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Rationale: Mycobacterial species contain high concentrations of mycolic acids in their cell wall. Mycobacteria can pose a threat to both human health and the environment. Mass spectrometry lipidomic characterization can identify bacterial species and suggest targets for microbiological interventions. Due to the complex structures of mycolic acids and the possibility of isobaric isomers, multiple levels of separation are required for complete characterization. In this study, cyclic ion mobility (cIM) mass spectrometry (MS) was used for the analysis, separation and fragmentation of mycolic acids isomers from the bacterial species Gordonia amarae and Mycobacterium bovis., Methods: Mycolic acid isomers were interrogated from cultured G. amarae biomass and commercially available M. bovis mycolic acid extracts. These were infused into a cIM-enabled quadrupole time-of-flight MS. Ions of interest were non-simultaneously selected with the quadrupole and passed around the cyclic ion mobility device multiple times. Fragment ion analysis was then performed for the resolved isomers of the quadrupole-selected ions., Results: Repeated passes of the cIM device successfully resolved otherwise overlapping MA isomers, allowing isomer isolation and producing an ion-specific post-mobility fragmentation spectrum without isomeric interference., Conclusions: Mycolic acids (MA) isomers from G. amarae and M. bovis were resolved, resulting in a high mobility resolution and low interference fragmentation analysis. These revealed varying patterns of MA isomers in the two species: G. amarae's most abundant ion of each set of MA has 1-2 conformations, while the MA + 2 m/z the most abundant ion of each set has 3-6 conformations. These were resolved after 70 passes of the cyclic device. M. bovis' most abundant ion of each keto-MA set has 2 conformations, while the keto-MA + 2 m/z has 1-2 conformations. These were resolved after 75 passes., (© 2024 The Author(s). Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

The Problem: Early and rapid diagnosis of bovine tuberculosis remains an issue of great interest., Aim: The aim of this study was to evaluate the use of synthetic lipid antigens for diagnosis of tuberculosis in red deer (Cervus elaphus). The proposition: Synthetic mycolic acid derivatives, identical to components of mycobacterial cells, bind to antibodies to lipids produced in active human tuberculosis. Experimental infection studies in red deer (Cervus elaphus) allow the evaluation of such antigens for the serodiagnosis of bovine tuberculosis., Results: Antibody levels in plasma from deer experimentally infected with Mycobacterium bovis were evaluated in ELISA using synthetic antigens based on several classes of mycolic acid, using protein G as conjugate. All antigens gave significantly increased responses 60 days post-infection, when all animals had active disease. A significantly increased response was also observed with four antigens 15 days after infection., Conclusion: ELISA using synthetic lipid antigens not only detects antibodies in the plasma of deer experimentally infected with M. bovis, but a strong response occurs early in the infection. With a full analysis of responses with naturally infected animals, this may offer a useful supplement to current diagnostic methods., Competing Interests: Declaration of Competing Interest MSB is a Director of Diagnostig Ltd; the company holds background patents in the applications of synthetic mycolic acids and their esters in therapy and diagnosis. JRAD has an interest in those patents. DAT is employed by Diagnostig, and PSM was employed by the company at the time this work was done., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Mycobacteria possess unique and robust lipid profile responsible for their pathogenesis and drug resistance. Mycolic acid (MA) represents an attractive diagnostic biomarker being absent in humans, inert and known to modulate host-pathogen interaction. Accurate measurement of MA is significant to design efficient therapeutics. Despite considerable advances in Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) based approaches, quantification of mycobacterial lipids including MA is still challenging mainly because of ion suppression effects due to complex matrix and non-availability of suitable internal standards for MA. The current study demonstrates the use of standard addition method (SAM) to circumvent this problem and provides a reliable and exhaustive analytical method to quantify mycobacterial MA based on reversed-phase ultra-high-performance liquid chromatography- mass spectrometry data acquisition. In this method, multiple reaction monitoring (MRM) has been applied, wherein 16 MRM channels or transitions have been chosen for quantification of alpha-, methoxy- and keto-MAs with C-24 and C-26 hydrocarbon chains that are actually best suited for TB diagnostics. We found that the overall methodological limit of detection and limit of quantification were in the range 0.05-0.71 ng/µl and 0.16-2.16 ng/µl. Taken together, SAM quantitative technique could serve as promising alternative for relative concentration determination of MA to aid medical research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis , the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

