Synthesis of chemical tools to label the mycomembrane of corynebacteria using modified iron(III) chloride-mediated protection of trehalose.

Trehalose-based probes are useful tools that allow the detection of the mycomembrane of mycobacteria through the metabolic labeling approach. Trehalose analogues conjugated to fluorescent probes can be used, and other probes are functionalized with a bioorthogonal chemical reporter for a two-step labeling approach. The synthesis of such trehalose-based probes mainly relies on the desymmetrization of natural trehalose using a large number of regioselective protection-deprotection steps to differentiate the eight hydroxyl groups. Herein, in order to avoid these time-consuming steps, we reinvestigated our previously reported tandem protocol mediated by FeCl3·6H2O, with the aim of modifying the ratio of the products to allow the challenging desymmetrization of the C2-symmetrical disaccharide trehalose. We demonstrate the usefulness of this method in providing easy access to trehalose analogues with a bioorthogonal moiety or a fluorophore in C-2, and also present their use in a one-step and two-step labeling approach, either of which can be used to study the mycomembrane in live mycobacteria.

The C-terminal domain of Corynebacterium glutamicum mycoloyltransferase A is composed of five repeated motifs involved in cell wall binding and stability.

The genomes of Corynebacteriales contain several genes encoding mycoloyltransferases (Myt) that are specific cell envelope enzymes essential for the biogenesis of the outer membrane. MytA is a major mycoloyltransferase of Corynebacterium glutamicum, displaying an N-terminal domain with esterase activity and a C-terminal extension containing a conserved repeated Leu-Gly-Phe-Pro (LGFP) sequence motif of unknown function. This motif is highly conserved in Corynebacteriales and found associated with cell wall hydrolases and with proteins of unknown function. In this study, we determined the crystal structure of MytA and found that its C-terminal domain is composed of five LGFP motifs and forms a long stalk perpendicular to the N-terminal catalytic α/ß-hydrolase domain. The LGFP motifs are composed of a 4-stranded ß-fold and occupy alternating orientations along the axis of the stalk. Multiple acetate binding pockets were identified in the stalk, which could correspond to putative ligand-binding sites. By using various MytA mutants and complementary in vitro and in vivo approaches, we provide evidence that the C-terminal LGFP domain interacts with the cell wall peptidoglycan-arabinogalactan polymer. We also show that the C-terminal LGFP domain is not required for the activity of MytA but rather contributes to the overall integrity of the cell envelope.

Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in Corynebacterium glutamicum.

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys+1 residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified the lgt and lspA genes from Corynebacterium glutamicum and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a C. glutamicum maltose-binding lipoprotein, and LppX, a Mycobacterium tuberculosis lipoprotein. However, Lgt is not required for these proteins' signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in C. glutamicum the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.

Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales.

Mycobacterium and Corynebacterium are important genera of the Corynebacteriales order, the members of which are characterized by an atypical diderm cell envelope. Indeed the cytoplasmic membrane of these bacteria is surrounded by a thick mycolic acid-arabinogalactan-peptidoglycan (mAGP) covalent polymer. The mycolic acid-containing part of this complex associates with other lipids (mainly trehalose monomycolate (TMM) and trehalose dimycolate (TDM)) to form an outer membrane. The metabolism of mycolates in the cell envelope is governed by esterases called mycoloyltransferases that catalyze the transfer of mycoloyl chains from TMM to another TMM molecule or to other acceptors such as the terminal arabinoses of arabinogalactan or specific polypeptides. In this review we present an overview of this family of Corynebacteriales enzymes, starting with their expression, localization, structure and activity to finally discuss their putative functions in the cell. In addition, we show that Corynebacteriales possess multiple mycoloyltransferases encoding genes in their genome. The reason for this multiplicity is not known, as their function in mycolates biogenesis appear to be only partially redundant. It is thus possible that, in some species living in specific environments, some mycoloyltransferases have evolved to gain some new functions. In any case, the few characterized mycoloyltransferases are very important for the bacterial physiology and are also involved in adaptation in the host where they constitute major secreted antigens. Although not discussed in this review, all these functions make them interesting targets for the discovery of new antibiotics and promising vaccines candidates. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Perception of vowel length by Japanese- and English-learning infants.

