Mycobacterial Stationary Phase Induced by Low Oxygen Tension: Cell Wall Thickening and Localization of the 16-Kilodalton α-Crystallin Homolog

Organisms and culture conditions.

M. bovis BCG (Statens Seruminstitut strain ST1077), M. tuberculosis H37Rv (ATCC 9360), and Mycobacterium smegmatis mc²155 were grown in Middlebrook 7H9 medium supplemented with albumin-dextrose-catalase (ADC; Difco Laboratories Ltd., West Molesey, United Kingdom) and 0.05% (vol/vol) Tween 80 (Sigma, Poole, United Kingdom). In some experiments Tween 80 was replaced, where stated, with 0.2% (vol/vol) glycerol. Either sterile polystyrene universals or 100-ml glass bottles containing 10 or 50 ml of 7H9–ADC–Tween 80 broth, respectively, were inoculated with bacilli and cultured in a shaking 37°C incubator until slight turbidity was achieved (usually at an optical density at 600 nm [OD₆₀₀] of ∼0.4). The culture vessels were then placed in a standard nonshaking 37°C incubator (the M. tuberculosis cultures were placed in a nonshaking CO₂ incubator with 5% CO₂) under either microaerobic or anaerobic conditions. To obtain a microaerobic environment, a self-induced oxygen gradient was permitted to form as the bacilli settled to the bottom of the culture vessel. The screw caps were loosened to allow gaseous exchange between the environment and the media. For anaerobic conditions, the culture was overlaid with filter-sterilized mineral oil (molecular biology grade; Sigma) and the tube caps were tightened. In some experiments M. bovis BCG was cultured in an anaerobic cabinet in the absence of mineral oil. The microaerobic and anaerobic cultures were left undisturbed for periods of up to 6 months before harvesting. Controls comprised bacilli cultured aerobically with agitation to an OD₆₀₀ of 0.07 to 0.4 (low-OD control) and bacilli cultured aerobically with shaking for 6 weeks to an OD₆₀₀ of >1.0 (nutrient-limited stationary-phase control [NLSPC]). Aerobic cultures were coprocessed with the anaerobic samples. Cultures were routinely checked for contaminants and were examined by Ziehl-Neelsen staining.

TEM.

Cells were harvested by low-speed centrifugation, washed in sterile phosphate-buffered saline (PBS), and fixed for a minimum of 1 h in 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium phosphate. Cells were then pelleted, the supernatant was removed, and the pellet was resuspended in 1% (wt/vol) osmium tetroxide for 1 h. Cells were then dehydrated through a graded ethanol series (50, 70, 90, and 100%; each level was applied twice for 5 min each time) and propylene oxide (twice for 15 min) and infiltrated with, unless otherwise stated, agar 100 (Agar, Stansted, United Kingdom). Resin blocks were polymerized at 60°C for 24 h. Sections were cut on a Reichert-Jung Ultracut E ultratome to silver/gold interference, picked up on Formvar-coated grids, and stained with 30% (wt/vol) uranyl acetate in 70% (vol/vol) methanol and counterstained with Reynold’s lead citrate (34) before examination on a Jeol JEM-100CXII TEM at an acceleration voltage of 80 kV. Under some circumstances, where stated, one or more of the above fixation or staining steps were omitted. Cells were also prepared for TEM by the method of progressive lowering of temperature (PLT) (see below), following which grids were stained with osmium tetroxide vapor by placing them in a petri dish for 3 h with an osmium tetroxide crystal. Qualitative X-ray microprobe analysis was used to help determine which heavy metal was responsible for the contrast observed in the thickened cell wall. Briefly, routinely prepared sections were analyzed in the electron microscope fitted with a high-resolution scanning attachment, a LaB₆ filament, and a Link ISIS 30-mm² Si(Li) detector, atmosphere thin window, and multichannel analyzer (Oxford Instruments, High Wycombe, United Kingdom).

Protein extraction and SDS-PAGE analysis.

