Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis

https://doi.org/10.1016/j.mimet.2004.02.012Get rights and content

Abstract

Ruthenium red is a polycationic stain used to visualize acid polysaccharides on the outer surface of cells. Ruthenium red staining followed by electron microscopic analysis was used to demonstrate the presence of an external glycoprotein layer surrounding the spore of both Bacillus anthracis and Bacillus subtilis. This layer is less apparent with traditional staining methods used for electron microscopy. Renografin gradients were used to purify B. subtilis spores. These purified spores displayed greatly enhanced staining with ruthenium red, indicating nonspecific binding of renografin, which has a major carbohydrate constituent, methylglucamine. For B. anthracis, staining with ruthenium red was sufficiently intense that it was not significantly enhanced by renografin purification. In addition to demonstrating a previously undiscovered layer surrounding the spores of B. subtilis, the results help explain a long-standing controversy as to ultrastructural differences among these genetically closely related organisms. Ruthenium red staining provides an important addition to the identification of surface glycoproteins in studies to define similarities and differences in the exosporium layers of Bacillus species.

Introduction

The biological function of the exosporium is unknown. It appears that the presence of surface glycoproteins is a conserved feature of Bacillus spores, regardless of whether a true exosporium is present. The conserved nature of these molecules suggests they play an important role in the biology of the endospore. It is also likely that the surface carbohydrates may play an important role in the architecture of the spore, the spore's resistance properties, and initial interactions with the human host tissues or the germination process. The following report describes the use of ruthenium red staining prior to ultrastructural observation for demonstration of an external glycoprotein layer surrounding spores of both Bacillus anthracis and Bacillus subtilis. Ruthenium red is a polycationic stain widely used for visualization of acid polysaccharides on the outer surface of cells. This methodology has been widely used for staining external capsules in vegetative bacteria Jones et al., 1969, Roth, 1977 but its use for visualization of an exosporium is a novel application. Indeed, as discussed below, the methodology allows greatly improved visualization of the glycoprotein nap component of the exosporium of B. anthracis and for the first time demonstrates the presence of such a glycoprotein nap surrounding the B. subtilis spore. Prior reports have questioned whether B. subtilis has an exosporium, although this organism is closely related genetically to B. anthracis Souza et al., 1976, Souza et al., 1978, Henriques and Moran, 2000, Turnbough, 2003. Previous methodology for the examination of spore ultrastructure did not allow visualization of a glycoprotein nap for B. subtilis. This may explain the conundrum concerning previous difficulties in observing a noticeable nap for B. subtilis.

The spore of B. anthracis is characterized by the presence of an external exosporium that consists of a basal layer surrounded by a glycoprotein nap Hachisuka et al., 1966, Kramer and Roth, 1968. The exosporium surrounds a coat layer Driks, 2002, Lai et al., 2003. However, the glycoprotein nap can be difficult to observe without additional staining (Fox et al., 2003). Kramer and Roth (1968) obtained weak staining with uranyl acetate and found potassium permanganate and lead citrate vital to enhance staining of the exosporium. The component of the exosporium stained in these earlier studies was not identified. Recently, it has been demonstrated that the exosporium consists largely of the BclA glycoprotein Sylvestre et al., 2002, Sylvestre et al., 2003, although other recently described glycoproteins including Exs J are also found in the exosporium Garcia-Patrone and Tandecarz, 1995, Charlton et al., 1999, Todd et al., 2003, Steichen et al., 2003.

There have been reports that B. subtilis does not possess an exosporium layer, since neither the basal layer nor glycoprotein nap are visible on electron microscopic examination Henriques and Moran, 2000, Turnbough, 2003. However, Souza et al., 1976, Souza et al., 1978 demonstrated that on urea–mercaptoethanol treatment, a layer is detached from the underlying outer coat that resembles the basal membrane of the exosporium of B. anthracis. The observed membrane could be a component of the outer coat, and an artifact of the chemical treatment, or may represent a distinct exosporium.

Spore-specific carbohydrates exist in both B. anthracis and B. subtilis. These carbohydrates display structural similarity, consisting of deoxyhexoses (rhamnose [deoxy-mannose] and quinovose [deoxy-glucose], respectively) Matz et al., 1970, Wunschel et al., 1994, Fox et al., 1993. Recently, it has been hypothesized that these carbohydrates are components of the exosporium glycoprotein nap (Fox et al., 2003). Mutations in the cgeAB and cgeCDE operons of B. subtilis produce spores with altered surface properties and it was speculated that this is due to changes in glycosylation of surface proteins. However, the focus of this study was on genetic regulation rather than function, and thus direct evidence was not provided (Roels and Losick, 1995).

