RESUMO
The physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules, and foreign particles of different sizes, we find that the bacterial cytoplasm displays properties that are characteristic of glass-forming liquids and changes from liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology, as the glassy behavior of the cytoplasm impacts all intracellular processes involving large components.
Assuntos
Caulobacter crescentus/citologia , Caulobacter crescentus/metabolismo , Escherichia coli/citologia , Fenômenos Biofísicos , Caulobacter crescentus/química , Cromossomos Bacterianos/metabolismo , Citoplasma/química , Escherichia coli/química , Escherichia coli/metabolismo , Plasmídeos/metabolismoRESUMO
Technical developments are unifying molecular and cellular biology. A recent electron cryotomography study by von Kügelgen et al. highlights the bright future for such studies, seamlessly integrating near-atomic resolution protein structures, organism-scale architecture, native mass spectrometry, and molecular dynamic simulations to clarify how the Caulobacter crescentus S-layer assembles on the lipopolysaccharides (LPS) of the cell surface.
Assuntos
Caulobacter crescentus/química , Lipopolissacarídeos/química , Configuração de Carboidratos , Espectrometria de Massas/métodos , Simulação de Dinâmica MolecularRESUMO
vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imagem Individual de Molécula/métodos , Realidade Virtual , Algoritmos , Animais , Células COS , Caulobacter crescentus/química , Linhagem Celular , Membrana Celular/química , Chlorocebus aethiops , Clatrina/química , Humanos , Células Jurkat , Microtúbulos/química , Poro Nuclear/química , SoftwareRESUMO
We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.
Assuntos
Proteínas de Bactérias , Caulobacter crescentus , Fator Proteico 1 do Hospedeiro , RNA Bacteriano , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/química , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Cristalografia por Raios X , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/metabolismo , Modelos Moleculares , Chaperonas Moleculares , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismoRESUMO
Spatial control of intracellular signaling relies on signaling proteins sensing their subcellular environment. In many cases, a large number of upstream signals are funneled to a master regulator of cellular behavior, but it remains unclear how individual proteins can rapidly integrate a complex array of signals within the appropriate spatial niche within the cell. As a model for how subcellular spatial information can control signaling activity, we have reconstituted the cell pole-specific control of the master regulator kinase/phosphatase CckA from the asymmetrically dividing bacterium Caulobacter crescentus CckA is active as a kinase only when it accumulates within a microdomain at the new cell pole, where it colocalizes with the pseudokinase DivL. Both proteins contain multiple PAS domains, a multifunctional class of sensory domains present across the kingdoms of life. Here, we show that CckA uses its PAS domains to integrate information from DivL and its own oligomerization state to control the balance of its kinase and phosphatase activities. We reconstituted the DivL-CckA complex on liposomes in vitro and found that DivL directly controls the CckA kinase/phosphatase switch, and that stimulation of either CckA catalytic activity depends on the second of its two PAS domains. We further show that CckA oligomerizes through a multidomain interaction that is critical for stimulation of kinase activity by DivL, while DivL stimulation of CckA phosphatase activity is independent of CckA homooligomerization. Our results broadly demonstrate how signaling factors can leverage information from their subcellular niche to drive spatiotemporal control of cell signaling.
Assuntos
Proteínas de Bactérias/química , Caulobacter crescentus/enzimologia , Proteínas Quinases/química , Multimerização Proteica , Caulobacter crescentus/química , Domínios Proteicos , Estrutura Quaternária de ProteínaRESUMO
The nucleoid-associated protein GapR found in Caulobacter crescentus is crucial for DNA replication, transcription, and cell division. Associated with overtwisted DNA in front of replication forks and the 3' end of highly-expressed genes, GapR can stimulate gyrase and topo IV to relax (+) supercoils, thus facilitating the movement of the replication and transcription machines. GapR forms a dimer-of-dimers structure in solution that can exist in either an open or a closed conformation. It initially binds DNA through the open conformation and then undergoes structural rearrangement to form a closed tetramer, with DNA wrapped in the central channel. Here, we show that the DNA binding domain of GapR (residues 1-72, GapRΔC17) exists as a dimer in solution and adopts the same fold as the two dimer units in the full-length tetrameric protein. It binds DNA at the minor groove and reads the spatial distribution of DNA phosphate groups through a lysine/arginine network, with a preference towards AT-rich overtwisted DNA. These findings indicate that the dimer unit of GapR has an intrinsic DNA binding preference. Thus, at the initial binding step, the open tetramer of GapR with two relatively independent dimer units can be more efficiently recruited to overtwisted regions.
