Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.549
Filtrar
1.
Adv Exp Med Biol ; 1267: 59-80, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894477

RESUMO

The internal spatial organization of prokaryotic organisms, including Escherichia coli, is essential for the proper functioning of processes such as cell division. One source of this organization in E. coli is the nucleoid, which causes the exclusion of macromolecules - e.g. protein aggregates and the chemotaxis network - from midcell. Similarly, following DNA replication, the nucleoid(s) assist in placing the Z-ring at midcell. These processes need to be efficient in optimal conditions and robust to suboptimal conditions. After reviewing recent findings on these topics, we make use of past data to study the efficiency of the spatial constraining of Z-rings, chemotaxis networks, and protein aggregates, as a function of the nucleoid(s) morphology. Also, we compare the robustness of these processes to nonoptimal temperatures. We show that Z-rings, Tsr clusters, and protein aggregates have temperature-dependent spatial distributions along the major cell axis that are consistent with the nucleoid(s) morphology and the volume-exclusion phenomenon. Surprisingly, the consequences of the changes in nucleoid size with temperature are most visible in the kurtosis of these spatial distributions, in that it has a statistically significant linear correlation with the mean nucleoid length and, in the case of Z-rings, with the distance between nucleoids prior to cell division. Interestingly, we also find a negative, statistically significant linear correlation between the efficiency of these processes at the optimal condition and their robustness to suboptimal conditions, suggesting a trade-off between these traits.


Assuntos
Escherichia coli/citologia , Escherichia coli/metabolismo , Organelas/metabolismo , Divisão Celular , Replicação do DNA
2.
PLoS One ; 15(8): e0228525, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822344

RESUMO

The toxic effect of strained hydrocarbon 2,2'-bis (bicyclo[2.2.1]heptane) (BBH) was studied using whole-cell bacterial lux-biosensors based on Escherichia coli cells in which luciferase genes are transcriptionally fused with stress-inducible promoters. It was shown that BBH has the genotoxic effect causing bacterial SOS response however no alkylating effect has been revealed. In addition to DNA damage, there is an oxidative effect causing the response of OxyR/S and SoxR/S regulons. The most sensitive to BBH lux-biosensor was E. coli pSoxS-lux which reacts to the appearance of superoxide anion radicals in the cell. It is assumed that the oxidation of BBH leads to the generation of reactive oxygen species, which provide the main contribution to the genotoxicity of this substance.


Assuntos
Compostos Bicíclicos com Pontes/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Mutagênicos/toxicidade , Alquilação/efeitos dos fármacos , Técnicas Biossensoriais , Dano ao DNA , Relação Dose-Resposta a Droga , Escherichia coli/citologia , Escherichia coli/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Regulon/efeitos dos fármacos , Regulon/genética
3.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
4.
Nat Commun ; 11(1): 2262, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385264

RESUMO

Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 µm-1 and acetoin titer to 67.2 g·L-1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 µm3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L-1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories.


Assuntos
Divisão Celular/efeitos da radiação , Escherichia coli/citologia , Escherichia coli/efeitos da radiação , Luz , Acetoína/metabolismo , Reatores Biológicos/microbiologia , Escherichia coli/genética , Escherichia coli/ultraestrutura , Genes Bacterianos , Poliésteres/metabolismo
5.
J Biosci Bioeng ; 130(3): 260-264, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32456985

RESUMO

Vanillin is a well-known fragrant, flavoring compound. Previously, we established a method of coenzyme-independent vanillin production via an oxygenase from Caulobacter segnis ATCC 21756, called Cso2, that converts 4-vinylguaiacol to vanillin and formaldehyde using oxygen. In this study, we found that reactive oxygen species inhibited the catalytic activity of Cso2, and the addition of catalase increased vanillin production. Since Escherichia coli harbors catalases, we used E. coli cells expressing Cso2 to produce vanillin. Cell immobilization in calcium alginate enabled the long-term use of the E. coli cells for vanillin production. Thus, we demonstrate the possibility of using immobilized E. coli cells for both continuous and repeated batch vanillin production without any coenzymes.


