Abstract
Haloferax mediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.
Highlights
Polyhydroxyalkanoates (PHAs) are biopolymers produced and accumulated in many prokaryotic microbes as carbon and energy storage materials, ensuring their survival under stress conditions [1]
We show that double-staining with SYBR
Staining with Nile red was performed with the cells of a known producer of PHBV, Haloferax mediterranei, growing in an aqueous and high-salt-grown medium (3.3 M NaCl)
Summary
Polyhydroxyalkanoates (PHAs) are biopolymers produced and accumulated in many prokaryotic microbes (bacteria or archaea) as carbon and energy storage materials, ensuring their survival under stress conditions [1]. PHA biosynthesis is favored by limiting an essential nutrient—such as nitrogen, phosphorous, or oxygen—for microbial growth, with a parallel high availability of an exogenous carbon source [2]. They are water insoluble polymers and stored in the cell cytoplasm as granules. PHAs are biodegradable, biocompatible, and thermoplastic polymers, and due to these features, they have recently attracted increasing attention for their industrial application in the production of biodegradable plastic used for packaging or in the biomedical field [3,4] In this context, extremophilic microbes in general, and the haloarchaea group (Archaea domain), have attracted the attention of the scientific community due to their peculiar metabolic capabilities. Other biocompounds and biomolecules of significant interest are gas vesicles, which are protein-based buoyancy organelles produced by some halophilic archaea with a potential role in immunology for the generation of vaccines due to their role as scaffolds during the presentation of epitopes [6]; ether-linked lipids as novel drug delivery systems [8]; bacteriorhodopsin, a photochemical material for bioelectronics and photochemical processes [5]; and enzymes with high activity and stability at high temperatures, and high ionic strength in industrial applications [9]
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