1 Institut de Pharmacologie et de Biologie Structurale, Unité Mixte de Recherche du Centre National de la Recherche Scientifique et de l'Université Paul Sabatier (UMR5089), Département Mécanismes Moléculaires des Infections Mycobactériennes, 205 route de Narbonne, 31077 Toulouse cedex 04, France
2 Unidade de Micobactérias, Instituto Nacional de Saúde Dr Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisboa, Portugal
Correspondence
Gilles Etienne
gilles.etienne{at}ipbs.fr
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ABSTRACT |
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INTRODUCTION |
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While the original report claimed that the ept mutation does not improve the DNA uptake abilities of the cell (Snapper et al., 1990), suggesting that no cell envelope change has occurred, the transformable character of mc2155 may be related to variations in M. smegmatis cell surface properties, e.g. hydrophobicity, sliding motility or clumping. These properties have been previously demonstrated to reflect profound alterations of the cell envelope composition (Etienne et al., 2002
). The nature or the absence of C-mycoside glycopeptidolipids (GPLs) (Aspinall et al., 1995
; Chatterjee & Khoo, 2001
) has been correlated with the rough morphotype of mutants of M. smegmatis (Billman-Jacobe et al., 1999
; Recht et al., 2000
) or Mycobacterium avium (Barrow & Brennan, 1982
; Belisle et al., 1993a
, b
) and even in the distantly related Gordonia hydrophobica (Moormann et al., 1997
).
In this study, we compared the cell-surface properties of the wild-type strain of M. smegmatis ATCC 607 with those of its transformable mutant mc2155. We showed that strain mc2155 displays profound modifications of its cell envelope composition and structure, and we suggest that its transformable character may be in part explained by these alterations of its cell envelope.
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METHODS |
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Mycobacterial cell-surface properties.
The determination of mycobacterial cell surface properties was as described elsewhere (Etienne et al., 2002); it is summarized here. (i) The motility assay was adapted from Martinez et al. (1999)
. Middlebrook 7H9 base medium (Difco) was solidified with 0·4 % high-grade agarose (Eurogentec). Plates were inoculated in their centre with 10 µl single-cell suspension (OD650 1) and motility was evaluated by measuring the diameter of the halo of growth formed by the mycobacteria. (ii) Cellular aggregation was quantified by cultivating mycobacteria in TS broth without Tween 80. The unicellular mycobacteria were separated from the aggregates by differential centrifugation (Cougoule et al., 2002
) and the cellular aggregation was calculated as the percentage of aggregate-containing pellets versus total cell weight. (iii) A Congo red accumulation assay (Cangelosi et al., 1999
) was adapted as follows. Mycobacteria were cultivated in TS broth plus 100 µg Congo red ml1 and 0·05 % Tween 80. The cells were then washed extensively with distilled water and the Congo red that remained associated with the cells was extracted with acetone. The Congo red binding index was defined as the A488 of the acetone extracts divided by the dry weight of the cell pellet. (iv) The relative hydrophobicity was assessed by the hexadecane partition procedure (Rosenberg et al., 1980
): a single cell suspension of each strain (OD650 1) was mixed with 0·3 ml hexadecane (Avocado Research Chemicals). The hydrophobicity index was defined as the percentage reduction in the OD650 of the aqueous phase after complete separation of the two phases. (v) The bacterial cell surface charge (Bayer & Sloyer, 1990
) was determined by measuring the zeta-potential (
) of a single-cell suspension (OD650 1) in a zetameter Zetasizer 3000 (Malvern Instruments).
Fractionation and analysis of the extracellular, surface-exposed and cell-bound components.
Surface-exposed material (SXM) and extracellular compounds were extracted and analysed as previously described (Lemassu et al., 1996; Ortalo-Magné et al., 1996
). Briefly, surface pellicles of mycobacteria grown on Sauton's medium were treated with glass beads (Ortalo-Magné et al., 1995
) and suspended in distilled water; bacilli were removed by filtration on a 0·2 µm Nalgene filter. Similarly, the culture broth, containing the extracellular materials, was filter-sterilized. Portions of the crude filtrates were separately concentrated under vacuum, extensively dialysed against distilled water and analysed for their carbohydrate and protein contents by colorimetric assays (Dische, 1962
, and the Lowry method, respectively). Their glycosyl composition was also determined by acid hydrolysis followed by analysis of the trimethylsilylated sugar derivatives by gas chromatography using erythritol as internal standard (Lemassu et al., 1996
). Alternatively, the various enzyme activities detectable in the SXM or in the culture broth were assayed as previously described (Raynaud et al., 1998
).
