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 nonstop thrill ride from genes to the assembly of the T3SS injectisome

The type three secretion system (T3SS) is a membrane-anchored nano-machine utilized by many pathogenic bacteria to inject effector proteins and thus take control of host cells. In a recent article, Kaval et al. reveal a striking colocalization of a T3SS-encoding locus, its transcriptional activators, protein products, and the complete structure at the cell membrane, which they claim provides evidence for a mechanism known as ‘transertion’

Z-ring Structure and Constriction Dynamics in E. coli

The Z-ring plays a central role in bacterial division. It consists of FtsZ filaments, but the way these reorganize in the ring-like structure during septation remains largely unknown. Here, we measure the effective constriction dynamics of the ring. Using an oscillating optical trap, we can switch individual rod-shaped E. coli cells between horizontal and vertical orientations. In the vertical orientation, the fluorescent Z-ring image appears as a symmetric circular structure that renders itself to quantitative analysis. In the horizontal orientation, we use phase-contrast imaging to determine the extent of the cell constriction and obtain the effective time of division. We find evidence that the Z-ring constricts at a faster rate than the cell envelope such that its radial width (inwards from the cytoplasmic membrane) grows during septation. In this respect, our results differ from those recently obtained using photoactivated localization microscopy (PALM) where the radial width of the Z-ring was found to be approximately constant as the ring constricts. A possible reason for the different behavior of the constricting Z-rings could be the significant difference in the corresponding cell growth rates

DnaA constructs used in this study.

<p>The constructs were engineered as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036441#s4" target="_blank">Materials and Methods</a>. Amino acids flanking DnaA domains and fragments are shown. Domains I and II are in white, mCherry sequence is colored black, domain III is represented by subdomain IIIa (orange) and IIIb (red) connected by the linker (blue) to domain IV (in green). Point mutation in domain IV, L417P, is also marked.</p

Increase of DnaA-mCherry content in the membrane fraction as a function of its expression level.

<p><i>E. coli</i> BL21 harboring pBAD24(<i>dnaA-mCherry</i>) was induced by 0.2% arabinose for time periods shown on the graph in the inset. DnaA-mCherry content in cell fractions is presented in mCherry fluorescence intensity units, determined as described in Experimental procedures.</p