Biology subjects

de Silva, R. T.

Publications and source records attributed to de Silva, R. T..

4 recordsLinked to original sources

bioRxiv2024

Dynamic self-association of archaeal tubulin-like protein CetZ1 drives Haloferax volcanii morphogenesis

Tubulin superfamily (TSF) proteins include the well-known eukaryotic tubulin and bacterial FtsZ families, and lesser-known archaeal CetZ family. In eukaryotes and bacteria, GTP-dependent polymerization and self-association of tubulin and FtsZ protofilaments are integral to the formation of cytoskeletal structures with essential roles in cell division, growth, and morphology. Archaeal CetZs are implicated in the control of cell shape and motility through unknown mechanisms. Here, we reveal a sequence of subcellular localization patterns of CetZ1, the prototypical member of the CetZ family, during stages of Haloferax volcanii rod cell development, in which it plays an essential role. Like tubulin and FtsZ, we found that CetZ1 formed GTP-dependent polymers in vitro, which appear to associate laterally as irregular polymer bundles. Mutations targeting regions predicted to mediate self-association and dynamic turnover of CetZ1, including the longitudinal (GTPase T7 and T4 loops) and lateral assembly interfaces, perturbed or altered rod shape development and subcellular assembly and dynamics, and caused corresponding effects on polymerization in vitro. Remarkably, a conspicuous amphipathic protrusion in the large microtubule (M-) loop, a characteristic of the CetZ1 subfamily, also strongly influenced function and assembly. Our findings reveal the importance of dynamic CetZ1 self-association in cellular morphogenesis involving multiple regions of the TSF fold, including tubulin- and FtsZ-like structural characteristics and CetZ1-specific features. Furthermore, they support a mechanism involving CetZ1 dynamic guidance of cell envelope-associated structures that reshape the cell during morphogenesis.

molecular biology↗

bioRxiv2024

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.

microbiology↗

bioRxiv2023

Stringent Control Over Cytoplasmic and Membrane Densities Defines Regulation of Cell Geometry in Escherichia coli

Microbes exhibit precise control over their composition and geometry in order to adapt and grow in diverse environments. However, the mechanisms that orchestrate this simultaneous regulation, and how they are causally linked, remains poorly understood. In this work, we derive and experimentally test a biophysical model of cell size regulation in Escherichia coli which relates the cellular surface-to-volume ratio to the total macromolecular composition and partitioning of the proteome between cellular compartments. Central to this model is the observation that the macromolecular density of the cytoplasm and the protein density within the cell membranes are maintained at a constant ratio across growth conditions. Using quantitative mass spectrometry, single-cell microscopy, and biochemical assays, we show this model quantitatively predicts a non-linear relationship between the surface-to-volume ratio, proteome localization, and the total ribosome content of the cell. This model holds under perturbations of intracellular ppGpp concentrations-thereby changing the ribosomal content-demonstrating that cellular geometry is not strictly determined by the cellular growth rate. These findings provide a biophysical link between the coregulation of proteome organization and cellular geometry, offering a quantitative framework for understanding bacterial size regulation across conditions.

microbiology↗

bioRxiv2020

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 rods, as well as the pleomorphic rods and tubules were dependent on the function of the tubulin-like cytoskeletal protein, CetZ1. Our results have revealed 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.

microbiology↗