Based on the strong evidence for external glycoproteins occurring in the spore of B. anthracis and circumstantial evidence for their occurrence in B. subtilis, an ultrastructural study was undertaken. It is demonstrated here that a carbohydrate-rich nap is clearly observable for both organisms. Ruthenium red staining for ultrastructural analysis could be highly useful in future genetic and biochemical studies to further define the composition of the surface structures present in these organisms.

Section snippets

Growth conditions

B. anthracis, strain ΔSterne-1 (derived from the vaccine strain Sterne, and lacking pXO1 and pXO2) was a gift from Dr. Stephen H. Leppla (National Institutes of Health, Bethesda, MD) and B. subtilis, strain 168 (trypC2) was provided by Dr. George Stewart (Kansas State University, Manhattan, KS). Five colonies of bacilli were taken from a tryptic soy agar (Difco) plate, inoculated into 250 ml of nutrient broth (Difco) in a 3-l Fernbach flask, placed in an orbital shaker at 37 °C, 150–200 rpm and

Results

On staining B. anthracis spores with uranyl acetate and osmium tetroxide, the external basement membrane of the exosporium is visible and readily distinguished from underlying coat layers. However, the glycoprotein nap is difficult to observe. The nap is largely composed of glycoproteins (Sylvestre et al., 2002) and it was hypothesized that EM stains that target carbohydrates would improve staining. Two stains were evaluated. Ruthenium red staining has been widely used for staining carbohydrate

Discussion

Ruthenium red staining improves ultrastructural observation of the external glycoprotein nap surrounding the spore of B. anthracis. Additionally, the current study demonstrates, using ruthenium red, for the first time that a glycoprotein nap is also present surrounding the spore of B. subtilis. The uniqueness of the morphology of the spores of the two organisms is confirmed, since a basement membrane is not clearly observable for B. subtilis using the mild ruthenium red staining procedure.

Acknowledgements

A. Fox (AF) and K. Fox (KF) were supported by funds from the University of South Carolina Center for Public Health Preparedness provided by the Centers for Disease Control. This work was also funded by USC School of Medicine/School of Mathematics and Science and USC School of Medicine/School of Public Health Collaborative Incentive Awards (AF and KF). Jeff Davis assisted in processing samples for electron microscopy.

References (24)

  • H.C Jones et al.

    Electron microscopic study of a slime layer

    J. Bacteriol.

    (1969)
  • M.J Kramer et al.

    Ultrastructural differences in the exosporium of the Sterne and Vollum strains of Bacillus anthracis

    Can. J. Microbiol.

    (1968)
  • Cited by (93)

    • Metalloglycomics of tris(2,2′-bipyridyl) cobalt and ruthenium compounds

      2022, Journal of Inorganic Biochemistry
      Citation Excerpt :

      In this paper, we expand our studies to substitution-inert octahedral cobalt(III) and ruthenium(II) complexes bearing the non‑hydrogen-donor ligand 2,2′-bipyridine (bpy). Earlier papers have described the application of the fluorescent properties of [Ru(bpy)3]2+ to detect heparin and HS, and even bacterial heparinase activity [8,9]. The fluorescence of the fluorophore [Ru(phen)2(dppz-idzo)]2+ (phen = 1,10-phenanthroline; dppz-idzo = dipyrido[3,2-a:2′,3′-c]phenazine-10,11-imidazolone-2) is significantly enhanced in the presence of heparin, allowing a simple but sensitive “switch-on” assay for heparin quantification even in presence of fetal bovine serum [10].

    • Medicinal inorganic chemistry: New perspectives and targets for the periodic table

      2020, Advances in Inorganic Chemistry
      Citation Excerpt :

      Cisplatin itself has been conjugated with chondroitin sulfate and heparin in formulations.57,58 The dye Ruthenium Red (Ru red) has long been used as a polycationic stain to visualize polysaccharides on the outer surface of cells .59–62 Indeed, Ru red staining followed by electron microscopic analysis demonstrated the presence of an external glycoprotein layer surrounding the spore of both Bacillus anthracis and Bacillus subtilis.59

    • Endocytosis of GM-CSF receptor β is essential for signal transduction regulating mesothelial-macrophage transition

      2019, Biochimica et Biophysica Acta - Molecular Cell Research
      Citation Excerpt :

      Both in vivo and in vitro mesentery samples were fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PBS), pH: 7.4 (1 h at room temperature) or in a mixture of 2% glutaraldehyde (GA) and 2% OsO4 (1:1) in 0.1 M cacodylate buffer, pH 7.4 (1 h on ice). In some cases the mesentery was fixed with 0.5 mg/ml Ruthenium red (Ru-red) (Honeywell Fluka™, Bucharest, Romania) [15,16] containing 1% OsO4-GA-fixatives. Ru-red binds the carbohydrate side chains, so it labels all the surface-connected vesicles.

    View all citing articles on Scopus
    View full text