Tuberculosis (TB) treatment becomes challenging due to the unique cell wall structure of Mycobacterium tuberculosis (M. tb). Among various components of the M.tb cell wall, mycolic acid (MA) is of particular interest because it is speculated to exhibit extremely low permeability for most of the drug molecules, thus helping M.tb to survive against medical treatment. However, no quantitative assessment of the thermodynamic barrier encountered by various well-known TB drugs in the mycolic acid monolayer has been performed so far using computational tools. On this premise, our present work aims to probe the permeability of some first and second line TB drugs, namely ethambutol, ethionamide, and isoniazid, through the modelled mycolic acid monolayer, using molecular dynamics (MD) simulation with two sets of force field (FF) parameters, namely GROMOS 54A7-ATB (GROMOS) and CHARMM36 (CHARMM) FFs. Our findings indicate that both FFs provide consistent results in terms of the mode of drug-monolayer interactions but significantly differ in the drug permeability through the monolayer. The mycolic acid monolayer generally exhibited a higher free energy barrier of crossing with CHARMM FF, while with GROMOS FF, better stability of drug molecules on the monolayer surface was observed, which can be attributed to the greater electrostatic potential at the monolayer-water interface, found for the later. Although both the FF parameters predicted the highest resistance against ethambutol (permeability values of 8.40 × 10 -34 cm s -1 and 9.61 × 10 -31 cm s -1 for the CHARMM FF and the GROMOS FF, respectively), results obtained using GROMOS were found to be consistent with the water solubility of drugs, suggesting it to be a slightly better FF for modelling drug-mycolic acid interactions. Therefore, this study enhances our understanding of TB drug permeability and highlights the potential of the GROMOS FF in simulating drug-mycolic acid interactions.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O -fatty acylation in Corynebacterium , termed protein O -mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O -mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O -mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O -acylated peptides. Using these tools in the model organism Corynebacterium glutamicum , we identified approximately 30 candidate O -mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O -mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O -mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae , Mycobacterium tuberculosis ), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

The emergence of new drug-resistant strains of the tuberculosis pathogen Mycobacterium tuberculosis (Mtb) is a new challenge for modern medicine. Its resistance capacity is closely related to the properties of the outer membrane of the Mtb cell wall, which is a bilayer membrane formed by mycolic acids (MAs) and their derivatives. To date, the molecular mechanisms of the response of the Mtb outer membrane to external factors and, in particular, elevated temperatures have not been sufficiently studied. In this work, we consider the temperature-induced changes in the structure, ordering, and molecular mobility of bilayer MA membranes of various chemical and conformational compositions. Using all-atom long-term molecular dynamics simulations of various MA membranes, we report the kinetic parameters of temperature-dependent changes in the MA self-diffusion coefficients and conformational compositions, including the apparent activation energies of these processes, as well as the characteristic times of ordering changes and the features of phase transitions occurring over a wide range of elevated temperatures. Understanding these effects could be useful for the prevention of drug resistance and the development of membrane-targeting pharmaceuticals, as well as in the design of membrane-based materials.

Lipidic adjuvant formulations consisting of immunomodulatory mycobacterial cell wall lipids interact with host cells following administration. The impact of this cross-talk on the host membrane's structure and function is rarely given enough consideration but is imperative to rule out nonspecific perturbation underlying the adjuvant. In this work, we investigated changes in the plasma membranes of live mammalian cells after exposure to mycobacterial mycolic acid (MA) and phenolic glycolipids, two strong candidates for lipidic adjuvant therapy. We found that phenolic glycolipid 1 softened the plasma membrane, lowering membrane tension and stiffness, but MA did not significantly change the membrane characteristics. Further, phenolic glycolipid 1 had a fluidizing impact on the host plasma membrane, increasing the fluidity and the abundance of fluid-ordered-disordered coexisting lipid domains. Notably, lipid diffusion was not impacted. Overall, MA and, to a lesser extent, phenolic glycolipid 1, due to minor disruption of host cell membranes, may serve as appropriate lipids in adjuvant formulations.