Assuntos
Benzaldeídos/metabolismo , Células Imobilizadas/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Guaiacol/análogos & derivados , Oxigenases/genética , Biotecnologia , Caulobacter/enzimologia , Expressão Gênica , Guaiacol/metabolismo
6.
Nucleic Acids Res ; 48(11): 6053-6067, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374866

RESUMO

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.


Assuntos
Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Fluorescência , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/química , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Resposta SOS em Genética
7.
Nat Commun ; 11(1): 1305, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161270

RESUMO

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Divisão Celular , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Lipoproteínas/metabolismo , Peptidoglicano/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana , Proteínas Periplásmicas/metabolismo
8.
Phys Rev Lett ; 124(4): 048102, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32058787

RESUMO

Experiments have suggested that bacterial mechanosensitive channels separate into 2D clusters, the role of which is unclear. By developing a coarse-grained computer model we find that clustering promotes the channel closure, which is highly dependent on the channel concentration and membrane stress. This behaviour yields a tightly regulated gating system, whereby at high tensions channels gate individually, and at lower tensions the channels spontaneously aggregate and inactivate. We implement this positive feedback into the model for cell volume regulation, and find that the channel clustering protects the cell against excessive loss of cytoplasmic content.


Assuntos
Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Biológicos , Modelos Químicos , Escherichia coli/citologia , Escherichia coli/metabolismo , Ativação do Canal Iônico , Mecanotransdução Celular
9.
Nature ; 578(7796): 588-592, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32076271

RESUMO

Elucidating elementary mechanisms that underlie bacterial diversity is central to ecology1,2 and microbiome research3. Bacteria are known to coexist by metabolic specialization4, cooperation5 and cyclic warfare6-8. Many species are also motile9, which is studied in terms of mechanism10,11, benefit12,13, strategy14,15, evolution16,17 and ecology18,19. Indeed, bacteria often compete for nutrient patches that become available periodically or by random disturbances2,20,21. However, the role of bacterial motility in coexistence remains unexplored experimentally. Here we show that-for mixed bacterial populations that colonize nutrient patches-either population outcompetes the other when low in relative abundance. This inversion of the competitive hierarchy is caused by active segregation and spatial exclusion within the patch: a small fast-moving population can outcompete a large fast-growing population by impeding its migration into the patch, while a small fast-growing population can outcompete a large fast-moving population by expelling it from the initial contact area. The resulting spatial segregation is lost for weak growth-migration trade-offs and a lack of virgin space, but is robust to population ratio, density and chemotactic ability, and is observed in both laboratory and wild strains. These findings show that motility differences and their trade-offs with growth are sufficient to promote diversity, and suggest previously undescribed roles for motility in niche formation and collective expulsion-containment strategies beyond individual search and survival.


Assuntos
Escherichia coli/fisiologia , Interações Microbianas , Movimento , Biodiversidade , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Flagelos/fisiologia , Modelos Biológicos , Análise Espacial
10.
Biochim Biophys Acta Biomembr ; 1862(6): 183214, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32081704

RESUMO

Structural data on membrane proteins in a lipid membrane environment is challenging to obtain but needed to provide information on the, often essential, protein-lipid interplay. A common experimental bottleneck in obtaining such data is providing samples in sufficient amounts and quality required for structural studies. We developed a new production protocol for the single-pass transmembrane protein (SPTMP) tissue factor (TF), exploiting the high expression level in E. coli inclusion bodies and subsequent refolding. This provided more than 5 mg of functional TF per liter bacterial culture. This is substantially more than what was obtained by the classical approaches for expressing TF in the membrane-anchored configuration. We optimized reconstitution into circularized nanodiscs enabling the formation of stable, TF loaded nanodiscs with different lipid compositions and with a limited material waste. The blood coagulation cascade is initiated by the complex formation between TF and Factor VIIa (FVIIa), and we probed this interaction by a functional assay and SPR measurements, which revealed similar activity and binding kinetics as TF produced by other protocols, demonstrating that high-yield production does not compromise TF function. Furthermore, the amounts of sample produced permitted initial small angle X-ray scattering studies providing the first structural information about TF and its binding to FVIIa in a lipid environment. This strategy possibly allows for probing the multicomponent complex TF:FVIIa together with its substrate Factor X on a lipid bilayer, but may also be relevant as a production strategy for other SPTMP for which structural information, in general, is limited.


