Ultrastructural Analysis of the Extracellular Matter Secreted by the Psychrotolerant Bacterium Pseudoalteromonas antarctica NF3

Abstract

The psychrotolerant strain Pseudoalteromonas antarctica NF3, a Gram-negative bacterium isolated from muddy soil samples of Antarctica, secretes large amounts of a mucoid exopolymer with a high protein content. It has self-assembly properties and capacity to coat and protect liposomes against surfactants. We examined the ultrastructure of P. antarctica and the extracellular matter it secretes by transmission electron microscopy (TEM) after high-pressure freezing, freeze substitution (HPF-FS), and Epon embedding, and compared this with information obtained by conventional methods. The improvements brought about by HPF-FS to the ultrastructural preservation of the extracellular matter allowed us to establish for the first time, in P. antarctica NF3, the presence of two components: a large amount of cell-derived outer membrane vesicles containing proteins and a capsular polymer around the cells.

Membrane Vesicles Released by a hypervesiculating Escherichia coli Nissle 1917 tolR Mutant Are Highly Heterogeneous and Show Reduced Capacity for Epithelial Cell Interaction and Entry

“…Some studies have described vesicle morphology by scanning electron microscopy (SEM) or thin-section TEM of chemically fixed and dehydrated samples at room temperature [14,15,51–53]. However, in both methods the extracellular matter, which includes MVs, has a marked propensity to collapse and be removed during sample preparation [2,54]. Surprisingly, few studies have used TEM of cryo-immobilized specimens by HPF followed by FS, or cryo-TEM to visualize MV samples, although both have proven very useful for observing detailed structures of MVs and their producing strains [26,27,34,55,56].…”

Therapeutic Efficacy of Stable Analogues of Vasoactive Intestinal Peptide against Pathogens

“…Electron Microscopy-E. coli (10 6 ; incubated with 5 M VIP51 or VIP51(6 -30) at 37°C) or log phase promastigotes of L. major (5 ϫ 10 6 ; incubated with 12 M VIP51 or VIP51(6 -30) at 28°C) were harvested by centrifugation (2,000 ϫ g, 15 min, at 4°C) after different incubation time periods and then subjected to the high pressure freezing method (25) and embedded in Lowicryl HM20 resin. Ultrathin cryosections (65 nm) were stained with 2% uranyl acetate (pH 7.0); collected with 2% methylcellulose, 4% uranyl acetate (pH 7.0); and observed on a JEM-1010 electron microscope (Jeol).…”

Brine Assemblages of Ultrasmall Microbial Cells within the Ice Cover of Lake Vida, Antarctica

“…For example, Lake Vida brine contains a network of uncharacterized fiber-like filaments connecting cells and particles ( Fig. 2A and C) (2), and we detected microorganisms (e.g., Pseudoalteromonas antarctica NF 3 ) (46,47) that are recognized EPS producers. Microbial cells enriched with exopolysaccharides (which carry a net negative electric charge and have the capacity to bind Fe 2ϩ ions) are known to serve as nucleation sites for Fe(III) encrustation formed from oxidation reactions (48,49).…”

“…Protein misfolding in cold and freezing environments is a crucial obstacle that cells need to overcome for survival. Cold-adapted bacteria from the genera Marinobacter, Psychrobacter, and Pseudoalteromonas have been described as producing large numbers of vesicles (20 to 100 nm) attached to cells and extracellular polymeric substances (EPS) in cultures grown at temperatures from 4°C to 15°C (46,53). Alternatively, the nanoparticles could also be representative of inclusion bodies (aggregated proteins associated with cell aging) (54,55) or viral particles which could serve as nucleation sites precipitating amorphous Fe,O material.…”