Bacilli were harvested, washed, and resuspended in 1 ml of PBS containing 0.4 ml of sterile glass beads (0.1 mm in diameter) before disruption in a Mini Beadbeater (Stratech Scientific, Luton, United Kingdom) by three 60-s bursts at 50,000 rpm. Protein concentration was determined by the bicinchoninic acid assay (BCA kit; Pierce and Warriner, Chester, United Kingdom). Proteins were separated on a 12% denaturing polyacrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (26) using a Mini-Protean II minigel apparatus (Bio-Rad, Hemel Hempstead, United Kingdom) and were visualized by 0.5% Coomassie brilliant blue staining. A protein band of interest was excised, and the N-terminal sequence was determined at the Glaxo Wellcome Medicines Research Centre, Stevenage, United Kingdom.

Cloning and sequencing of the M. bovis BCG 16-kDa α-crystallin homolog.

Oligonucleotide primers based on the N-terminal sequence of the M. bovis BCG 16-kDa protein and the nucleotide sequence of the gene encoding the M. tuberculosis 16-kDa α-crystallin homolog (GenBank accession no., S79751) were designed. Two primers, 5′-ATGGCCACCACCCTTCCCGT-3′ and 5′-CAGTTGGTGGACCGGATCTG-3′, were used to amplify the M. bovis BCG 16-kDa α-crystallin gene with TaqI polymerase (Boehringer Mannheim, Lewes, United Kingdom) by PCR. The PCR product was purified (Geneclean Spin kit; Anachem, Luton, United Kingdom), polished with PfuI polymerase, blunt-end cloned into pCR-ScriptSK(+) (Stratagene, Cambridge, United Kingdom), and transformed into Escherichia coli SURE cells (Stratagene). Transformants were selected for ampicillin resistance with blue/white color selection. Plasmid DNA containing the appropriate insert was sequenced on an automated sequencer (ABI 373) by Alta Bioscience, University of Birmingham, United Kingdom, by the Taq Dye Deoxy terminator cycle method (Applied Biosystems Ltd., Warrington, United Kingdom).

Immunoelectron microscopy.

Bacilli were prepared for immunogold analysis by the PLT method (35). Samples were fixed for 60 min in 2% paraformaldehyde–0.05% glutaraldehyde in 0.1 M phosphate buffer. Samples were dehydrated through a graded ethanol series (from 30 to 100% ethanol) from 0 to −50°C. The samples were then infiltrated with Lowicryl HM20 for 24 h at −50°C prior to embedding and polymerization with ultraviolet light in fresh Lowicryl HM20, first for 48 h at −50°C and then for 24 h at 15°C. Silver/gold sections were cut as described above and labelled by blocking with 1.0% bovine serum albumin in PBS for 45 min at room temperature before exposure to a pooled polyclonal murine anti-M. tuberculosis recombinant 16-kDa α-crystallin antibody (a kind gift from J. Ivanyi [MRC Clinical Sciences Centre, Tuberculosis and Related Infections Unit, Imperial College School of Medicine, Hammersmith Campus, London, United Kingdom]) diluted in blocking solution for 3 h at 37°C. The grids were washed three times for 5 min in blocking solution before the addition of the immunogold conjugate, a 1:50 dilution of 10-nm colloidal gold-labelled goat anti-mouse antibody (British Biocell International, Cardiff, United Kingdom). The grids were washed a further three times for 5 min in blocking solution and three times for 1 min in distilled water before being stained with 30% uranyl acetate in 70% methanol. Controls of preimmune murine serum and conjugate, as well as conjugate only, were incorporated to ensure that low background labelling and high specificity of labelling were achieved. Sections were visualized with a Jeol JEM-100CXII TEM at an acceleration voltage of 80 kV.

Changes in cell wall electron density in M. bovis BCG and M. tuberculosis are associated with decreased oxygen tension.

Analysis by TEM of M. bovis BCG cultured under anaerobic conditions for 6 months revealed a marked thickening of the cell wall (Fig. 1A and B). The main feature of these cells was the extremely dense electron staining of the cell walls, which in most cases homogeneously covered the surfaces of the bacilli. In some cases the thickened cell wall appeared to have ruptured, to have “peeled” off some cells, and other cells appeared to be denuded of part of their thickened cell walls, probably due to the processing of the cells for TEM. Despite the long period of culture under these conditions, the internal architecture of the cells appeared to be preserved. The same cell wall thickening was also seen in microaerobic cultures, although less frequently. However, this phenomenon was not observed in aerobically grown (low-OD control) bacilli (Fig. 1C) or in bacilli cultured aerobically with continuous agitation for 6 weeks, which would have entered stationary phase (NLSPC) (Fig. 1D). The bacilli cultured under microaerobic and anaerobic conditions for 6 months could be resuscitated by inoculation into fresh media (7H9–ADC–glycerol broth) under aerobic growth conditions. Only a loopful of the 6-month cultures was inoculated, and the results were merely recorded as positive growth. We did not ascertain how many cells in the inoculum were viable.

