LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain.

BACKGROUND: Aminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region. RESULTS: Using genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human ß-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria. CONCLUSIONS: Overall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.

The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope.

Trehalose polyphleates (TPP) are high-molecular-weight, surface-exposed glycolipids present in a broad range of nontuberculous mycobacteria. These compounds consist of a trehalose core bearing polyunsaturated fatty acyl substituents (called phleic acids) and a straight-chain fatty acid residue and share a common basic structure with trehalose-based glycolipids produced by Mycobacterium tuberculosis TPP production starts in the cytosol with the formation of a diacyltrehalose intermediate. An acyltransferase, called PE, subsequently catalyzes the transfer of phleic acids onto diacyltrehalose to form TPP, and an MmpL transporter promotes the export of TPP or its precursor across the plasma membrane. PE is predicted to be an anchored membrane protein, but its topological organization is unknown, raising questions about the subcellular localization of the final stage of TPP biosynthesis and the chemical nature of the substrates that are translocated by the MmpL transporter. Here, using genetic, biochemical, and proteomic approaches, we established that PE of Mycobacterium smegmatis is exported to the cell envelope following cleavage of its signal peptide and that this process is required for TPP biosynthesis, indicating that the last step of TPP formation occurs in the outer layers of the mycobacterial cell envelope. These results provide detailed insights into the molecular mechanisms controlling TPP formation and transport to the cell surface, enabling us to propose an updated model of the TPP biosynthetic pathway. Because the molecular mechanisms of glycolipid production are conserved among mycobacteria, these findings obtained with PE from M. smegmatis may offer clues to glycolipid formation in M. tuberculosis.

Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane.

The mycobacterial envelope is unique, containing the so-called mycomembrane (MM) composed of very-long chain fatty acids, mycolic acids (MA). Presently, the molecular composition of the MM remains unproven, due to the diversity of methods used for determining its composition. The plasma membranes (PM) and the native MM-containing cell walls (MMCW) of two rapid-growing mycobacterial species, Mycobacterium aurum and M. smegmatis, were isolated from their cell lysates by differential ultracentrifugation. Transmission electron microscopy and biochemical analyses demonstrated that the two membranes were virtually pure. Bottom-up quantitative proteomics study indicated a different distribution of more than 2,100 proteins between the PM and MMCW. Among these, the mannosyltransferase PimB, galactofuranosyltransferase GlfT2, Cytochrome p450 and ABC transporter YjfF, were most abundant in the PM, which also contain lipoglycans, phospholipids, including phosphatidylinositol mannosides, and only a tiny amount of other glycolipids. Antigen85 complex proteins, porins and the putative transporters MCE protein family were mostly found in MMCW fraction that contains MA esterifying arabinogalactan, constituting the inner leaflet of MM. Glycolipids, phospholipids and lipoglycans, together with proteins, presumably composed the outer leaflet of the MM, a lipid composition that differs from that deduced from the widely used extraction method of mycobacterial cells with dioctylsulfosuccinate sodium.

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide.

Mycobacteria have a complex cell wall structure that includes many lipids; however, even within a single subspecies of Mycobacterium avium these lipids can differ. Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide (L5P), yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis as well as biochemical and physico-chemical approaches. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in ovine strains, compared to five modules in bovine strains (C-type). Sequence analysis predicted these modules would produce the tripeptide Phe-N-Methyl-Val-Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico-chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D-Phe-N-Methyl-L-Val-L-Ala-OMe attached in N-ter to a 20-carbon fatty acid chain. These data demonstrate that S-type strains, which are more adapted in sheep, produce a unique lipid. There is a dose-dependent effect observed for L3P on upregulation of CD25+ CD8 T cells from infected cows, while L5P effects were static. In contrast, L5P demonstrated a significantly stronger induction of CD25+ B cells from infected animals compared to L3P.

pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence.

Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii-like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host-pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis, which is known to lack lipooligosaccharides. Our results suggest that pks5-recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts.

Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

Structural elucidation and genomic scrutiny of the C60-C100 mycolic acids of Segniliparus rotundus.

