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. 1998 Feb;180(4):801-8.
doi: 10.1128/JB.180.4.801-808.1998.

Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton alpha-crystallin homolog

A F Cunningham  1 , C L Spreadbury
Affiliations

Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton alpha-crystallin homolog

A F Cunningham et al. J Bacteriol. 1998 Feb.

Abstract

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How Mycobacterium tuberculosis survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy. Mycobacterium bovis BCG and M. tuberculosis H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured Mycobacterium smegmatis. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or alpha-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in M. bovis BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of M. tuberculosis.

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Figures

FIG. 1
FIG. 1
Effect of anaerobic culture on the morphology of M. bovis BCG, M. tuberculosis, and M. smegmatis. (A) Longitudinal section of an M. bovis BCG bacillus cultured anaerobically for 6 months showing pronounced cell wall thickening (some cytoplasmic dehydration is evident). (B) Transverse sections of M. bovis BCG bacilli cultured anaerobically for 6 months. (C) Lack of cell wall thickening in low-OD aerobically cultured M. bovis BCG bacilli. (D) NLSPC M. bovis BCG bacilli showing the absence of cell wall thickening. (E and F) Cell wall thickening in M. tuberculosis following anaerobic culture for 1 month under mineral oil. (G) Aerobically cultured M. smegmatis. (H) M. smegmatis bacilli exhibiting cell lysis following 35 days of anaerobic culture. Bars, 200 nm except for panels C (100 nm) and G (400 nm). The bar in panel A is also valid for panel F; the bar in panel D is also valid for panel H.
FIG. 2
FIG. 2
Cell wall architecture of anaerobically cultured M. bovis BCG (A to C) compared to that of aerobically cultured M. bovis BCG (D) showing that only the outer electron-opaque layer is thickened in response to low oxygen tension. The numbers in panel A refer to the individual cell wall envelope components, as interpreted by Brennan and Nikaido (3). From the cytoplasm outwards, the numbers indicate the following: 1 and 2, the plasma membrane; 3, the electron-dense layer (peptidoglycan); 4, the electron-transparent layer; 5, the outer electron-opaque layer. The thickened outer electron-opaque layer and cell envelope appear to exhibit plasticity (B). Note the absence of the outer electron-opaque layer where two bacilli are in direct physical contact (C). (D) Low-OD aerobically cultured bacillus. Bar, 30 nm (for all panels).
FIG. 3
FIG. 3
Immunogold electron microscopy localization of the 16-kDa α-crystallin protein of M. bovis BCG. (A) Low level of prevalence of the 16-kDa protein in the low-OD aerobic control. (B) Bacillus from a 12-week anaerobic culture showing localization to the periphery of the cell. (C) Twenty-six-week anaerobically cultured bacillus showing localization of the 16-kDa protein to the cell wall (marked by the arrowheads). (D) Twenty-six-week anaerobically cultured bacillus showing the 16-kDa protein associated with a splayed arrangement of fibrous structures. (E) Bacillus cultured for 26 weeks microaerobically showing localization of the protein to fibrils thought to be peptidoglycan (indicated by arrowheads). (F) Four-week anaerobically cultured bacillus; lines of colloidal gold labelling are seen despite the absence of detailed fibril ultrastructure. (G) Four-week anaerobically cultured bacillus showing clusters both associated with the intracellular environment and at the cell periphery. (H) Bacillus cultured for 2 weeks anaerobically. Note the lack of localization to the chromosome in the center of the cell. Bars, 100 nm except for panel A (300 nm) and panels C and H (200 nm).
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