally regulated.
Gitai, Zemer. “New fluorescence microscopy methods for microbiology: sharper, faster, and quantitative.”. Curr Opin Microbiol 12.3 (2009): , 12, 3, 341-6. Web.Abstract
In addition to the inherent interest stemming from their ecological and human health impacts, microbes have many advantages as model organisms, including ease of growth and manipulation and relatively simple genomes. However, the imaging of bacteria via light microscopy has been limited by their small sizes. Recent advances in fluorescence microscopy that allow imaging of structures at extremely high resolutions are thus of particular interest to the modern microbiologist. In addition, advances in high-throughput microscopy and quantitative image analysis are enabling cellular imaging to finally take advantage of the full power of bacterial numbers and ease of manipulation. These technical developments are ushering in a new era of using fluorescence microscopy to understand bacterial systems in a detailed, comprehensive, and quantitative manner.
Werner, John N, et al.Quantitative genome-scale analysis of protein localization in an asymmetric bacterium.”. Proc Natl Acad Sci U S A 106.19 (2009): , 106, 19, 7858-63. Web.Abstract
Despite the importance of subcellular localization for cellular activities, the lack of high-throughput, high-resolution imaging and quantitation methodologies has limited genomic localization analysis to a small number of archival studies focused on C-terminal fluorescent protein fusions. Here, we develop a high-throughput pipeline for generating, imaging, and quantitating fluorescent protein fusions that we use for the quantitative genomic assessment of the distributions of both N- and C-terminal fluorescent protein fusions. We identify nearly 300 localized Caulobacter crescentus proteins, up to 10-fold more than were previously characterized. The localized proteins tend to be involved in spatially or temporally dynamic processes and proteins that function together and often localize together as well. The distributions of the localized proteins were quantitated by using our recently described projected system of internal coordinates from interpolated contours (PSICIC) image analysis toolkit, leading to the identification of cellular regions that are over- or under-enriched in localized proteins and of potential differences in the mechanisms that target proteins to different subcellular destinations. The Caulobacter localizome data thus represent a resource for studying both global properties of protein localization and specific protein functions, whereas the localization analysis pipeline is a methodological resource that can be readily applied to other systems.
Huang, Kerwyn Casey, et al.Cell shape and cell-wall organization in Gram-negative bacteria.”. Proc Natl Acad Sci U S A 105.49 (2008): , 105, 49, 19282-7. Web.Abstract
In bacterial cells, the peptidoglycan cell wall is the stress-bearing structure that dictates cell shape. Although many molecular details of the composition and assembly of cell-wall components are known, how the network of peptidoglycan subunits is organized to give the cell shape during normal growth and how it is reorganized in response to damage or environmental forces have been relatively unexplored. In this work, we introduce a quantitative physical model of the bacterial cell wall that predicts the mechanical response of cell shape to peptidoglycan damage and perturbation in the rod-shaped Gram-negative bacterium Escherichia coli. To test these predictions, we use time-lapse imaging experiments to show that damage often manifests as a bulge on the sidewall, coupled to large-scale bending of the cylindrical cell wall around the bulge. Our physical model also suggests a surprising robustness of cell shape to peptidoglycan defects, helping explain the observed porosity of the cell wall and the ability of cells to grow and maintain their shape even under conditions that limit peptide crosslinking. Finally, we show that many common bacterial cell shapes can be realized within the same model via simple spatial patterning of peptidoglycan defects, suggesting that minor patterning changes could underlie the great diversity of shapes observed in the bacterial kingdom.
Guberman, Jonathan M, et al.PSICIC: noise and asymmetry in bacterial division revealed by computational image analysis at sub-pixel resolution.”. PLoS Comput Biol 411 (2008): , 4, 11, e1000233. Web.Abstract
Live-cell imaging by light microscopy has demonstrated that all cells are spatially and temporally organized. Quantitative, computational image analysis is an important part of cellular imaging, providing both enriched information about individual cell properties and the ability to analyze large datasets. However, such studies are often limited by the small size and variable shape of objects of interest. Here, we address two outstanding problems in bacterial cell division by developing a generally applicable, standardized, and modular software suite termed Projected System of Internal Coordinates from Interpolated Contours (PSICIC) that solves common problems in image quantitation. PSICIC implements interpolated-contour analysis for accurate and precise determination of cell borders and automatically generates internal coordinate systems that are superimposable regardless of cell geometry. We have used PSICIC to establish that the cell-fate determinant, SpoIIE, is asymmetrically localized during Bacillus subtilis sporulation, thereby demonstrating the ability of PSICIC to discern protein localization features at sub-pixel scales. We also used PSICIC to examine the accuracy of cell division in Esherichia coli and found a new role for the Min system in regulating division-site placement throughout the cell length, but only prior to the initiation of cell constriction. These results extend our understanding of the regulation of both asymmetry and accuracy in bacterial division while demonstrating the general applicability of PSICIC as a computational approach for quantitative, high-throughput analysis of cellular images.
Gitai, Zemer. “Diversification and specialization of the bacterial cytoskeleton.”. Curr Opin Cell Biol 19.1 (2007): , 19, 1, 5-12. Web.Abstract
The past decade has witnessed the identification and characterization of bacterial homologs of the three major eukaryotic cytoskeletal families: actin, tubulin and intermediate filaments. These proteins play essential roles in organizing bacterial subcellular environments. Recently, the ParA/MinD superfamily has emerged as a new bacterial cytoskeletal class, and imaging studies hint at the existence of even more, as yet unidentified, cytoskeletal systems. Much as the cytoskeleton is used for different purposes in different eukaryotic cells, the specific identities, functions and regulatory mechanisms of cytoskeletal proteins can vary between different bacterial species. In addition, extensive cross-talk between bacterial cytoskeletal systems may represent an important mode of cytoskeletal regulation. These themes of diversity, species-specificity and crosstalk are emerging as central properties of cytoskeletal biology.
Gitai, Zemer. “Plasmid segregation: a new class of cytoskeletal proteins emerges.”. Curr Biol 16.4 (2006): , 16, 4, R133-6. Web.Abstract
The discovery that a plasmid-partitioning ATPase forms astral cytoskeletal structures both unveils a new family of cytoskeletal proteins and suggests that cytoskeletal involvement is a universal feature of DNA segregation.
Gitai, Zemer, Martin Thanbichler, and Lucy Shapiro. “The choreographed dynamics of bacterial chromosomes.”. Trends Microbiol 13.5 (2005): , 13, 5, 221-8. Web.Abstract
Despite decades of study, the exquisite temporal and spatial organization of bacterial chromosomes has only recently been appreciated. The direct visualization of specific chromosomal loci has revealed that bacteria condense, move and position their chromosomes in a reproducible fashion. The realization that bacterial chromosomes are actively translocated through the cell suggests the existence of specific mechanisms that direct this process. Here, we review bacterial chromosome dynamics and our understanding of the mechanisms that direct and coordinate them.
Gitai, Zemer. “The new bacterial cell biology: moving parts and subcellular architecture.”. Cell 120.5 (2005): , 120, 5, 577-86. Web.Abstract
Recent advances have demonstrated that bacterial cells have an exquisitely organized and dynamic subcellular architecture. Like their eukaryotic counterparts, bacteria employ a full complement of cytoskeletal proteins, localize proteins and DNA to specific subcellular addresses at specific times, and use intercellular signaling to coordinate multicellular events. The striking conceptual and molecular similarities between prokaryotic and eukaryotic cell biology thus make bacteria powerful model systems for studying fundamental cellular questions.