Mycolic acids, very long-chain α-alkyl, ß-hydroxylated fatty acids, occur in the members of the order Corynebacteriales where their chain lengths (C(26)-C(88)) and structural features (oxygen functions, cis or trans double bonds, cyclopropane rings and methyl branches) are genus- and species-specific. The molecular composition and structures of the mycolic acids of two species belonging to the genus Segniliparus were determined by a combination of modern analytical chemical techniques, which include MS and NMR. They consist of mono-ethylenic C(62-)C(64) (α'), di-ethylenic C(77)-C(79) (α) and extremely long-chain mycolic acids (α(+)) ranging from 92 to 98 carbon atoms and containing three unsaturations, cis and/or trans double bonds and/or cyclopropanes. The double bonds in each class of mycolic acids were positioned by oxidative cleavage and exhibit locations similar to those of α- and α'-mycolic acids of mycobacteria. For the ultralong chain α-mycolic acids, the three double bonds were located at equally spaced carbon intervals (C(13)-C(16)), with the methyl branches adjacent to the proximal and distal trans double bonds. Examination of the Segniliparus rotundus genome compared with those of other members of the Corynebacteriales indicated two obvious differences in genes encoding the elongation fatty acid (FAS-II) enzymes involved in the biosynthesis of mycolic acids: the organization of 3-ketoacyl-ACP synthases (KasA and KasB) and (3R)-hydroxyacyl-ACP dehydratases (HadAB/BC), on one hand, and the presence of two copies of the hadB gene encoding the catalytic domain of the latter enzyme type, on the other. This observation is discussed in light of the most recent data accumulated on the biosynthesis of this hallmark of Corynebacteriales.

A novel mycolic acid species defines two novel genera of the Actinobacteria, Hoyosella and Amycolicicoccus.

Corynebacterineae are characterized by the presence of long-chain lipids, notably mycolic acids (α-alkyl, ß-hydroxy fatty acids), the structures of which are genus-specific. Mycolic acids from two environmental strains, Amycolicicoccus subflavus and Hoyosella altamirensis, were isolated and their structures were established using a combination of mass spectrometry analysis, (1)H-NMR spectroscopy and chemical degradations. The C(2)-C(3) cleavage of these C(30)-C(36) acids led to the formation of two fragments: saturated C(9)-C(11) acids, and saturated and unsaturated C(20)-C(25) aldehydes. Surprisingly, the fatty acids at the origin of the two fragments making up these mycolic acids were present in only minute amounts in the fatty acid pool. Moreover, the double bond in the main C(24) aldehyde fragment was located at position ω16, whereas that found in the ethylenic fatty acids of the bacteria was at ω9. These data question the biosynthesis of these new mycolic acids in terms of the nature of the precursors, chain elongation and desaturation. Nevertheless, they are consistent with the occurrence of the key genes of mycolic acid biosynthesis, including those encoding proteins of the fatty acid synthase II system, identified in the genome of A. subflavus. Altogether, while the presence of mycolic acids and analysis of their 16S rDNA sequences would suggest that these strains belong to the Mycobacteriaceae family, the originality of their structures reinforces the recent description of the novel genera Amycolicicoccus and Hoyosella.

Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants.

Changes in the cell envelope composition of mycobacteria cause major changes in cytokine profiles of infected antigen presenting cells. We describe here the modulation of inflammatory responses by Mycobacterium abscessus, an emerging pathogen in cystic fibrosis. M. abscessus is able to switch from a smooth (S) to a rough (R) morphotype by the loss of a surface glycopeptidolipid. R variants are associated with severe clinical forms and a 'hyper-proinflammatory' response in ex vivo and in vivo models. Using partitioning of cell surface components we found that a complex fraction, more abundant in R variants than in S variants, made a major contribution to the TLR-2-dependent hyper-proinflammatory response induced by R variants. Lipoproteins were the main TLR-2 agonists in this fraction, consistent with the larger amounts of 16 lipoproteins in cell surface extracts from R variants; 15 out of 16 being more strongly induced in R variant than in S variant. Genetic interruption of glycopeptidolipid pathway in wild-type S variant resulted in R phenotype with similar induction of lipoprotein genes. In conclusion, R morphotype in M. abscessus is associated with increased synthesis/exposure at the cell surface of lipoproteins, these changes profoundly modifying the innate immune response through TLR-2-dependent mechanisms.

MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis.

The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram-negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single-chain antibodies against the surface-exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C-terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery.

Identification of the polyketide synthase involved in the biosynthesis of the surface-exposed lipooligosaccharides in mycobacteria.

