The <i>Escherichia coli</i> baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle

Bates, David; Epstein, Jessica; Boye, Erik; Fahrner, Karen; Berg, Howard C.; Kleckner, Nancy

doi:10.1111/j.1365-2958.2005.04693.x

Cited by 66 publications

(71 citation statements)

References 22 publications

(36 reference statements)

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“…with a His 6 tag at the N terminus were cloned into the expression vector pQE-30 (Qiagen) and transformed into E. coli M15 cells. The expression vector of BpHsp70 was also transformed into E. coli ⌬fliC Ϫ/Ϫ strain NK9375 kindly provided by Dr. N. Kleckner (15). The recombinant proteins were expressed and purified as described previously (16).…”

Section: Methodsmentioning

confidence: 99%

Flagellin Contamination of Recombinant Heat Shock Protein 70 Is Responsible for Its Activity on T Cells

Gan

2007

Journal of Biological Chemistry

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Heat shock proteins (Hsp) 60 and 70 have been intensively studied for their ability to activate innate immunity. Heat shock proteins had been shown to induce the activation of dendritic cells, T cells, and B cells. However, the possible contamination of endotoxin in heat shock protein preparations makes their function as an activator of immune system ambiguous. Here, we examined the ability of bacterial Hsp60 and Hsp70 to activate Jurkat T cells and primary T cells. We found that Burkholderia pseudomallei Hsp70 and Mycobacterium tuberculosis Hsp70 could costimulate Jurkat T cells to make IL-2 and signal through TLR5. This costimulatory activity is not due to endotoxin or contaminants signaling via TLR2 nor TLR4. However, recombinant Hsp70 expressed in Escherichia coli ⌬fliC strain completely lost its ability to costimulate T cells. Thus, the activation of T cells by recombinant Hsp70 is ascribed to flagellin contamination.Although heat shock proteins 60 and 70 have been touted to be potent activators of the innate immune system (1-3), recent studies have urged caution in the interpretation of these results due to the possibility of bacterial contaminants in the recombinant protein preparations. Recombinant human Hsp70 and Hsp60 cleaned of endotoxin contamination failed to induce activation and cytokine secretion from antigen-presenting cells such as macrophages (4 -6). There had also been reports on the ability of heat shock proteins to activate T cells and B cells. For example, human T cells were reported to directly respond to soluble human Hsp60 via TLR2 (7,8). The same group also reported activation of B cells by human Hsp60 via the TLR4-MyD88 pathway (9). Lipopolysaccharide (LPS) 2 -free eukaryotic Hsp60s were shown to enhance antigen-specific murine T cell activation (10).Besides mammalian Hsps, mycobacterial Hsp70 has been shown to stimulate the release of cytokines and chemokines from dendritic cells (11,12). Furthermore, a mycobacterial Hsp70 fusion protein with ovalbumin was shown to elicit a CD4ϩ T cell-independent, ovalbumin-specific cytotoxic T lymphocyte (CTL) response in mice (13). Harmala et al. (14) extended the finding by demonstrating that immunization with the fusion protein in mice was qualitatively and quantitatively superior to complete Freund's adjuvant and LPS in eliciting antigen-specific CTL responses in vivo and the adjuvant effect of the fusion protein was not due to LPS contamination. However, the mechanism of the adjuvanticity of the fusion protein was not addressed.To determine whether bacterial Hsps belonging to the 60 and 70 families have a direct effect on T cells as had been reported for mammalian Hsp60 (10), we examine the ability of mycobacterial Hsp65 and Hsp70, as well as Burkholderia pseudomallei Hsp60 and Hsp70 to activate the human Jurkat T cell line as well as primary CD4 ϩ T cells. Only Hsp70s are found to enhance T cell activation. However, this effect is completely abrogated when the recombinant proteins are made in Escherichia coli with the flagellin gene deleted...