Assuntos
Caulobacter crescentus/metabolismo , DNA Bacteriano/metabolismo , Transativadores/química , Transativadores/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Caulobacter crescentus/química , Caulobacter crescentus/genética , Cristalografia por Raios X , DNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Transativadores/genéticaRESUMO
The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod-shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme-resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD-crosslinks between meso-diaminopimelic acid and D-alanine residues, whereas stalk peptidoglycan had more LD-transpeptidation (meso-diaminopimelic acid-meso-diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD-transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD-crosslinking. Despite no obvious selection pressure for LD-crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP-DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.
Assuntos
Proteínas de Bactérias/química , Caulobacter crescentus/química , Peptidoglicano/química , Caulobacter crescentus/efeitos dos fármacos , Parede Celular/química , Muramidase/farmacologia , Fosfatos/metabolismoRESUMO
Single-molecule super-resolution fluorescence microscopy conducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of 4, attributable to reduced photobleaching.
Assuntos
Temperatura Baixa , Microscopia Crioeletrônica/métodos , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Caulobacter crescentus/química , Fotodegradação , Proteína Vermelha FluorescenteRESUMO
Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly ß-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified ß-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of ß-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a ß-helical architecture, in which 18 ß-strand segments are arranged in six consecutive windings of a ß-helix.
Assuntos
Proteínas de Bactérias/química , Caulobacter crescentus/química , Citoesqueleto/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/genética , Sequência Conservada , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Tomografia com Microscopia Eletrônica , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures. In vitro, the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiological temperatures and low divalent calcium ion concentrations. At higher concentrations, calcium ions stabilize monomeric RsaA, which can then transition to the two-dimensional crystalline state. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. These findings provide a biochemical and physiological basis for RsaA's calcium-binding behavior, which extends far beyond calcium's commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness.
Assuntos
Cálcio/metabolismo , Caulobacter crescentus/química , Caulobacter crescentus/metabolismo , Glicoproteínas de Membrana/química , Cálcio/química , Caulobacter crescentus/ultraestrutura , Dicroísmo Circular , Cristalização , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Agregados Proteicos , Dobramento de Proteína , Espalhamento a Baixo Ângulo , Estresse Fisiológico , Temperatura , Difração de Raios XRESUMO
We examine the localization patterns of ParA, ParB, PopZ, and MipZ, which are key division proteins in C. crescentus bacteria. While Par and PopZ proteins have been implicated in the physical segregation of the replicated chromosome, MipZ dimers control the placement of the cell division plane by preventing FtsZ proteins from assembling into a Z-ring. MipZ proteins generate bipolar gradients that are sensitive to Par protein localization, however, it is not understood how the MipZ gradient is shaped so as to allow for the correct Z-ring placement during asymmetric cell division in C. crescentus. In this paper, we develop and analyze a mathematical model that incorporates the known interactions between Par, PopZ, and MipZ proteins and use it to test mechanisms for MipZ gradient formation. Using our model, we show that gradient-dependent ParB advection velocities in conjunction with a ParA polar recycling mechanism are sufficient to maintain a robust new pole-directed ParA dimer gradient during segregation. A "saturation of binding site" hypothesis limiting access of ParA and MipZ to the ParB complex is then necessary and sufficient to generate time-averaged bipolar MipZ protein gradients with minima that are skewed toward ParA gradient peaks at the new pole, in agreement with data. By analyzing reduced versions of the model, we show the existence of oscillatory ParA localization regimes provided that cytoplasmic PopZ oligomers interact with ParA and ParA is over-expressed. We use our model to study mechanisms by which these protein patterns may simultaneously direct proper chromosome segregation and division site placement in C. crescentus.