To determine whether this cell wall thickening could be observed in a pathogenic mycobacterial species, M. tuberculosis bacilli were cultured under anaerobic conditions in the same way for 1 month. TEM analysis showed that M. tuberculosis also developed a thickened cell wall associated with a decreased availability of oxygen (Fig. 1E and F). This was not observed in aerobically grown M. tuberculosis. Morphologically, the thickened cell wall appeared to be similar in M. tuberculosis and M. bovis BCG. In contrast, the fast-growing saprophytic species, M. smegmatis, did not develop the thickened cell wall after 17 and 35 days of anaerobic culture. In the 35-day-old anaerobic culture less than 1% of the bacilli remained intact. Of those which were intact, none exhibited the cell wall thickening (Fig. 1H).

Confirmation of observations: elimination of influence of mineral oil and Tween 80.

It was not possible that the presence of the mineral oil used to induce anaerobic conditions was responsible for the thickening as it was also seen in microaerobic cultures (cultured in the absence of mineral oil). Bacilli cultured in an anaerobic cabinet without mineral oil developed the thickened cell wall from 2 weeks onwards. Moreover, when M. bovis BCG cells were grown as described for the aerobic cultures, overlaid with mineral oil prior to being washed and processed for TEM analysis, and treated in the same manner as for the anaerobic cultures, thickened cell walls were not observed (only M. bovis BCG was used in these experiments as facilities for standard anaerobic culture of M. tuberculosis were not available). To eliminate the possibility that this was a Tween 80-dependent phenomenon, M. bovis BCG cells were cultured under mineral oil for 6 weeks in 7H9–ADC broth with 0.2% (vol/vol) glycerol replacing Tween 80. The same thickened cell walls were observed, showing that Tween 80 was not necessary for the thickened cell walls to be formed.

Osmium tetroxide postfixation and staining is primarily responsible for the detection of the cell wall thickening.

Osmium tetroxide was shown, by a number of experiments, to be necessary for the postfixation of the cell wall in samples prepared for routine TEM and for enhancing the contrast of the cell wall in samples prepared for TEM by the PLT method. In one experiment, anaerobic cultures were prepared for routine TEM; however, half of the culture was not postfixed in osmium tetroxide but was treated identically in every other respect to the remainder of the culture. When sections from the nonpostfixed specimen were examined, it was found that the thick cell wall was not visualized. This contrasted starkly with the osmium tetroxide-postfixed cell preparation in which the thick cell wall was evident even in the absence of further staining with lead or uranium salts. To provide further evidence that osmium tetroxide was responsible for the staining, X-ray microprobe analysis was undertaken to compare the cell wall to the background. This analysis confirmed the presence of osmium in the cell wall. No common coprecipitants such as phosphate (as illustrated by the presence of phosphorus) were identified. To further confirm that osmium tetroxide was responsible for the staining, some cells were prepared for TEM by the PLT method. This method of preparation permits structural integrity to survive the preparation process but yields poor ultrastructural detail. Sections prepared in this manner were stained with osmium vapor after processing. The detection of the unusual cell wall morphology in cells prepared and stained in this manner suggests that the osmium was not altering the cell wall during processing for standard TEM analysis.

Cell wall thickness and cell size.

