An Oscillating MinD Protein Determines the Cellular Positioning of the Motility Machinery in Archaea.

MinD proteins are well studied in rod-shaped bacteria such as E. coli, where they display self-organized pole-to-pole oscillations that are important for correct positioning of the Z-ring at mid-cell for cell division. Archaea also encode proteins belonging to the MinD family, but their functions are unknown. MinD homologous proteins were found to be widespread in Euryarchaeota and form a sister group to the bacterial MinD family, distinct from the ParA and other related ATPase families. We aimed to identify the function of four archaeal MinD proteins in the model archaeon Haloferax volcanii. Deletion of the minD genes did not cause cell division or size defects, and the Z-ring was still correctly positioned. Instead, one of the deletions (ΔminD4) reduced swimming motility and hampered the correct formation of motility machinery at the cell poles. In ΔminD4 cells, there is reduced formation of the motility structure and chemosensory arrays, which are essential for signal transduction. In bacteria, several members of the ParA family can position the motility structure and chemosensory arrays via binding to a landmark protein, and consequently these proteins do not oscillate along the cell axis. However, GFP-MinD4 displayed pole-to-pole oscillation and formed polar patches or foci in H. volcanii. The MinD4 membrane-targeting sequence (MTS), homologous to the bacterial MinD MTS, was essential for the oscillation. Surprisingly, mutant MinD4 proteins failed to form polar patches. Thus, MinD4 from H. volcanii combines traits of different bacterial ParA/MinD proteins.

Positioning of the Motility Machinery in Halophilic Archaea.

Bacteria and archaea exhibit tactical behavior and can move up and down chemical gradients. This tactical behavior relies on a motility structure, which is guided by a chemosensory system. Environmental signals are sensed by membrane-inserted chemosensory receptors that are organized in large ordered arrays. While the cellular positioning of the chemotaxis machinery and that of the flagellum have been studied in detail in bacteria, we have little knowledge about the localization of such macromolecular assemblies in archaea. Although the archaeal motility structure, the archaellum, is fundamentally different from the flagellum, archaea have received the chemosensory machinery from bacteria and have connected this system with the archaellum. Here, we applied a combination of time-lapse imaging and fluorescence and electron microscopy using the model euryarchaeon Haloferax volcanii and found that archaella were specifically present at the cell poles of actively dividing rod-shaped cells. The chemosensory arrays also had a polar preference, but in addition, several smaller arrays moved freely in the lateral membranes. In the stationary phase, rod-shaped cells became round and chemosensory arrays were disassembled. The positioning of archaella and that of chemosensory arrays are not interdependent and likely require an independent form of positioning machinery. This work showed that, in the rod-shaped haloarchaeal cells, the positioning of the archaellum and of the chemosensory arrays is regulated in time and in space. These insights into the cellular organization of H. volcanii suggest the presence of an active mechanism responsible for the positioning of macromolecular protein complexes in archaea.IMPORTANCE Archaea are ubiquitous single cellular microorganisms that play important ecological roles in nature. The intracellular organization of archaeal cells is among the unresolved mysteries of archaeal biology. With this work, we show that cells of haloarchaea are polarized. The cellular positioning of proteins involved in chemotaxis and motility is spatially and temporally organized in these cells. This suggests the presence of a specific mechanism responsible for the positioning of macromolecular protein complexes in archaea.

Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging.

Using the haloarchaeon Haloferax volcanii as a model, we developed nascent DNA labeling and the functional GFP-labeled single-stranded binding protein RPA2 as novel tools to gain new insight into DNA replication and repair in live haloarchaeal cells. Our quantitative fluorescence microscopy data revealed that RPA2 forms distinct replication structures that dynamically responded to replication stress and DNA damaging agents. The number of the RPA2 foci per cell followed a probabilistic Poisson distribution, implying hitherto unnoticed stochastic cell-to-cell variation in haloarchaeal DNA replication and repair processes. The size range of haloarchaeal replication structures is very similar to those observed earlier in eukaryotic cells. The improved lateral resolution of 3D-SIM fluorescence microscopy allowed proposing that inhibition of DNA synthesis results in localized replication foci clustering and facilitated observation of RPA2 complexes brought about by chemical agents creating DNA double-strand breaks. Altogether our in vivo observations are compatible with earlier in vitro studies on archaeal single-stranded DNA binding proteins. Our work thus underlines the great potential of live cell imaging for unraveling the dynamic nature of transient molecular interactions that underpin fundamental molecular processes in the Third domain of life.

DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea.