Reevaluation of envelope profiles and cytoplasmic ultrastructure of mycobacteria processed by conventional embedding and freeze-substitution protocols

Abstract

The cell envelope architectures and cytoplasmic structures of Mycobactenium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-_4Cjacetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (-25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures.Tuberculosis remains a major health problem for humans, particularly in developing countries, with an estimated 8 million cases and 3 million deaths per year worldwide (7). In the United States, more than 22,000 new cases are being reported annually in particular populations. The epidemiology of tuberculosis has undergone a profound transition in recent years (21). An increase in the incidence of infection due to human immunodeficiency virus has resulted in a rise in the number of both typical and atypical mycobacterial infections. The Mycobacterium avium complex and M. kansasii are the most frequently encountered etiologic agents of atypical mycobacterial infections associated with AIDS (21); however, M. xenopi, M. fortuitum, and M. chelonae have also been reported previously (21).The molecular and structural compositions of the mycobacterial cell wall must play a significant role in its mechanisms of pathogenicity (18,22,23). Moreover, the intricate physical relationships among the diverse macromolecules within the wall appear to be responsible for properties such as drug resistance (4), acid fastness (1), hydrophobic interactions (25), and th...

The cell envelope structure and properties of Mycobacterium smegmatis mc2155: is there a clue for the unique transformability of the strain?

“…1a, b), composed of (i) a plasma membrane, (ii) a hypothetical 'periplasmic' space (Daffé & Draper, 1998), (iii) a thick internal electron-dense layer, (iv) a thin electron-transparent layer, difficult to distinguish in these sections from the electron-dense layer, and (v) an electrondense outer layer. This ultrastructural appearance is similar to that previously found in other mycobacterial species (Daffé & Draper, 1998;Draper, 1982;Paul & Beveridge, 1992;Rastogi et al, 1986) and particularly in M. smegmatis mc 2 155, where the staining of the electron-dense outer layer with ruthenium red has been attributed to GPLs (Etienne et al, 2002). The fact that the two strains displayed no marked difference in the electron density of their outer layer (Fig.…”

“…The method for preparing samples for transmission electron microscopy was based on procedures of Daffé et al (1989) and Paul & Beveridge (1992). Briefly, early-exponential-phase bacteria (TS medium) were fixed in 2?5 % (w/v) glutaraldehyde, 0?05 % (w/v) ruthenium red in cacodylate buffer for 2 h in the dark at room temperature.…”

Lipid domains of mycobacteria studied with fluorescent molecular probes

“…Mycobacteria, therefore, may well have what amounts to an external membrane, somewhat analogous to that in Gram-negative bacteria. Indeed, freeze-fracture studies clearly demonstrate two major cleavage planes through the envelope of certain mycobacteria (Benedetti et al, 1984;Paul and Beveridge, 1992) The great difference is that, in mycobacteria, the basal anchoring mycolic acid organelle is covalently bound to the cell wall polysaccharide structure. It is therefore very difficult to prepare an outer membrane unit for detailed study.…”

High-Resolution Visualization ofPseudomonas aeruginosaPAO1 Biofilms by Freeze-Substitution Transmission Electron Microscopy

“…Remarkably, the freeze-substitution method has even preserved the O-side chains of the lipopolysaccharide (LPS) on the outer face of the outer membrane of Pseudomonas aeruginosa (41). Even the cell walls of gram-positive cells show more detail; the walls of Bacillus subtilis revealed structural aspects of cell wall turnover (24) and mycobacterial walls showed more accurate distribution of their complex polymeric networks (54,55).…”