A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

Suboptimal resource allocation in changing environments constrains response and growth in bacteria.

To respond to fluctuating conditions, microbes typically need to synthesize novel proteins. As this synthesis relies on sufficient biosynthetic precursors, microbes must devise effective response strategies to manage depleting precursors. To better understand these strategies, we investigate the active response of Escherichia coli to changes in nutrient conditions, connecting transient gene expression to growth phenotypes. By synthetically modifying gene expression during changing conditions, we show how the competition by genes for the limited protein synthesis capacity constrains cellular response. Despite this constraint cells substantially express genes that are not required, trapping them in states where precursor levels are low and the genes needed to replenish the precursors are outcompeted. Contrary to common modeling assumptions, our findings highlight that cells do not optimize growth under changing environments but rather exhibit hardwired response strategies that may have evolved to promote fitness in their native environment. The constraint and the suboptimality of the cellular response uncovered provide a conceptual framework relevant for many research applications, from the prediction of evolution to the improvement of gene circuits in biotechnology.

Improved growth and morphological plasticity of Haloferax volcanii.

Some microbes display pleomorphism, showing variable cell shapes in a single culture, whereas others differentiate to adapt to changed environmental conditions. The pleomorphic archaeon Haloferax volcanii commonly forms discoid-shaped ('plate') cells in culture, but may also be present as rods, and can develop into motile rods in soft agar, or longer filaments in certain biofilms. Here we report improvement of H. volcanii growth in both semi-defined and complex media by supplementing with eight trace element micronutrients. With these supplemented media, transient development of plate cells into uniformly shaped rods was clearly observed during the early log phase of growth; cells then reverted to plates for the late log and stationary phases. In media prepared with high-purity water and reagents, without supplemental trace elements, rods and other complex elongated morphologies ('pleomorphic rods') were observed at all growth stages of the culture; the highly elongated cells sometimes displayed a substantial tubule at one or less frequently both poles, as well as unusual tapered and highly curved forms. Polar tubules were observed forming by initial mid-cell narrowing or tubulation, causing a dumbbell-like shape, followed by cell division towards one end. Formation of the uniform early log-phase rods, as well as the pleomorphic rods and tubules were dependent on the function of the tubulin-like cytoskeletal protein, CetZ1. Our results reveal the remarkable morphological plasticity of H. volcanii cells in response to multiple culture conditions, and should facilitate the use of this species in further studies of archaeal biology.

Archaeal cell biology: diverse functions of tubulin-like cytoskeletal proteins at the cell envelope.

The tubulin superfamily of cytoskeletal proteins is widespread in all three domains of life - Archaea, Bacteria and Eukarya. Tubulins build the microtubules of the eukaryotic cytoskeleton, whereas members of the homologous FtsZ family construct the division ring in prokaryotes and some eukaryotic organelles. Their functions are relatively poorly understood in archaea, yet these microbes contain a remarkable diversity of tubulin superfamily proteins, including FtsZ for division, a newly described major family called CetZ that is involved in archaeal cell shape control, and several other divergent families of unclear function that are implicated in a variety of cell envelope-remodelling contexts. Archaeal model organisms, particularly halophilic archaea such as Haloferax volcanii, have sufficiently developed genetic tools and we show why their large, flattened cells that are capable of controlled differentiation are also well suited to cell biological investigations by live-cell high-resolution light and electron microscopy. As most archaea only have a glycoprotein lattice S-layer, rather than a peptidoglycan cell wall like bacteria, the activity of the tubulin-like cytoskeletal proteins at the cell envelope is expected to vary significantly, and may involve direct membrane remodelling or directed synthesis or insertion of the S-layer protein subunits. Further studies of archaeal cell biology will provide fresh insight into the evolution of cells and the principles in common to their fundamental activities across the full spectrum of cellular life.