Chloroform and methanol were added to the remaining portions of the crude filtrates to obtain partition mixtures composed of chloroform/methanol/water (3 : 4 : 3, by vol.); the organic phases were washed with water and evaporated to dryness to yield crude lipid extracts. The mycolate-containing lipids were separated from the other types of lipids by precipitation with methanol (Villeneuve et al., 2003). Both kinds of lipids were further fractionated on a Florisil column irrigated with chloroform and then with a stepwise gradient of increasing concentrations of methanol and water in chloroform (Ortalo-Magné et al., 1996
). Finally, the various classes of lipids were identified by TLC analysis as previously described (Etienne et al., 2002
) and by matrix-assisted laser-desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (see below). The N-acylphenylalanyl moiety of GPLs was produced as methyl ester from native GPLs by methanolysis with anhydrous 1·5 M CH3OH/HCl for 16 h at 80 °C; a portion of the products was further perdeuteroacetylated in anhydrous 1 : 1 (CD3CO)2O/pyridine (100 °C, 1 h). The resulting products were analysed by MALDI-TOF mass spectrometry.
The cell-bound fatty acids were released by saponification of the delipidated cells for 16 h at 80 °C in 5 % (w/v) KOH in methanol/benzene (8 : 2, v/v), extracted with diethyl ether and methylated. They were further analysed by TLC (Jackson et al., 1999) or MALDI-TOF mass spectrometry as previously described. Quantification of the mycolic acids resulted from the saponification of at least 300 mg (dry weight) of delipidated cells in triplicate.
Spectrometric methods.
MALDI-TOF mass spectrometry analysis of lipids was performed as previously described (Laval et al., 2001). Sample solutions (1 mM) were directly applied onto the sample plate as a 1 µl droplet, followed by the addition of 0·5 µl of the matrix solution [10 mg 2,5-dihydroxybenzoic acid ml1 in chloroform/methanol (1 : 1, v/v)]. After crystallization of the samples, MALDI-TOF spectra were acquired on a Voyager-DE STR mass spectrometer (PerSeptive Biosystems) equipped with a pulsed nitrogen laser emitting at 337 nm and were analysed in the reflectron mode using an extraction delay time set at 100 ns and an accelerating voltage operating in positive-ion mode of 20 kV. An external mass spectrum calibration was performed using calibration mixture 1 of the Sequazyme Peptide Mass Standards Kit (PerSeptive Biosystems), including known peptide standards in a mass range from 900 to 1600 Da.
Labelling of lipids.
The various classes of extractable and cell surface lipids were quantified by labelling: 1·2 MBq sodium [1-14C]acetate (Amersham) was added to 100 ml (Sauton broth) of 2-day-old cultures containing mid-exponential-phase bacteria. After 16 h incubation, the reaction was stopped by centrifugation and the SXMs were isolated by extraction with glass beads (Ortalo-Magné et al., 1995). Lipids from these latter materials and those from bead-treated cells were extracted with chloroform/methanol (1 : 2, v/v). Both types of lipid extracts were analysed by TLC using the solvent mixtures described above; the radioactivity was located and measured on plates using an automatic TLC linear analyser (Berthold LB 2832). Then, the lipid spots were visualized by spraying with the appropriate reagents, with charring when necessary.
Permeability assays.
The permeability of the strains of M. smegmatis to chenodeoxycholate was assessed as previously described (Bardou et al., 1998; Jackson et al., 1999
). Exponentially growing cells were first labelled for 16 h with [5,6-3H]uracil (2x105 M, 1·85 TBq mol1; DuPont NEN) to quantify the biomass and aliquots of labelled cells were used to measure radioactivity, then dried and weighed to correlate 3H-labelling with cell dry weight. Accumulation assays were performed under continuous agitation. [14C]Chenodeoxycholate (2x105 M, 1·8 GBq mmol1; DuPont NEN) was added to 1 ml 10 mM HEPES pH 7·2 buffer containing about 40 mg 3H-labelled cells. Aliquots (0·1 ml) were removed at different time intervals and added at the top of an Eppendorf centrifuge tube containing 0·25 ml silicone oil/paraffin oil (1 : 0·2, v/v). Cells were separated from the accumulation medium by centrifugation (13 000 g, 1 min). Centrifuged tubes were frozen on dry ice and the pellets were dropped into counting flasks by cutting the cone top. Scintillation solution (Aqualuma) was added and the vials were sonicated for 30 min in a water bath to disperse the cells.