This study investigated vowel length discrimination in infants from 2 language backgrounds, Japanese and English, in which vowel length is either phonemic or nonphonemic. Experiment 1 revealed that English 18-month-olds discriminate short and long vowels although vowel length is not phonemically contrastive in English. Experiments 2 and 3 revealed that Japanese 18-month-olds also discriminate the pairs but in an asymmetric manner: They detected only the change from long to short vowel, but not the change in the opposite direction, although English infants in Experiment 1 detected the change in both directions. Experiment 4 tested Japanese 10-month-olds and revealed a symmetric pattern of discrimination similar to that of English 18-month-olds. Experiment 5 revealed that native adult Japanese speakers, unlike Japanese 18-month-old infants who are presumably still developing phonological perception, ultimately acquire a symmetrical discrimination pattern for the vowel contrasts. Taken together, our findings suggest that English 18-month-olds and Japanese 10-month-olds perceive vowel length using simple acoustic?phonetic cues, whereas Japanese 18-month-olds perceive it under the influence of the emerging native phonology, which leads to a transient asymmetric pattern in perception.

First access to a mycolic acid-based bioorthogonal reporter for the study of the mycomembrane and mycoloyltransferases in Corynebacteria.

In this study, we report the first synthesis of an alkyne-based trehalose monomycolate probe containing a ß-hydroxylated fatty acid and an α-branched chain similar to those of the natural mycolic acid. We demonstrate its utility for the labeling of the mycomembrane of Corynebacteria as well as for the study of mycoloyltransferases.

Infant-directed speech supports phonetic category learning in English and Japanese.

Across the first year of life, infants show decreased sensitivity to phonetic differences not used in the native language [Werker, J. F., & Tees, R. C. (1984). Cross-language speech perception: evidence for perceptual reorganization during the first year of life. Infant Behaviour and Development, 7, 49-63]. In an artificial language learning manipulation, Maye, Werker, and Gerken [Maye, J., Werker, J. F., & Gerken, L. (2002). Infant sensitivity to distributional information can affect phonetic discrimination. Cognition, 82(3), B101-B111] found that infants change their speech sound categories as a function of the distributional properties of the input. For such a distributional learning mechanism to be functional, however, it is essential that the input speech contain distributional cues to support such perceptual learning. To test this, we recorded Japanese and English mothers teaching words to their infants. Acoustic analyses revealed language-specific differences in the distributions of the cues used by mothers (or cues present in the input) to distinguish the vowels. The robust availability of these cues in maternal speech adds support to the hypothesis that distributional learning is an important mechanism whereby infants establish native language phonetic categories.

Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum.

Corynebacterium glutamicum is a biotin-auxotrophic bacterium and some strains efficiently produce glutamic acid under biotin-limiting conditions. In an effort to understand C. glutamicum metabolism under biotin limitation, growth of the type strain ATCC 13032 was investigated in batch cultures and a time-course analysis was performed. A transient excretion of organic acids was observed and we focused our attention on lactate synthesis. Lactate synthesis was due to the ldh-encoded l-lactate dehydrogenase (Ldh). Features of Ldh activity and ldh transcription were analysed. The ldh gene was shown to be regulated at the transcriptional level by SugR, a pleiotropic transcriptional repressor also acting on most phosphotransferase system (PTS) genes. Electrophoretic mobility shift assays (EMSAs) and site-directed mutagenesis allowed the identification of the SugR-binding site. Effector studies using EMSAs and analysis of ldh expression in a ptsF mutant revealed fructose 1-phosphate as a highly efficient negative effector of SugR. Fructose 1,6-bisphosphate also affected SugR binding.

Native language governs interpretation of salient speech sound differences at 18 months.

One of the first steps infants take in learning their native language is to discover its set of speech-sound categories. This early development is shown when infants begin to lose the ability to differentiate some of the speech sounds their language does not use, while retaining or improving discrimination of language-relevant sounds. However, this aspect of early phonological tuning is not sufficient for language learning. Children must also discover which of the phonetic cues that are used in their language serve to signal lexical distinctions. Phonetic variation that is readily discriminable to all children may indicate two different words in one language but only one word in another. Here, we provide evidence that the language background of 1.5-year-olds affects their interpretation of phonetic variation in word learning, and we show that young children interpret salient phonetic variation in language-specific ways. Three experiments with a total of 104 children compared Dutch- and English-learning 18-month-olds' responses to novel words varying in vowel duration or vowel quality. Dutch learners interpreted vowel duration as lexically contrastive, but English learners did not, in keeping with properties of Dutch and English. Both groups performed equivalently when differentiating words varying in vowel quality. Thus, at one and a half years, children's phonological knowledge already guides their interpretation of salient phonetic variation. We argue that early phonological learning is not just a matter of maintaining the ability to distinguish language-relevant phonetic cues. Learning also requires phonological interpretation at appropriate levels of linguistic analysis.