Lipooligosaccharides (LOS) are highly antigenic glycolipids produced by a number of Mycobacterium species, which include "M. canettii," a member of the M. tuberculosis complex, and the opportunistic pathogens M. marinum and M. kansasii. The various LOS share a core composed of trehalose esterified by at least 1 mole of polymethyl-branched fatty acid (PMB-FA) and differ from one another by their oligosaccharide extensions. In this study, we identified a cluster of genes, MSMEG_4727 through MSMEG_4741, likely involved in the synthesis of LOS in M. smegmatis. Disruption of MSMEG_4727 (the ortholog of pks5 of M. tuberculosis), which encodes a putative polyketide synthase, resulted in the concomitant abrogation of the production of both PMB-FA and LOS in the mutant strain. Complementation of the mutant with the wild-type gene fully restored the phenotype. We also showed that, in contrast to the case for "M. canettii" and M. marinum, LOS are located in deeper compartments of the cell envelope of M. smegmatis. The availability of two mycobacterial strains differing only in LOS production should help in defining the biological role(s) of this important glycolipid.

The cell surface-exposed glycopeptidolipids confer a selective advantage to the smooth variants of Mycobacterium smegmatis in vitro.

The cell surface of mycobacteria is quite rich in lipids. Glycopeptidolipids, surface-exposed lipids that typify some mycobacterial species, have been associated with a phenotypic switch between rough and smooth colony morphotypes. This conversion in Mycobacterium smegmatis is correlated with the absence/presence of glycopeptidolipids on the cell surface and is due to insertion sequence mobility. Here, we show that the occurrence of a high amount of glycopeptidolipids in the smooth variant leads to lower invasion abilities and lower internalization by macrophages. We further show that the high production of glycopeptidolipids on the cell surface can confer a selective advantage to the smooth variant when grown in vitro. This higher fitness under the laboratory condition might explain the selection of smooth variants in several independent laboratories. The implications of these findings are discussed.

SigF controls carotenoid pigment production and affects transformation efficiency and hydrogen peroxide sensitivity in Mycobacterium smegmatis.

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that sigma(F) is required for carotenoid pigment production in Mycobacterium smegmatis. We further show that a sigF mutant exhibits a transformation efficiency 10(4)-fold higher than that of the parental strain, suggesting that sigma(F) regulates the production of components affecting cell wall permeability. In addition, a sigF mutant showed an increased sensitivity to hydrogen peroxide. An in silico search of the M. smegmatis genome identified a number of SigF consensus sites, including sites upstream of the carotenoid synthesis locus, which explains its SigF regulation.

Spontaneous transposition of IS1096 or ISMsm3 leads to glycopeptidolipid overproduction and affects surface properties in Mycobacterium smegmatis.

Natural modification of the colony appearance is a phenomenon that has not been fully understood in mycobacteria. Here, we show that Mycobacterium smegmatis ATCC607 displays a low-frequency spontaneous morphological variation that correlates with the acquisition of a panel of new phenotypes, such as aggregation, biofilm formation and sliding motility. These variants produce larger amounts of glycopeptidolipid (GPL), a cell-surface component, than did the wild-type strain. This conversion results from the transposition of two types of insertion sequences, IS1096 and ISMsm3, into two loci. One locus is the promoter region of the mps operon, the GPL biosynthesis gene cluster, leading to the overexpression of these genes. The other locus is the lsr2 gene, which encodes a small basic histone-like protein that likely plays a regulatory role at the mps promoter and also controls pigment production. This study demonstrates that insertion sequence mobility play a crucial role in the acquisition of new phenotypes.

Lipopentapeptide induces a strong host humoral response and distinguishes Mycobacterium avium subsp. paratuberculosis from M. avium subsp. avium.

BACKGROUND: Many non-tuberculous mycobacteria synthesize abundant glycopeptidolipids (GPLs). These surface-located GPLs are involved in pathogenicity by interfering with the host immune system. In Mycobacterium avium subsp. avium (Mav), GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes. Whereas it is very obvious that most, if not all, of the Mav isolates produce GPLs, the picture is not as clear for M. avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease in cattle, and several conflicting data have been produced. METHODS: Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the recently published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. We also showed that the peptidyl moiety of the L5P is a target for a strong specific humoral response in Map infected animals. CONCLUSIONS: These genomic and biochemical differences may help to unambiguously distinguish Map from Mav and also from M. bovis, to reclassify related strains of the Map species and to allow the convenient and specific diagnosis of paratuberculosis.

Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae.