Transmission electron microscopy.
The method for preparing samples for transmission electron microscopy (Etienne et al., 2002) 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. Cells were washed three times, postfixed for 2 h in the dark in 1 % (w/v) osmium tetroxide, 0·05 % (w/v) ruthenium red and then washed twice each in cacodylate buffer and then water. Cells were dehydrated by exposure to increasing ethanol concentrations for 5 min each, washed twice in 100 % ethanol and then twice in propylene oxide. Cells were suspended in 1 : 1 propylene oxide/Spurr resin for 2 h. After infiltration overnight, samples were transferred to 100 % Spurr resin and left overnight. Resin was replenished the next morning and samples were left to cure at 60 °C overnight. Blocks were thin-sectioned on a ReichertJung microtome and mounted on copper grids. Sections were post-stained with uranyl acetate and Reynold's lead citrate. Microscopy was performed on a JEOL 120 EX electron microscope.
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RESULTS |
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Surface lipids represent a very minor fraction (usually less than 5 % of the dry weight) of mycobacteria grown on Sauton's medium (Lemassu et al., 1996; Ortalo-Magné et al., 1996
). Therefore the lipids of exponential-phase cultures from the two strains were metabolically labelled with sodium [14C]acetate before extracting the SXMs and the bead-treated cells to make it easier to quantify and compare the surface-exposed and the cell lipids. Triacylglycerols (TAGs) were the predominant class of lipids, representing 25·6 % and 57·2 % of the total [14C]acetate incorporated in the bead-treated bacterial and the surface-exposed lipids, respectively, of the wild-type strain of M. smegmatis grown on the glycerol-rich Sauton's medium (Fig. 1a, b
). The other major lipids identified in the ATCC 607 strain were glycopeptidolipids (GPLs), trehalose monomycolates (TMMs) and phospholipids; the trehalose dimycolates (TDMs) were not detected in the surface-exposed lipids, as previously observed (Ortalo-Magné et al., 1996
; Etienne et al., 2002
). The major classes of lipids found in the wild-type strain were also predominant in the mc2155 mutant strain, but their relative distribution between the surface and the deeper layers of the envelope was dramatically different from that found in the parent strain (Fig. 2
). While the bacterial surface of the wild-type strain was 2·2 times enriched in TAGs, compared to the deeper layers of the envelope, TMMs and phospholipids were respectively 14·6 and 1·6 times more abundant in the bead-treated cells compared to the outermost layer. In contrast, the mc2155 cellular envelope seemed to have lost this differentiation: the TAGs and phospholipids displayed comparable amounts in both compartments (Fig. 2
). The difference in the TMMs between the outermost and the deeper layers of the envelope was also reduced to 1·8 times. These observations may account for the difference in negative net charge observed between the surfaces of the two strains (Table 1
), although this can not be precisely correlated to a distinctive lipid species.
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The glycopeptidolipids (GPLs)
GPLs have been previously largely implicated in biological and cell-surface properties of M. smegmatis, e.g. receptors for mycobacteriophages (Furuchi & Tokunaga, 1972; Goren et al., 1972
), colony morphology (Etienne et al., 2002
; Billman-Jacobe et al., 1999
) or binding to human monocyte-derived macrophages (Villeneuve et al., 2003
). They are also involved in the sliding motility of mc2155, as shown by the non-motility of GPL-deficient mutants (Martinez et al., 1999
; Etienne et al., 2002
). Therefore, we addressed the question of a possible difference in the GPL content of strains ATCC 607 and mc2155. GPLs are among the characteristic lipids of mycobacteria (Daffé & Lemassu 2000
) and share a common lipopeptidyl core consisting of a mixture of 3-hydroxy and 3-methoxy C2634 fatty acids amidated by the tripeptide D-phe-D-allo-thr-D-ala and terminated by L-alaninol (Daffé et al., 1983b
). The diversity of GPLs has been recently extended in M. smegmatis (Ojha et al., 2002
; Villeneuve et al., 2003
). The major and first-described GPL of M. smegmatis (GPL I) is composed of, in addition to the core, a di-O-acetyl-6-deoxytalosyl residue linked to allo-threoninyl and a tri-O-methylrhamnosyl unit attached to the alaninol residue (Brennan, 1988
