Characterization of Escherichia coli nucleoids released by osmotic shock

Wegner, Anna S.; Alexeeva, Svetlana; Odijk, Theo; Woldringh, Conrad L.

doi:10.1016/j.jsb.2012.03.007

Cited by 30 publications

(42 citation statements)

References 52 publications

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“…More expanded nucleoids in high salt were generally observed to have multiple-lobed structures (Fig. 1C) that may reflect the replication or segregation state of the chromosome (21).…”

Section: Isolation and Visualization Of Nucleoidsmentioning

confidence: 97%

“…Following the protocol used by Wegner et al (21), cells were incubated with lysozyme for less than 10 min. Osmotic shock was applied within 5 min of transferring the cells onto the microscope slide by gently adding 500 l of hypotonic buffer (10 mM NaCl) into the well, resulting in a 100-fold dilution of the cells (21). While some cells were spherical before hypotonic treatment, many rounded up only after addition of hypotonic buffer.…”

Section: Bacterial Strain Expressing Gfp-fis Proteinmentioning

confidence: 99%

“…In short, we isolated nucleoids from Escherichia coli cells modified to express a green fluorescent protein (GFP) fusion of Fis (14). Osmotic shock (20,21) was used to release the nucleoid, which avoids detergents that can destabilize proteins bound to DNA (22,23) and which is optimal for maintaining in vivo nucleoid organization (20). We monitored the dynamics of the GFP-Fis fluorescence as a function of time in the presence of varied concentrations of bulk nonfluorescent wild-type Fis (WT Fis) protein, the heterotypic nucleoid protein HU, and varied salt concentrations.…”

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confidence: 99%

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Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

2016

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Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or "facilitated dissociation." Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an "exchange" rate constant (k exch ) of Ϸ10 4 M ؊1 s ؊1 , comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo. A ll aspects of chromosome dynamics involve the binding and unbinding of proteins from the DNA double helix. The rate of binding for a given species of protein to a location along a DNA is usually controlled by concentration of that species, but it is widely assumed that unbinding times, or equivalently off-rates, are controlled by the strength of interactions inside the DNA-protein complex and are independent of other nearby molecules in the nucleoplasm (1). However, this picture has been challenged by a number of recent in vitro experiments which have shown off-rates of proteins from duplex DNA that depend on bulk protein concentrations (2-5). Similar effects have been observed for singlestranded DNA (ssDNA)-binding proteins (6, 7), antibody-ligand binding (8), and ssDNA-ssDNA interactions (9).A straightforward explanation for this effect is that macroscopic dissociation (complete dissociation followed by diffusive motion of a protein to a location far away from the initial binding site) occurs via one or more partially dissociated intermediate ("microdissociated") states. A protein in bulk solution could impact dissociation events in a variety of ways (5, 9). This interaction can affect the rate of macroscopic dissociation of the original protein, for example, by having the second "invading" protein "block" rebinding of the first protein (5, 10, 11). Alternatively, both the initially bound and invading proteins might bind at adjacent sites, with the second protein accelerating the off-rate of the first protein via allosteric interactions (12). In either case, formation of a transient ternary complex (initially bound protein plus DNA site plus invading protein) may lead to appreciable increases in off-rates as the bulk protein concentration is increased. In the molecularly crowded cell interior, the rates of replacement of proteins on DNA may well be very different from what might be predicted from dilute-solution studies of binding kinetics, a fact that may help reconcile observations of rapid turnover on DNA in vivo but slower kinetics observed in vitro (13)(14)(15)(16)(17).An example o...

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“…More expanded nucleoids in high salt were generally observed to have multiple-lobed structures (Fig. 1C) that may reflect the replication or segregation state of the chromosome (21).…”

Section: Isolation and Visualization Of Nucleoidsmentioning

confidence: 97%

Section: Bacterial Strain Expressing Gfp-fis Proteinmentioning

confidence: 99%

mentioning

confidence: 99%

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Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

2016

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“…The small size of the cell prevented the use of micropipettes (20) or microneedles (21) that have been successful in probing much larger eukaryotic chromosomes and spindles. Instead, we started from previous work that demonstrated the proof of principle of bacterial chromosome extraction in a microfluidic device (26) and lysis in suspension (27). As illustrated in Fig.…”

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confidence: 99%

“…1 C-D is driven mainly by the release of micromechanical energy stored in the in vivo chromosome, rather than by dissociation of nucleoid-associated proteins. We cannot exclude the possibility that fast dissociation of unlabeled nucleoid-associated proteins or Mg 2þ contributed to chromosome expansion or modulated the unbinding rates of other nucleoid-associated proteins (37); however, lysed in suspension (27), chromosomes expanded to comparable in vitro volumes in the presence and absence of Mg 2þ and adenosine-5′-triphosphate (ATP) (see SI Appendix, Section I D).…”

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confidence: 99%

Physical manipulation of the Escherichia coli chromosome reveals its soft nature

Pelletier

Halvorsen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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193

270

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Replicating bacterial chromosomes continuously demix from each other and segregate within a compact volume inside the cell called the nucleoid. Although many proteins involved in this process have been identified, the nature of the global forces that shape and segregate the chromosomes has remained unclear because of limited knowledge of the micromechanical properties of the chromosome. In this work, we demonstrate experimentally the fundamentally soft nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment. We developed a unique "micropiston" and measured the force-compression behavior of single Escherichia coli chromosomes in confinement. Our data show that forces on the order of 100 pN and free energies on the order of 10 5 k B T are sufficient to compress the chromosome to its in vivo size. For comparison, the pressure required to hold the chromosome at this size is a thousand-fold smaller than the surrounding turgor pressure inside the cell. Furthermore, by manipulation of molecular crowding conditions (entropic forces), we were able to observe in real time fast (approximately 10 s), abrupt, reversible, and repeatable compaction-decompaction cycles of individual chromosomes in confinement. In contrast, we observed much slower dissociation kinetics of a histone-like protein HU from the whole chromosome during its in vivo to in vitro transition. These results for the first time provide quantitative, experimental support for a physical model in which the bacterial chromosome behaves as a loaded entropic spring in vivo.chromosome segregation | depletion forces | polymer physics | mother machine | optical trap

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New horizons in developing cell lysis methods: A review

Danaeifar

2022

Biotech & Bioengineering

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Cell lysis is an essential step in many studies related to biology and medicine. Based on the scale and medium that cell lysis is carried out, there are three main types of the cell lysis: (1) lysis of the cells in the surrounding environment, (2) lysis of the isolated or cultured cells, and (3) single cell lysis. Conventionally, several cell lysis methods have been developed, such as freeze‐thawing, bead beating, incursion in liquid nitrogen, sonication, and enzymatic and chemical‐based approaches. In recent years, various novel technologies have been employed to develop new methods of cell lysis. The aim of studies in this field is to introduce more precise and efficient tools or to reduce the costs of cell lysis procedures. Nanostructure‐based lysis methods, acoustic oscillation, electrical current, irradiation, bacteria‐mediated cell lysis, magnetic ionic liquids, bacteriophage genes, monolith columns, hydraulic forces, and steam explosion are some examples of newly developed cell lysis methods. Besides the significant advances in this field, there are still many challenges and tools must be further improved.

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Characterization of Escherichia coli nucleoids released by osmotic shock

Cited by 30 publications

References 52 publications

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

Physical manipulation of the Escherichia coli chromosome reveals its soft nature

New horizons in developing cell lysis methods: A review

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