Assuntos
Fator VIIa/metabolismo , Bicamadas Lipídicas/química , Complexos Multiproteicos/química , Dobramento de Proteína , Tromboplastina/química , Animais , Escherichia coli/citologia , Escherichia coli/metabolismo , Fator X/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Métodos , Nanoestruturas , Ligação Proteica , Tromboplastina/metabolismo
11.
Biochem Biophys Res Commun ; 524(2): 484-489, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32007271

RESUMO

DNA-binding proteins from starved cells (Dps) in Escherichia coli protects DNA from multiple stresses during the stationary phase by forming a stable Dps-DNA complex. In contrast, Dps cannot bind to DNA during the exponential phase and it has not been clear why Dps conditionally binds to DNA depending on the growth phase. In this study, we show that DNA-free Dps in the exponential phase can also bind to RNA and the preemptive binding of RNA precludes DNA from interacting with Dps. The critical role of RNA in modulating the stability and functional competence of Dps and their morphology, leads us to propose a two-state model of Dps in executing stress responses. In the exponential phase, Dps is present predominantly as ribonucleoprotein complex. Under starvation, RNAs are degraded by up-regulated RNases, activating Dps to bind with chromosomal DNAs protecting them from diverse stresses. A dual role of RNA as an inhibitor of DNA binding and chaperone to keep dynamic functional status of Dps would be crucial for operating an immediate protection of chromosomal DNAs on starvation. The holdase-type chaperoning role of RNA in Dps-mediated stress responses would shed light on the role of RNAs as chaperone (Chaperna).


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/citologia , Infecções por Escherichia coli/microbiologia , Humanos , Estresse Fisiológico
12.
J Basic Microbiol ; 60(2): 136-148, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32011760

RESUMO

Histone-like nucleoid-structuring protein (H-NS) and integration host factor (IHF) are major nucleoid-associated proteins, and DnaA, a replication initiator, may also be related with nucleoid compaction. It has been shown that protein-dependent DNA compaction is related with many aspects of bacterial physiology, including transcription, DNA replication, and site-specific recombination. However, the mechanism of bacterial physiology resulting from nucleoid compaction remains unknown. Here, we show that H-NS is important for correct nucleoid compaction in a medium-independent manner. H-NS-mediated nucleoid compaction is not required for correct cell division, but the latter is dependent on H-NS in rich medium. Further, it is found that the IHFα-mediated nucleoid compaction is needed for correct cell division, and the effect is dependent on medium. Also, we show that the effects of H-NS and IHF on nucleoid compaction are cumulative. Interestingly, DnaA also plays an important role in nucleoid compaction, and the effect of DnaA on nucleoid compaction appears to be related to cell division in a medium-dependent manner. The results presented here suggest that scrambled initiation of replication, improper cell division, and slow growth is likely associated with disturbances in nucleoid organization directly or indirectly.


Assuntos
Proteínas de Bactérias/genética , Divisão Celular , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/citologia , Proteínas de Fímbrias/genética , Fatores Hospedeiros de Integração/genética , Cromossomos Bacterianos/genética , Meios de Cultura , DNA Bacteriano/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica
13.
Proc Natl Acad Sci U S A ; 117(5): 2326-2331, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964833

RESUMO

Suspending self-propelled "pushers" in a liquid lowers its viscosity. We study how this phenomenon depends on system size in bacterial suspensions using bulk rheometry and particle-tracking rheoimaging. Above the critical bacterial volume fraction needed to decrease the viscosity to zero, [Formula: see text], large-scale collective motion emerges in the quiescent state, and the flow becomes nonlinear. We confirm a theoretical prediction that such instability should be suppressed by confinement. Our results also show that a recent application of active liquid-crystal theory to such systems is untenable.