BACKGROUND: The outermost layer of the bacterial surface is of crucial importance because it is in constant interaction with the host. Glycopeptidolipids (GPLs) are major surface glycolipids present on various mycobacterial species. In the fast-grower model organism Mycobacterium smegmatis, GPL biosynthesis involves approximately 30 genes all mapping to a single region of 65 kb. RESULTS: We have recently sequenced the complete genomes of two fast-growers causing human infections, Mycobacterium abscessus (CIP 104536T) and M. chelonae (CIP 104535T). We show here that these two species contain genes corresponding to all those of the M. smegmatis "GPL locus", with extensive conservation of the predicted protein sequences consistent with the production of GPL molecules indistinguishable by biochemical analysis. However, the GPL locus appears to be split into several parts in M. chelonae and M. abscessus. One large cluster (19 genes) comprises all genes involved in the synthesis of the tripeptide-aminoalcohol moiety, the glycosylation of the lipopeptide and methylation/acetylation modifications. We provide evidence that a duplicated acetyltransferase (atf1 and atf2) in M. abscessus and M. chelonae has evolved through specialization, being able to transfer one acetyl at once in a sequential manner. There is a second smaller and distant (M. chelonae, 900 kb; M. abscessus, 3 Mb) cluster of six genes involved in the synthesis of the fatty acyl moiety and its attachment to the tripeptide-aminoalcohol moiety. The other genes are scattered throughout the genome, including two genes encoding putative regulatory proteins. CONCLUSION: Although these three species produce identical GPL molecules, the organization of GPL genes differ between them, thus constituting species-specific signatures. An hypothesis is that the compact organization of the GPL locus in M. smegmatis represents the ancestral form and that evolution has scattered various pieces throughout the genome in M. abscessus and M. chelonae.

A derivative of Mycobacterium smegmatis mc(2)155 that lacks the duplicated chromosomal region.

The genome of Mycobacterium smegmatis mc(2)155 contains a 56kb duplicated region. We isolated a mutant of mc(2)155 lacking this duplication (DeltaDRKIN). This mutation did not affect the growth rate, surface properties or transformation efficiency of the organism, confirming the potential utility of DeltaDRKIN for the study of genes contained within the duplicated region.

Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface.

The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.

A glycosyltransferase involved in biosynthesis of triglycosylated glycopeptidolipids in Mycobacterium smegmatis: impact on surface properties.

The cell envelope of mycobacteria is a complex structure that plays an important role in the interactions of the cell with its environment and in the protection against the antimicrobial activity of the immune system. Glycopeptidolipids (GPLs) are species- or type species-specific glycolipids that are present at the surface of a number of mycobacteria and that are characterized by a high variability in glycosylation patterns. These GPLs possess various biological activities that depend mostly on the sugars capping the core molecule. In Mycobacterium smegmatis, the GPL core can be substituted by either two or three deoxyhexoses. In this study, we show that Gtf3 is a glycosyltransferase responsible for the synthesis of the triglycosylated GPLs. Biochemical analysis of these molecules, with a combination of mass spectrometry and chemical degradation methods, has shown that they contain three deoxyhexose moieties. The presence of the triglycosylated GPLs is associated with cell surface modifications that lead to a decrease in sliding motility as well as a modification in cellular aggregation and colony appearance on Congo red. Phylogenetic analysis indicated that Gtf3 is a member of a yet-uncharacterized glycosyltransferase family conserved among the mycobacteria.

The cell envelope structure and properties of Mycobacterium smegmatis mc(2)155: is there a clue for the unique transformability of the strain?

Mycobacterium smegmatis is often used as a surrogate host for pathogenic mycobacteria, especially since the isolation of the transformable smooth morphotype strain mc(2)155 from the isogenic rough wild-type strain ATCC 607. Biochemical analysis of the cell envelope components revealed a lack of polar glycolipids, namely the lipooligosaccharides and the polar subfamilies of glycopeptidolipids, in the mc(2)155 strain. In addition, the latter strain differs from its parent by the distribution of various species of glycolipids and phospholipids between the outermost and deeper layers of the cell envelope. The presence of filamentous and rope-like structures at the cell surface of mc(2)155 cells grown in complex media further supported an ultrastructural change in the cell envelope of the mutant. Importantly, a significantly more rapid uptake of the hydrophobic chenodeoxycholate was observed for the mutant compared to wild-type cells. Taken together, these data indicate that the nature of the surface-exposed and envelope constituents is crucial for the surface properties, cell wall permeability and bacterial phenotype, and suggest that the transformable character of the mc(2)155 strain may be in part explained by these profound modifications of its cell envelope.