). Other components of this family include the deacetylated GPLs I (or GPLs IIa) and the hyperglycosylated GPLs I (or GPLs IIb), which possess a second di-O-methylrhamnosyl unit linked to alaninol (Ojha et al., 2002
; Villeneuve et al., 2003
). The succinylated GPLs (or GPLs IIIa and IIIb, with one and two rhamnosyl units, respectively) carry a succinyl substituent on the terminal di-O-methylrhamnosyl unit linked to the alaninol end (Villeneuve et al., 2003
). Crude cellular GPLs were prepared by methanol precipitation of the total cellular lipids and analysed by TLC (Fig. 3a
). Polar GPLs were more abundant in strain ATCC 607 than in strain mc2155; it was not possible, however, to determine with certainty by this method whether GPLs II and GPLs III coexist in the mixtures, as the two types of GPLs co-migrated in TLC (Villeneuve et al., 2003
). The MALDI-TOF mass spectra of the crude GPLs of strains ATCC 607 and mc2155 are depicted Fig. 3(b)
. GPLs I were the prominent compounds, with the major pseudomolecular masses (M+Na) at m/z 1283·1 and 1257·3 for strains ATCC 607 and mc2155, respectively. GPLs IIb (m/z at 1429·1, 1443·1), IIIa (m/z at 1369·0, 1383·0) and IIIb (m/z at 1543·0) were readily detectable in the mass spectrum of strain ATCC 607. In contrast, pseudomolecular masses corresponding to GPLs IIb and GPLs IIIb were not detected in the mass spectrum of strain mc2155; in addition, the relative intensity of the peak corresponding to GPLs IIIa (m/z at 1343·3) appeared to be considerably diminished in the mass spectrum of this strain. When the ratio of the peaks corresponding to GPLs II/I or III/I were estimated from the MALDI-TOF spectra (Fig. 3b
) and compared, it appeared that while GPLs IIIa represented 51 % of the GPLs I in strain ATCC 607, they accounted for only 13 % in strain mc2155. The percentages of GPLs IIa, IIb and IIIb in strain ATCC 607 were, respectively, 3·5, 14 and 10 %, whereas they were below 1 % in strain mc2155. Thus, the MALDI-TOF mass spectrometry data correlated well with the results of TLC and showed that both GPLs II and III were present in very small amounts in strain mc2155.
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The lipooligosaccharides (LOSs)
LOSs are alkali-labile trehalose-containing lipids (Daffé & Lemassu, 2000) which have been characterized in some mycobacterial species, including M. smegmatis (Saada & Ballou, 1983
; Kamisango et al., 1985
). They were hardly detectable in the total lipid extracts by either TLC or MALDI-TOF mass spectrometry analyses. They were eluted from the Florisil column in the same fractions as the GPLs II subfamily, i.e. chloroform/methanol 80 : 20 (v/v), but co-migrated on TLC with these latter compounds (Fig. 4
, lane 1). MALDI-TOF mass spectrometry analysis of these fractions revealed, besides the presence of GPLs II in both strains, the characteristic pseudomolecular ions [M+Na]+ (Aspinall et al., 1995
) of the major species of LOSs A (Fig. 5
) in the mass spectra of fractions from strain ATCC 607 at m/z 1689 and 1717 (LOSs A), but not in those from strain mc2155. Another species of LOSs, LOSs B1, previously described in M. smegmatis, was also detected with the [M+Na]+ expected at m/z 1429/1457 in ATCC 607, but not in strain mc2155. The third species of LOSs (LOSs B2 with the [M+Na]+ at m/z 1591/1619) were absent from the fractions isolated from both strains (Fig. 5
). As LOSs B1 were expected to co-elute with GPLs II and to exhibit identical molecular masses, the GPLs/LOSs-containing fractions of strain ATCC 607 were saponified and re-examined by both TLC and mass spectrometry. TLC analysis showed, upon saponification, the disappearance of two spots with respective RF values of 0·45 and 0·39 (Fig. 4
, lanes 1 and 2). The two notable spots of low RF, which appeared upon saponification of the ATCC 607 lipids (Fig. 4
, lane 2), were attributable to deacylated GPLs II. Concomitantly, the characteristic pseudomolecular ion [M+Na]+ peaks at m/z 1689 and 1717 a.m.u. disappeared from the MALDI-TOF spectra of the alkali-treated fractions of strain ATCC 607 (data not shown). It was thus likely that only strain ATCC 607 produces LOSs A. In contrast, no change was observed in the TLC pattern of glycolipids from strain mc2155 after the KOH treatment (Fig. 4
, lanes 3 and 4), indicating that all the spots visible on the TLC of native lipids consisted of non-acylated alkali-stable GPLs. These data confirmed that LOSs were absent from strain mc2155.
