16 research outputs found
An Easy-To-Use Simulation Program Demonstrates Variations in Bacterial Cell Cycle Parameters Depending on Medium and Temperature
Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results
Association of the Chromosome Replication Initiator DnaA with the Escherichia coli Inner Membrane In Vivo: Quantity and Mode of Binding
DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein’s domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication
A Coarse-Grained Biophysical Model of E. coli and Its Application to Perturbation of the rRNA Operon Copy Number
We propose a biophysical model of Escherichia coli that predicts growth rate
and an effective cellular composition from an effective, coarse-grained
representation of its genome. We assume that E. coli is in a state of balanced
exponential steadystate growth, growing in a temporally and spatially constant
environment, rich in resources. We apply this model to a series of past
measurements, where the growth rate and rRNA-to-protein ratio have been
measured for seven E. coli strains with an rRNA operon copy number ranging from
one to seven (the wild-type copy number). These experiments show that growth
rate markedly decreases for strains with fewer than six copies. Using the
model, we were able to reproduce these measurements. We show that the model
that best fits these data suggests that the volume fraction of macromolecules
inside E. coli is not fixed when the rRNA operon copy number is varied.
Moreover, the model predicts that increasing the copy number beyond seven
results in a cytoplasm densely packed with ribosomes and proteins. Assuming
that under such overcrowded conditions prolonged diffusion times tend to weaken
binding affinities, the model predicts that growth rate will not increase
substantially beyond the wild-type growth rate, as indicated by other
experiments. Our model therefore suggests that changing the rRNA operon copy
number of wild-type E. coli cells growing in a constant rich environment does
not substantially increase their growth rate. Other observations regarding
strains with an altered rRNA operon copy number, such as nucleoid compaction
and the rRNA operon feedback response, appear to be qualitatively consistent
with this model. In addition, we discuss possible design principles suggested
by the model and propose further experiments to test its validity
Stability of bacterial toggle switches is enhanced by cell-cycle lengthening by several orders of magnitude
Copy-number control of the Escherichia coli chromosome: a plasmidologist's view
The homeostatic system that sets the copy number, and corrects over-replication and under-replication, seems to be different for chromosomes and plasmids in bacteria. Whereas plasmid replication is random in time, chromosome replication is tightly coordinated with the cell cycle such that all origins are initiated synchronously at the same cell mass per origin once per cell cycle. In this review, we propose that despite their apparent differences, the copy-number control of the Escherichia coli chromosome is similar to that of plasmids. The basic mechanism that is shared by both systems is negative-feedback control of the availability of a protein or RNA positive initiator. Superimposed on this basic mechanism are at least three systems that secure the synchronous initiation of multiple origins; however, these mechanisms are not essential for maintaining the copy number
Evolution of the Clock from Yeast to Man by Period-Doubling Folds in the Cellular Oscillator
Inflating bacterial cells by increased protein synthesis
Understanding how the homeostasis of cellular size and composition is accomplished by different organisms is an outstanding challenge in biology. For exponentially growing Escherichia coli cells, it is long known that the size of cells exhibits a strong positive relation with their growth rates in different nutrient conditions. Here, we characterized cell sizes in a set of orthogonal growth limitations. We report that cell size and mass exhibit positive or negative dependences with growth rate depending on the growth limitation applied. In particular, synthesizing large amounts of "useless" proteins led to an inversion of the canonical, positive relation, with slow growing cells enlarged 7- to 8-fold compared to cells growing at similar rates under nutrient limitation. Strikingly, this increase in cell size was accompanied by a 3- to 4-fold increase in cellular DNA content at slow growth, reaching up to an amount equivalent to similar to 8 chromosomes per cell. Despite drastic changes in cell mass and macromolecular composition, cellular dry mass density remained constant. Our findings reveal an important role of protein synthesis in cell division control.SCI(E)[email protected]; [email protected]