Assuntos
Fenômenos Fisiológicos Bacterianos , Suspensões/química , Bactérias/citologia , Rastreamento de Células , Escherichia coli/citologia , Escherichia coli/fisiologia , Locomoção , Reologia , Resistência ao Cisalhamento , Viscosidade
14.
Can J Microbiol ; 66(4): 313-327, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31971820

RESUMO

In Escherichia coli, the N-terminal domain of the essential protein FtsK (FtsKN) is proposed to modulate septum formation through the formation of dynamic and essential protein interactions with both the Z-ring and late-stage division machinery. Using genomic mutagenesis, complementation analysis, and in vitro pull-down assays, we aimed to identify protein interaction partners of FtsK essential to its function during division. Here, we identified the cytoplasmic Z-ring membrane anchoring protein FtsA as a direct protein-protein interaction partner of FtsK. Random genomic mutagenesis of an ftsK temperature-sensitive strain of E. coli revealed an FtsA point mutation (G50E) that is able to fully restore normal cell growth and morphology, and further targeted site-directed mutagenesis of FtsA revealed several other point mutations capable of fully suppressing the essential requirement for functional FtsK. Together, this provides insight into a potential novel co-complex formed between these components during division and suggests FtsA may directly impact FtsK function.


Assuntos
Divisão Celular , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Mutagênese , Mutação de Sentido Incorreto , Ligação Proteica
15.
Proc Natl Acad Sci U S A ; 117(4): 1902-1909, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932440

RESUMO

Executing gene circuits by cell-free transcription-translation into cell-sized compartments, such as liposomes, is one of the major bottom-up approaches to building minimal cells. The dynamic synthesis and proper self-assembly of macromolecular structures inside liposomes, the cytoskeleton in particular, stands as a central limitation to the development of cell analogs genetically programmed. In this work, we express the Escherichia coli gene mreB inside vesicles with bilayers made of lipid-polyethylene glycol (PEG). We demonstrate that two-dimensional molecular crowding, emulated by the PEG molecules at the lipid bilayer, is enough to promote the polymerization of the protein MreB at the inner membrane into a sturdy cytoskeleton capable of transforming spherical liposomes into elongated shapes, such as rod-like compartments. We quantitatively describe this mechanism with respect to the size of liposomes, lipid composition of the membrane, crowding at the membrane, and strength of MreB synthesis. So far unexplored, molecular crowding at the surface of synthetic cells emerges as an additional development with potential broad applications. The symmetry breaking observed could be an important step toward compartment self-reproduction.


Assuntos
Células Artificiais/metabolismo , Membrana Celular/metabolismo , Forma Celular , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Lipossomos/metabolismo , Membrana Celular/química , Citoesqueleto/química , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lipossomos/química , Polimerização , Biossíntese de Proteínas , Conformação Proteica
16.
Soft Matter ; 16(5): 1359-1365, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31934708

RESUMO

We confine a dense suspension of motile Escherichia coli inside a spherical droplet in a water-in-oil emulsion, creating a "bacterially" propelled droplet. We show that droplets move in a persistent random walk, with a persistence time τ∼ 0.3 s, a long-time diffusion coefficient D∼ 0.5 µm2 s-1, and an average instantaneous speed V∼ 1.5 µm s-1 when the bacterial suspension is at the maximum studied concentration. Several droplets are analyzed, varying the drop radius and bacterial concentration. We show that the persistence time, diffusion coefficient and average speed increase with the bacterial concentration inside the drop, but are largely independent of the droplet size. By measuring the turbulent-like motion of the bacteria inside the drop, we demonstrate that the mean velocity of the bacteria near the bottom of the drop, which is separated from a glass substrate by a thin lubrication oil film, is antiparallel to the instantaneous velocity of the drop. This suggests that the driving mechanism is a slippery rolling of the drop over the substrate, caused by the collective motion of the bacteria. Our results show that microscopic organisms can transfer useful mechanical energy to their confining environment, opening the way to the assembly of mesoscopic motors composed of microswimmers.