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Analysis of the extracellular materials
GPLs are major components of the outermost layer of M. smegmatis (Etienne et al., 2002). One possible consequence of the absence of GPLs II and III from the mutant cells may be the release of surface-exposed substances into the culture medium. Moreover, the apparent disorganization of the cell envelope in strain mc2155 (Etienne et al., 2002
) may also lead to the release of some of its components. To test this hypothesis, the composition of the extracellular material of the mutant was compared with that of the parent strain. The amounts of materials released by the two strains in the culture broth were similar: 3·1±0·4 and 2·9±0·4 mg per 100 mg dry cells for strains ATCC 607 and mc2155, respectively (means±SD). The composition of the culture-broth substances was also comparable in the two strains. Extracellular material from strain ATCC 607 consisted of 6·7±2·8 % lipid, 53·5±15·5 % protein and 39·8±29·7 % carbohydrate, whereas these values were 7·9±0·4 %, 52·0±4·1 % and 40·1±31·4 %, respectively, in strain mc2155. In addition, both strains exhibited the same sugar composition (data not shown), which was similar to that previously found for M. smegmatis (Lemassu et al., 1996
). Another approach used to detect the possible release of surface-exposed substances was to examine extracellular enzyme activities in the culture broth of strain mc2155. We have previously detected 14 extracellular enzyme activities in M. smegmatis ATCC 607; all of them were also surface-exposed, along with 11 additional activities which were more or less deeply located in the cell envelope of the bacteria (Raynaud et al., 1998
). Examination of the enzyme activities present in the culture broth or surface-exposed in strain mc2155 revealed no difference between mc2155 and the ATCC 607 strains (data not shown). Thus, the absence of GPLs II and III from the mutant strain and the apparent disorganization of its cell envelope does not induce the release of surface components into the culture fluid.
Cell wall permeability of the M. smegmatis strains
GPLs are major lipids of M. smegmatis and have been shown to significantly contribute to the permeability barrier of the cell envelope of the bacteria (Etienne et al., 2002). Considering the low level of GPLs II and III in strain mc2155, it was interesting to address the question of the importance of the GPL types in the permeability of the mycobacterial outer barrier. To determine whether it was altered in strain mc2155, the uptake of the hydrophobic chenodeoxycholate by cells of the wild-type strain ATCC 607 and the mutant mc2155 was compared. Chenodeoxycholate is a negatively charged hydrophobic molecule that has been previously used to evaluate the fluidity of mycobacterial cell wall lipids (Dubnau et al., 2000
; Etienne et al., 2002
; Jackson et al., 1999
; Liu et al., 1996
; Yuan et al., 1997
). Strain mc2155 showed an initial rate of uptake and a final accumulation of chenodeoxycholate significantly higher than those of the parent strain (Fig. 6
).
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DISCUSSION |
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One of the main differences revealed in the cell envelope composition of the two strains of M. smegmatis examined here is a marked dissimilarity in strain ATCC 607 of the lipid distribution between the outermost layer and the deeper compartments. One additional difference between the two strains is the formation of filamentous and rope-like structures at the surface of the mc2155 cells in complex media, when large amounts of GPLs are present on the cell surface. These structures were absent from a GPL-deficient mutant cultivated in the same conditions (Etienne et al., 2002), and therefore can be attributed to GPLs budding from the surface. It seems that, in strain mc2155, a particular stratified organization of the envelope has been lost; the reason for such a loss deserves consideration, as the precise nature of this stratification remains elusive. It is worth noting, however, that the disturbance of the outermost layer was not the reflection of a marked disorganization of the envelope. Indeed, neither specific release of surface constituents into the culture broth nor the exposure at the cell surface of a compound located in deeper compartments of the mycobacterial cell envelope such as TDM (Ortalo-Magné et al., 1996
) were observed in the mutant.