Assuntos
Divisão Celular Assimétrica/fisiologia , Caulobacter crescentus/química , Modelos Teóricos , Análise Espaço-Temporal , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos , Proteínas do Citoesqueleto/metabolismoRESUMO
In the environment, most bacteria form surface-attached cell communities called biofilms. The attachment of single cells to surfaces involves an initial reversible stage typically mediated by surface structures such as flagella and pili, followed by a permanent adhesion stage usually mediated by polysaccharide adhesives. Here, we determine the absolute and relative timescales and frequencies of reversible and irreversible adhesion of single cells of the bacterium Caulobacter crescentus to a glass surface in a microfluidic device. We used fluorescence microscopy of C. crescentus expressing green fluorescent protein to track the swimming behavior of individual cells prior to adhesion, monitor the cell at the surface, and determine whether the cell reversibly or irreversibly adhered to the surface. A fluorescently labeled lectin that binds specifically to polar polysaccharides, termed holdfast, discriminated irreversible adhesion events from reversible adhesion events where no holdfast formed. In wild-type cells, the holdfast production time for irreversible adhesion events initiated by surface contact (23 s) was 30-times faster than the holdfast production time that occurs through developmental regulation (13 min). Irreversible adhesion events in wild-type cells (3.3 events/min) are 15-times more frequent than in pilus-minus mutant cells (0.2 events/min), indicating the pili are critical structures in the transition from reversible to irreversible surface-stimulated adhesion. In reversible adhesion events, the dwell time of cells at the surface before departing was the same for wild-type cells (12 s) and pilus-minus mutant cells (13 s), suggesting the pili do not play a significant role in reversible adhesion. Moreover, reversible adhesion events in wild-type cells (6.8 events/min) occur twice as frequently as irreversible adhesion events (3.3 events/min), demonstrating that most cells contact the surface multiple times before transitioning from reversible to irreversible adhesion.
Assuntos
Aderência Bacteriana , Caulobacter crescentus/metabolismo , Técnicas Analíticas Microfluídicas , Biofilmes , Caulobacter crescentus/química , Vidro , Proteínas de Fluorescência Verde/metabolismo , Microscopia de FluorescênciaRESUMO
Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10-20 nm.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Caulobacter crescentus/química , Imageamento Tridimensional , Microscopia , Rodaminas/química , Compostos de Espiro/química , Sobrevivência Celular , Coloração e RotulagemRESUMO
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.
Assuntos
Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/química , Proteínas de Bactérias/genética , Caulobacter crescentus/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Cromossomos Bacterianos/metabolismo , Dicroísmo Circular , Mutação Puntual , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Caulobacter crescentus/química , Caulobacter crescentus/metabolismo , Sequência Conservada , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/genética , Citoesqueleto/química , Citoesqueleto/metabolismo , Proteínas de Ligação às Penicilinas/análise , Filogenia , Transporte ProteicoRESUMO
We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.
Assuntos
Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/química , CorRESUMO
The bacterial second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which induce specific phenotypic responses critical for bacterial survival. One class of enzymes responsible for regulating the intracellular concentration of c-di-GMP, and therefore the physiological behavior of the cell, consists of the EAL domain phosphodiesterases, which degrade the second messenger to its linear form, pGpG. Here, we investigate how base and backbone modifications of c-di-GMP affect the rate of cyclic dinucleotide degradation by an EAL domain protein (CC3396 from Caulobacter crescentus). The doubly substituted thiophosphate analogue is highly resistant to hydrolysis by this metabolizing enzyme but can still bind c-di-GMP riboswitch targets. We used these findings to develop a novel ribosyl phosphate-modified derivative of c-di-GMP containing 2'-deoxy and methylphosphonate substitutions that is charge neutral and demonstrate that this analogue is also resistant to EAL domain-catalyzed degradation. This suggests a general strategy for designing c-di-GMP derivatives with increased enzymatic stability that also possess desirable properties for development as chemical probes of c-di-GMP signaling.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , GMP Cíclico/análogos & derivados , Diester Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/química , Caulobacter crescentus/química , GMP Cíclico/química , GMP Cíclico/metabolismo , Hidrólise , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/química , Estrutura Terciária de Proteína , Ribose/química , Ribose/metabolismo , Riboswitch , Sistemas do Segundo Mensageiro , Especificidade por SubstratoRESUMO