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Section: Methodsmentioning

confidence: 99%

Flagellin Contamination of Recombinant Heat Shock Protein 70 Is Responsible for Its Activity on T Cells

Gan

2007

Journal of Biological Chemistry

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“…The cell cycle with the DNA content per cell as a readout [¡G 1 ¡S¡G 2 ¡D(M)¡] is invariant across kingdoms, with the two critical stages, S and D(M), corresponding to two critical chromosomal transactions, replication and segregation (141)(142)(143). Because of the necessary ϳ1,000ϫ degree of intracellular compaction (reviewed in reference 101), chromosomal DNA has to decompact and recompact in order to replicate and segregate.…”

Section: The Two Chromosome Cyclesmentioning

confidence: 99%

The Precarious Prokaryotic Chromosome

Kuzminov

2014

J Bacteriol

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Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the two distinct ways to organize chromosomes are driven by the differences between the global-consecutive chromosome cycle of eukaryotes and the local-concurrent chromosome cycle of prokaryotes. Specifically, progressive chromosome segregation in prokaryotes demands a single duplicon per chromosome, while other "precarious" features of the prokaryotic chromosomes can be viewed as compensations for this severe restriction.

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“…We previously defined E. coli chromosome dynamics at high temporal resolution in synchronous cultures in the same conditions used here (1,2). Cells tethered to a glass bead column release newly divided (newborn) cells.…”

Section: Sister Splitting Times Define Two Late-splitting Intersistermentioning

confidence: 99%

“…We previously examined sister chromosome relationships over time in the Escherichia coli cell cycle (1). High temporal resolution (5-10 min) was achieved using synchronous populations (2). Sister relationships at individual loci were evaluated as in other studies.…”

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Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps

Joshi

Bourniquel

Fisher

et al. 2011

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

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110

176

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The basis for segregation of sister chromosomes in bacteria is not established. We show here that two discrete~150-kb regions, both located early in the right replichore, exhibit prolonged juxtaposition of sister loci, for 20 and 30 min, respectively, after replication. Flanking regions, meanwhile, separate. Thus, the two identified regions comprise specialized late-splitting intersister connections or snaps. Sister snap loci separate simultaneously in both snap regions, concomitant with a major global nucleoid reorganization that results in emergence of a bilobed nucleoid morphology. Split snap loci move rapidly apart to a separation distance comparable with one-half the length of the nucleoid. Concomitantly, at already split positions, sister loci undergo further separation to a comparable distance. The overall consequence of these and other effects is that thus far replicated sister chromosomes become spatially separated (individualized) into the two nucleoid lobes, while the terminus region (and likely, all unreplicated portions of the chromosome) moves to midcell. These and other findings imply that segregation of Escherichia coli sister chromosomes is not a smooth continuous process but involves at least one and likely, two major global transition(s). The presented patterns further suggest that accumulation of internal intranucleoid forces and constraining of these forces by snaps play central roles in global chromosome dynamics. They are consistent with and supportive of our previous proposals that individualization of sisters in E. coli is driven primarily by internally generated pushing forces and is directly analogous to sister individualization at the prophase to prometaphase transition of the eukaryotic cell cycle.E. coli chromosome | chromosome segregation | bacterial nucleoid I n bacteria, sister chromosomes segregate concomitant with DNA replication. We previously examined sister chromosome relationships over time in the Escherichia coli cell cycle (1). High temporal resolution (5-10 min) was achieved using synchronous populations (2). Sister relationships at individual loci were evaluated as in other studies. Also, uniquely, at a larger scale, whole nucleoid disposition and morphology were defined. This analysis identified a discrete transition, occurring part way through the replication process, in which the nucleoid becomes bilobed. This morphological change is accompanied by reciprocal repositioning of the replication origin (oriC) and terminus region (ter) and by strongly delayed splitting of one particular locus located near the replication origin (gln). These coordinate effects pointed to global reorganization of the nucleoid. We proposed that this reorganization resulted in spatial segregation of sister chromosomes and that it is analogous to the prophase to prometaphase transition of the eukaryotic cell cycle. We further proposed that, in both cases, sister individualization results from internally generated forces, more specifically, mechanical pushing effects (1, 3).Other models for seg...

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The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle

Cited by 66 publications

References 22 publications

Flagellin Contamination of Recombinant Heat Shock Protein 70 Is Responsible for Its Activity on T Cells

Flagellin Contamination of Recombinant Heat Shock Protein 70 Is Responsible for Its Activity on T Cells

The Precarious Prokaryotic Chromosome

Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps

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