Image enhancement of electron micrographs taken of the thickened cell wall (Fig. 2) showed that the thickening was restricted to the cell wall outer electron-opaque layer. The other components of the cell wall appeared not to be enhanced in any observable way. The thickness of the cell wall was measured for anaerobically cultured bacilli. Measurements were only taken on transverse sections of the cell wall at the thinnest point. Anaerobic culture of M. bovis BCG in the presence or absence of mineral oil did not alter the degree of thickening of the cell wall. The cell wall was thicker in anaerobically cultured M. tuberculosis than in anaerobically cultured M. bovis BCG (21.2 ± 2.04 nm [n = 9] versus 16.1 ± 1.03 nm [n = 49], respectively). The internal diameters of the bacilli (i.e., excluding the cell wall) were also measured by measuring the diameters of the cells at the narrowest axes of transverse sections. The cell diameter was used as opposed to the cell length as it was easier to measure and more likely to reflect a valid and reproducible measurement. The anaerobically cultured M. bovis BCG cells were much thinner (255 ± 4.35 nm [n = 52]) than the aerobically grown controls (284 ± 6.20 nm [n = 29]), and these differences were highly significant (P < 0.001). The same pattern was also observed for M. tuberculosis, where the anaerobically cultured bacilli were thinner (278 ± 22.5 nm [n = 9]) than the aerobically grown bacilli (338 ± 18.9 nm [n = 10]; P < 0.05).

Identification of the M. bovis BCG 16-kDa α-crystallin homolog.

SDS-PAGE analysis of protein extracted from M. bovis BCG cultured microaerobically and anaerobically for 1, 2, 4, 12, and 26 weeks (6 months) showed the presence of a protein of approximately 18 kDa (data not shown). This protein appeared to reach maximal expression, as a proportion of total protein, after 2 weeks of either microaerobic or anaerobic culture, and this level of expression was maintained for up to 26 weeks of culture. The protein appeared to be poorly expressed in the low-OD aerobic controls, but greater expression was noted in the NLSPC and probably reflects its induction by oxygen deficiency due to cell clumping. The N-terminal sequence of this protein—ATTLPVQRHPRSLFP—had 100% homology with the previously described M. tuberculosis 16-kDa small heat shock protein (smHSP) or α-crystallin homolog (27, 40).

A partial nucleotide sequence of the gene encoding the M. bovis BCG 16-kDa α-crystallin protein is 100% identical to that of the equivalent region in the M. tuberculosis homolog.

PCR amplification of M. bovis BCG genomic DNA with primers based on the M. tuberculosis gene sequence encoding the 16-kDa protein generated a product 434 bp in size. Excluding the primer sequences, 394 bases were sequenced and were found to be 100% identical to those of the equivalent region in M. tuberculosis.

Immunolocalization of the M. bovis BCG 16-kDa α-crystallin protein.

Immunoelectron microscopy with a specific polyclonal antiserum to the 16 kDa protein was used to determine the distribution of the protein. An examination of aerobically cultured low-OD bacilli revealed poor expression of the 16-kDa protein (Fig. 3A). However, a high degree of specific labelling was observed for the microaerobic and anaerobic cultures from 2 to 26 weeks, and three patterns of 16-kDa protein distribution were observed.

Sixteen-kilodalton protein pattern of distribution I: localization to the cell periphery.

The protein was frequently found to be localized to the peripheral regions of M. bovis BCG bacilli (Fig. 3B), where the label could clearly be seen to follow the contours of the cell. The 16-kDa protein was also located to the cell membrane and cell wall skeleton and less frequently the outer regions of the cell wall (Fig. 3C), suggesting that the 16-kDa protein may be associated with maintaining the structural integrity of the cell.

Sixteen-kilodalton protein pattern of distribution II: localization to fibrous structures.

The second type of label distribution showed that there were internal line structures along which the 16-kDa protein was associated (Fig. 3D and E). These lines could be frequently visualized as structural details of varying electron density on the electron micrographs and probably represent peptidoglycan located in the cell wall (1, 22). These fibrous structures were most frequently seen in longitudinal sections, probably due to the plane of sectioning. However, this pattern of label distribution was also seen in the absence of detailed fibril ultrastructure (Fig. 3F).

Sixteen-kilodalton protein pattern of distribution III: localization to intracellular and peripheral clusters.

The 16-kDa protein was observed to form clusters (Fig. 3G and H) which did not exceed 40 nm in width. These clusters were located both inside the cell and at its periphery, but which cellular components the clusters were associated with could not be determined. However, little or no label appeared to be associated with chromosomal DNA (Fig. 3H).