Efforts to characterize proteins found in the outer membrane (OM) of Gram-negative bacteria have been steadily increasing due to the promise of expanding our understanding of fundamental bacterial processes such as cell adhesion or cell wall biogenesis as well as the promise of finding potential vaccine- or drug-targets for virulent bacteria. We have developed a mass spectrometry-compatible experimental strategy that resulted in increased coverage of the OM proteome of a model organism, Caulobacter crescentus. The specificity of the OM enrichment step was improved by using detergent solubilization of the protein pellet, low-density cell culture conditions, and a surface-layer deficient cell line. Additionally, efficient gel-assisted digestion, high-resolution RP/RP-MS/MS, and rigorous bioinformatic analysis led to the identification of 234 proteins using strict identification criteria (≥ two unique peptides per protein; peptide false discovery rate <2%). Eighty-four of the detected proteins were predicted to localize to the OM or extracellular space. These results represent ~70% coverage of the predicted OM/extracellular proteome of C. crescentus. This analytical approach, which considers important experimental variables not previously explored in published OM protein studies, can be applied to other OM proteomic endeavors "as is" or with slight modification and should improve the large-scale study of this especially challenging subproteome.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Caulobacter crescentus/química , Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Western Blotting/métodos , Carbonatos/química , Cromatografia Líquida de Alta Pressão/métodos , Biologia Computacional , Detergentes/química , Eletroforese em Gel de Poliacrilamida , Proteoma/química , Proteoma/isolamento & purificação , Sarcosina/análogos & derivados , Sarcosina/química , Sensibilidade e Especificidade , SolubilidadeRESUMO
The binding of the soluble cytoplasmic protein FliG to the transmembrane protein FliF is one of the first interactions in the assembly of the bacterial flagellum. Once established, this interaction is integral in keeping the flagellar cytoplasmic ring, responsible for both transmission of torque and control of the rotational direction of the flagellum, anchored to the central transmembrane ring on which the flagellum is assembled. Here we isolate and characterize the interaction between the N-terminal domain of Thermotoga maritima FliG (FliG(N)) and peptides corresponding to the conserved C-terminal portion of T. maritima FliF. Using nuclear magnetic resonance (NMR) and other techniques, we show that the last ~40 amino acids of FliF (FliF(C)) interact strongly (upper bound K(d) in the low nanomolar range) with FliG(N). The formation of this complex causes extensive conformational changes in FliG(N). We find that T. maritima FliG(N) is homodimeric in the absence of the FliF(C) peptide but forms a heterodimeric complex with the peptide, and we show that this same change in oligomeric state occurs in full-length T. maritima FliG, as well. We relate previously observed phenotypic effects of FliF(C) mutations to our direct observation of binding. Lastly, on the basis of NMR data, we propose that the primary interaction site for FliF(C) is located on a conserved hydrophobic patch centered along helix 1 of FliG(N). These results provide new detailed information about the bacterial flagellar motor and support efforts to understand the cytoplasmic ring's precise molecular structure and mechanism of rotational switching.
Assuntos
Proteínas de Bactérias/química , Flagelos/química , Proteínas de Membrana/química , Proteínas Motores Moleculares/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/química , Flagelos/genética , Flagelos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Thermotoga maritima/químicaRESUMO
Upon initiating replication of the Caulobacter chromosome, one copy of the parS centromere remains at the stalked pole; the other moves to the distal pole. We identified the segregation dynamics and compaction characteristics of newly replicated Caulobacter DNA during transport (highly variable from cell to cell) using time-lapse fluorescence microscopy. The parS centromere and a length (also highly variable) of parS proximal DNA on each arm of the chromosome are segregated with the same relatively slow transport pattern as the parS locus. Newly replicated DNA further than about 100 kb from parS segregates with a different and faster pattern, while loci at 48 kb from parS segregate with the slow pattern in some cells and the fast pattern in others. The observed parS-proximal DNA compaction characteristics have scaling properties that suggest the DNA is branched. HU2-deletion strains exhibited a reduced compaction phenotype except near the parS site where only the ΔHU1ΔHU2 double mutant had a compaction phenotype. The chromosome shows speed-dependent extension during translocation suggesting the DNA polymer is under tension. While DNA segregation is highly reliable and succeeds in virtually all wild-type cells, the high degree of cell to cell variation in the segregation process is noteworthy.