Assuntos
Escherichia coli/química , Óleos/química , Fenômenos Biomecânicos , Emulsões/química , Escherichia coli/citologia , Propriedades de Superfície , Tensoativos/química , Água/química
17.
PLoS One ; 15(1): e0227601, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978064

RESUMO

The diversity of living cells, in both size and internal complexity, calls for imaging methods with adaptable spatial resolution. Soft x-ray tomography (SXT) is a three-dimensional imaging technique ideally suited to visualizing and quantifying the internal organization of single cells of varying sizes in a near-native state. The achievable resolution of the soft x-ray microscope is largely determined by the objective lens, but switching between objectives is extremely time-consuming and typically undertaken only during microscope maintenance procedures. Since the resolution of the optic is inversely proportional to the depth of focus, an optic capable of imaging the thickest cells is routinely selected. This unnecessarily limits the achievable resolution in smaller cells and eliminates the ability to obtain high-resolution images of regions of interest in larger cells. Here, we describe developments to overcome this shortfall and allow selection of microscope optics best suited to the specimen characteristics and data requirements. We demonstrate that switchable objective capability advances the flexibility of SXT to enable imaging cells ranging in size from bacteria to yeast and mammalian cells without physically modifying the microscope, and we demonstrate the use of this technology to image the same specimen with both optics.


Assuntos
Imageamento Tridimensional/métodos , Análise de Célula Única/métodos , Tomografia por Raios X/instrumentação , Tomografia por Raios X/métodos , Linfócitos B/citologia , Desenho de Equipamento , Escherichia coli/citologia , Humanos , Schizosaccharomyces/citologia , Análise de Célula Única/instrumentação
18.
Nat Commun ; 10(1): 5744, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848350

RESUMO

During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Citocinese/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Escherichia coli/citologia , Processamento de Imagem Assistida por Computador , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula
19.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848269

RESUMO

Bacillus subtilis and Escherichia coli are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, the two organisms follow the same phenomenological principle, known as the adder principle, for cell size homeostasis. We thus asked to what extent B. subtilis and E. coli share common physiological principles in coordinating growth and the cell cycle. We measured physiological parameters of B. subtilis under various steady-state growth conditions with and without translation inhibition at both the population and single-cell levels. These experiments revealed core physiological principles shared between B. subtilis and E. coli Specifically, both organisms maintain an invariant cell size per replication origin at initiation, under all steady-state conditions, and even during nutrient shifts at the single-cell level. Furthermore, the two organisms also inherit the same "hierarchy" of physiological parameters. On the basis of these findings, we suggest that the basic principles of coordination between growth and the cell cycle in bacteria may have been established early in evolutionary history.IMPORTANCE High-throughput, quantitative approaches have enabled the discovery of fundamental principles describing bacterial physiology. These principles provide a foundation for predicting the behavior of biological systems, a widely held aspiration. However, these approaches are often exclusively applied to the best-known model organism, E. coli In this report, we investigate to what extent quantitative principles discovered in Gram-negative E. coli are applicable to Gram-positive B. subtilis We found that these two extremely divergent bacterial species employ deeply similar strategies in order to coordinate growth, cell size, and the cell cycle. These similarities mean that the quantitative physiological principles described here can likely provide a beachhead for others who wish to understand additional, less-studied prokaryotes.


Assuntos
Bacillus subtilis/fisiologia , Fenômenos Fisiológicos Bacterianos , Divisão Celular , Replicação do DNA , Origem de Replicação , Bacillus subtilis/citologia , Ciclo Celular , Escherichia coli/citologia , Escherichia coli/fisiologia
20.
Sensors (Basel) ; 20(1)2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31881651

RESUMO

Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of E. coli between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (Kd) of 63 ± 6 µM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM-1493 µM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries.


Assuntos
Técnicas Biossensoriais , Sistemas Computacionais , Isoleucina/análise , Nanopartículas/química , Escherichia coli/citologia , Transferência Ressonante de Energia de Fluorescência , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Viabilidade Microbiana , Simulação de Acoplamento Molecular , Mutação/genética , Saccharomyces cerevisiae/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA