| Abstract: | Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum. AftB catalyzes the transfer of Araf (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal β(1 → 2)-linked Araf residues. Here we show that ΔaftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a ΔaftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum, but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae, and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the ΔaftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum.Corynebacterineae is a specific suborder of Gram-positive bacteria that includes medically and economically important species, such as Mycobacterium tuberculosis, Mycobacterium leprae, and Corynebacterium glutamicum. The ultrastructure of cell envelopes in the Corynebacterineae has been intensively studied during recent decades and has revealed a completely unexpected scheme. Indeed, they are composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. This outer membrane is made up of mycolic acids that either esterify trehalose (free mycolates) or are terminal Araf residues of AG skeleton (bound mycolates) (38-40). The disclosure of this very atypical structure initially came from functional studies in which pore-forming proteins were identified in almost all members of the Corynebacterineae (41, 62), pointing out the presence of an unexpected additional hydrophobic barrier in the cell envelope of these Gram-positive bacteria. Its presence was recently clearly visualized by cryo-electron microscopy of vitreous sections (CEMOVIS) (29, 67), but the exact physiological properties of this barrier are poorly documented because only a few integral outer membrane proteins (OMPs) are yet characterized. In mycobacteria, two examples of integral OMPs are well documented: OmpA of M. tuberculosis and MspA of Mycobacterium smegmatis. MspA is a major porin involved in mycolate outer membrane permeability (58, 64) and has a very specific structure based on a β-barrel core reminiscent of that found in Gram-negative porins (25). In C. glutamicum, four different porins of very low molecular mass (PorA [36, 42], PorH [30], and PorB and PorC [18]) have been identified. Very recently it was shown that a heterooligomeric structure composed of PorA and PorH is needed to form the major cell wall channel of C. glutamicum (6). Although these proteins have been extensively characterized in vitro, their physiological importance remains elusive. Surprisingly, preliminary structural studies of PorB suggested that it could be organized in an α-helical structure, in contrast to all the “canonical” porins, and thus could represent a new class of outer membrane proteins (66).Because Gram-negative outer membrane proteins and MspA from M. smegmatis are organized in β-barrel structures, two studies aimed to identify M. tuberculosis OMPs by a bioinformatic approach using β-barrel prediction algorithms (43, 57). Although very attractive, this approach has several limitations. Indeed, these algorithms generally produce a high number of false-positive proteins and are not able to predict amphipathic α-helical structures such as those expected for PorB. Nevertheless, this approach led to the identification of a new channel-forming outer membrane protein of M. tuberculosis, Rv1698, which is conserved among all members of the Corynebacterineae (55). This protein increases the susceptibility of bacteria to hydrophilic antibiotics and increases the rate of glucose uptake. Its structure has not yet been characterized.Isolation of the outer membrane of mycobacteria or corynebacteria using standard biochemical methods is difficult due to the covalent links between mycolic acids and the underlying heteropolymer hampering the identification of new OMPs. In order to overcome this problem, an alternative approach could come from outer membrane-derived vesicles (OMVs), whose release in the medium is a conserved mechanism among Gram-negative bacteria and has been observed in many environments (34). Increased OMV release has been reported for mutants lacking either components of the Tol-Pal system (8, 63) or Lpp, the major lipoprotein involved in noncovalent interactions between the outer membrane and the peptidoglycan (8, 14, 15, 22, 31). This suggested that OMV production in Gram-negative bacteria is controlled through specific domains that promote outer membrane protein-peptidoglycan and outer membrane protein-inner membrane interactions (22). OMVs from Gram-negative bacteria are composed of lipopolysaccharide (LPS), phospholipids, and outer membrane proteins but lack inner membrane proteins. In some cases, elementary units of peptidoglycan and periplasmic proteins are also recovered in these vesicles (35, 37).C. glutamicum is the prototype of amino acid producers and has been widely used in biotechnology for several decades. More recently it was also shown to be a very attractive model for depicting cell wall biosynthesis of the Corynebacterineae (45). Indeed, an extensive library of mutants involved in the different steps of mycolic acid and arabinogalactan synthesis is available for C. glutamicum but not for mycobacteria, where these components are strictly essential for viability. In this work, we wanted to screen different C. glutamicum cell wall mutants for their ability to release outer membrane-derived vesicles in the external medium. More particularly, we focused on mutants with altered covalent linkage between the mycolate outer membrane and the arabinogalactan. Accordingly, we constructed an arabinosyltransferase mutant unable to catalyze the transfer of Araf onto the arabinan domain of AG to form terminal β(1 → 2)-linked Araf residues. This abnormal AG structure results in both a decrease in mycoloylation sites on arabinogalactan and a concomitant appearance of outer membrane-derived fragments in the external medium. |