We investigated the consequence of these differences in envelope composition on cell wall properties, especially their impact on the outer permeability barrier. In all currently proposed models, the outer permeability barrier of mycobacteria consists of a monolayer of mycoloyl residues covalently linked to the cell wall arabinogalactan and includes other lipids which are probably arranged to form a bilayer with the mycoloyl residues (Brennan & Nikaido, 1995; Daffé & Draper, 1998
; Minnikin, 1982
; Rastogi, 1991
). Although cell-wall-linked mycolates certainly contribute to this barrier (Jackson et al., 1999
), the involvement of non-covalently bound lipids in the wall bilayer has been demonstrated to date for only phthiocerol dimycocerosates of M. tuberculosis MT103 (Camacho et al., 2001
) and GPLs of M. smegmatis mc2155 (Etienne et al., 2002
). The present work demonstrates that the events that have led to M. smegmatis mc2155 have a profound effect on the uptake of chenodeoxycholate, a hydrophobic molecule that diffuses through lipid domains of the mycobacterial cell wall (Dubnau et al., 2000
; Etienne et al., 2002
; Jackson et al., 1999
; Liu et al., 1996
; Yuan et al., 1997
). Considering the implication of lipids in the establishment of the outer permeability barrier of mycobacteria and knowing that the two strains examined elaborated comparable amounts of GPLs, the increased permeability of the cell envelope of strain mc2155 may originate either from the absence of LOSs or from a change in the chemical structure of the GPLs exposed by this strain. Although LOSs are hardly quantifiable by mass spectrometry or TLC analysis, they appeared not to be as major a component as GPLs or phospholipids in the cell envelope of strain ATCC 607. It follows that variations in the proportions of GPL subfamilies are likely to be the origin of the alterations of the envelope properties of strain mc2155. The fact that subtle modifications of some cell envelope constituents may lead to a dramatic phenotypic change is not without precedent. Indeed, it has been previously reported that the chemical structures of mycolic acids play a role in determining the fluidity and permeability of the mycobacterial cell wall (George et al., 1995
; Liu et al., 1996
; Dubnau et al., 2000
). Moreover, the O-succinylation of GPLs is critical in their ability to inhibit mycobacterial phagocytosis by human macrophages (Villeneuve et al., 2003
). Finally, should the fact that strain mc2155 displays an enhanced cellular permeability be correlated with its transformablity? Snapper et al., (1990)
, who isolated the strain, looked for a possible alteration of the cell wall as an alternative hypothesis for the ept phenotype. Electroporation, in contrast to plasmid transformation, of mc2155 and its parental strain mc26 derived from strain ATCC 607 by DNA from mycobacteriophage D29 resulted in no significant difference between the two strains. In addition, transformation by an integrative plasmid yielded similar numbers of transformants for both strains mc2155 and mc26, at frequencies that were four orders of magnitude lower than for transformation with replicative plasmids (Snapper et al., 1990
). Thus, it appears that the alteration of the cell envelope of mc2155, although enough to affect the uptake of small hydrophobic molecules, may not be sufficient to completely explain its transformable character.
An alternative explanation would be that genomic and phenotypic rearrangements have occurred as the result of the subcloning of strain ATCC 607 which has led to mc26, the parental strain of mc2155 (Jacobs, 2000). In this connection, PFGE analysis has revealed that a large duplication exists in the mc2155 genome (Galamba et al., 2001
) but which is differently located in the ATCC 607 genome (A. Galamba & J. Content, unpublished results). In addition, it appears that another rearrangement of the genetic material, possibly a deletion, has occurred in the ATCC 607 chromosome to generate mc2155 (A. Galamba & J. Content, unpublished results). To our best knowledge, this would be the first report where the isolation of a mutant strain in M. smegmatis could be correlated with a major chromosomic rearrangement. But such events are not uncommon in mycobacteria. Although members of the M. tuberculosis complex display an unusually high degree of conservation in their housekeeping genes (Sreevatsan et al., 1997
), whole-genome comparison of strains has revealed a large polymorphism due to numerous insertiondeletion events (Behr et al., 1999
; Gordon et al., 1999
; Brosch et al., 2002
; Tsolaki et al., 2004
). These events have often been correlated with a smooth-to-rough variation in species of the M. avium complex (Belisle et al., 1993b
; Eckstein et al., 2000
; O'Shea et al., 2004
) or in the fast-growing Mycobacterium abscessus (Howard et al., 2002
). Thus, M. smegmatis strains appear to be, like other mycobacterial species, subject to chromosome rearrangement. Taken together, these data indicate that the nature of the surface-exposed compounds and of the envelope constituents is crucial for the mycobacterial phenotype, and suggest that the transformable character of the mc2155 strain may be in part explained by a profound modification of its cell envelope.
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ACKNOWLEDGEMENTS |
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Received 5 January 2005;
revised 8 March 2005;
accepted 9 March 2005.
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