Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD

Bergmiller, Tobias; Peña‐Miller, Rafael; Boehm, Alexander; Ackermann, Martin

doi:10.1186/1471-2180-11-118

Cited by 20 publications

(14 citation statements)

References 41 publications

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“…In E. coli, a decrease in cell size was found to be associated with a reduced growth during the stringent response (34,35,41). We noticed that C. crescentus cells accumulating (p)ppGpp often appeared smaller under the microscope (see Fig.…”

Section: Resultsmentioning

confidence: 85%

Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

González

Collier

2014

J Bacteriol

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Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra-or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.

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Section: Resultsmentioning

confidence: 85%

Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

González

Collier

2014

J Bacteriol

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“…To be able to reconstruct a proper PALM image, it is of utmost importance that cells remain immobile during the whole acquisition time. This has been ordinarily achieved by immobilizing cells in agarose pads, which have the added advantage that they allow for time-lapse imaging under conditions that promote normal growth [13], [14], [27], [28].…”

Section: Resultsmentioning

confidence: 99%

Super-Resolution Imaging of Bacteria in a Microfluidics Device

et al. 2013

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Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency, accuracy, adaptability, and redundancy. Super-resolution fluorescence microscopy methods are ideally suited to investigate the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. These techniques require the long-term stability of samples, high signal-to-noise-ratios, low chromatic aberrations and surface flatness, conditions difficult to meet with traditional immobilization methods. We present a method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages, as it permits the long-term immobilization of cells and proper correction of drift, avoids chromatic aberrations caused by the use of different filter sets, and allows for the flat immobilization of cells on the surface. In addition, we show that different surface chemistries can be used to image bacteria at different time-scales, and we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level.

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“…Together these observations imply that cells require an intact EKC/KEOPS complex to work properly and if this is not achieved, cells experience growth problems as a result of improper translation. The only ortholog of components of the KEOPS complex in Escherichia coli , Kae1 (YgjD) is essential 24 and using an inducible construct it has been shown that YgjD is also required for cell growth 25 . These results suggest that t 6 A synthesis is the most primitive function of Kae1, incorporated during evolution into the EKC/KEOPS complex to fulfill physiological requirements of eukaryotic and archeal cells.…”

Section: The Ekc/keops Complex Is Required For Trna Modification and mentioning

confidence: 98%

TheDrosophilaEKC/KEOPS complex

Rojas-Benítez

Ibar²,

Glavic³

2013

Fly

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The TOR signaling pathway is crucial in the translation of nutritional inputs into the protein synthesis machinery regulation, allowing animal growth. We recently identified the Bud32 (yeast)/PRPK (human) ortholog in Drosophila, Prpk (p53-related protein kinase), and found that it is required for TOR kinase activity. Bud32/PRPK is an ancient and atypical kinase conserved in evolution from Archeae to humans, being essential for Archeae. It has been linked with p53 stabilization in human cell culture and its absence in yeast causes a slow-growth phenotype. This protein has been associated to KEOPS (kinase, putative endopeptidase and other proteins of small size) complex together with Kae1p (ATPase), Cgi-121 and Pcc1p. This complex has been implicated in telomere maintenance, transcriptional regulation, bud site selection and chemical modification of tRNAs (tRNAs). Bud32p and Kae1p have been related with N6-threonylcarbamoyladenosine (t6A) synthesis, a particular chemical modification that occurs at position 37 of tRNAs that pair A-starting codons, required for proper translation in most species. Lack of this modification causes mistranslations and open reading frame shifts in yeast. The core constituents of the KEOPS complex are present in Drosophila, but their physical interaction has not been reported yet. Here, we present a review of the findings regarding the function of this complex in different organisms and new evidence that extends our recent observations of Prpk function in animal growth showing that depletion of Kae1 or Prpk, in accordance with their role in translation in yeast, is able to induce the unfolded protein response (UPR) in Drosophila. We suggest that EKC/KEOPS complex could be integrating t6A-modified tRNA availability with translational rates, which are ultimately reflected in animal growth.

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Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD

Cited by 20 publications

References 41 publications

Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

Super-Resolution Imaging of Bacteria in a Microfluidics Device

TheDrosophilaEKC/KEOPS complex

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