The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Corynebacterium glutamicum has been widely utilized for the industrial production of various amino acids. Trehalose is a prerequisite for the biosynthesis of mycolates which are structurally important constituents of the cell envelope in C. glutamicum. In this study, C. glutamicum mutant ΔSYA, which is unable to synthesize trehalose was constructed by deleting genes treS, treY and otsA in the three pathways of trehalose biosynthesis. In the fermentation medium, ΔSYA grew as well as the control C. glutamicum ATCC13869, synthesized similar levels of glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids to ATCC13869, but produced 12.5 times more L-glutamate than ATCC13869. Transcriptional levels of the genes relevant to L-arginine biosynthesis, encoding ODHC and relevant to the biosynthesis of sulfur-containing amino acids were down-regulated in ΔSYA. In minimal medium with different concentrations of glucose, ΔSYA grew worse than ATCC13869 but excreted more L-glutamate. When grown in minimal medium, phospholipids are the major lipid in ΔSYA, while glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids are the major lipid in ATCC13869. The transcriptional levels of mscCG in ΔSYA was significantly up-regulated when grown in minimal medium., Competing Interests: Disclosures The authors have no conflicts of interest., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Bilayers of mycolic acids (MAs) form the outer membrane of Mycobacterium tuberculosis that has high strength and extremely low permeability for external molecules (including antibiotics). For the first time, we were able to study them using the all-atom long-term molecular dynamic simulations (from 300 ns up to 1.2 μs) in order to investigate the conformational changes and most favorable structures of the mycobacterial membranes. The structure and properties of the membranes are crucially dependent on the initial packing of the α-mycolic acid (AMA) molecules, as well as on the presence of the secondary membrane components, keto- and methoxy mycolic acids (KMAs and MMAs). In the case of AMA-based membranes, the most labile conformation is W while other types of conformations (sU as well as sZ, eU, and eZ) are much more stable. In the multicomponent membranes, the presence of the KMA and MMA components (in the W conformation) additionally stabilizes both the W and eU conformations of AMA. The membrane in which AMA prevails in the eU conformation is much thicker and, at the same time, much denser. Such a packing of the MA molecules promotes the formation of a significantly stronger outer mycobacterial membrane that should be much more resistant to the threatening external factors.

Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis ( Mtb ). Mutations in compound 1 -resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb , in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.

Background: Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure-function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis., Results: We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility., Conclusions: The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself., (© 2022. The Author(s).)

Mycobacterium tuberculosis (Mtb) cells are known to synthesize very long chain (C60-90) structurally complex mycolic acids with various functional groups. In this study, we applied linear ion-trap (LIT) multiple-stage mass spectrometry (MS n ), combined with high-resolution mass spectrometry to study the mechanisms underlying the fragmentation processes of mycolic acid standards desorbed as lithiated adduct ions by ESI. This is followed by structural characterization of a Mtb mycolic acid family (Bovine strain). Using the insight fragmentation processes gained from the study, we are able to achieve a near complete characterization of the whole mycolic acid family, revealing the identity of the α-alkyl chain, the location of the functional groups including methyl, methoxy, and keto groups along the meroaldehyde chain in each lipid species. This study showcased the power of LIT MS n toward structural determination of complex lipids in a mixture, which would be otherwise very difficult to define using other analytical techniques.

Tuberculosis is one of the leading causes of death across the world. The treatment regimens for tuberculosis are well established, but still the control of the disease faces many challenges such as lengthy treatment protocols, drug resistance and toxicity. In the present work, mycolic acid methyl transferase (MmaA1), a protein involved in the maturation of mycolic acids in the biochemical pathway of the Mycobacterium, was studied for novel drug discovery. The homology model of the MmaA1 protein was built and validated by using computational techniques. The MmaA1 protein has 286 amino acid residues consisting of 10 α-helices and 7 β-sheets. The active site of the MmaA1 protein was identified using CASTp, SiteMap and PatchDock. Virtual screening studies were performed with two small molecule ligand databases: Asinex synergy and Diverse_Elite_Gold_Platinum databases having a total of 43,446 molecules and generated 1,30,814 conformers against the predicted and validated active site of the MmaA1 protein. Binding analysis showed that the residues ASP 19, PHE 22, TRP 30, TYR 32, TRP 74 and ALA 77 of MmaA1 protein have consistent interactions with the ligands. The hit ligands were further filtered by in silico ADME properties to eliminate potentially toxic molecules. Of the top 10 molecules, 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl) benzamide was synthesised and screened for in vitro anti-TB activity against Mtb H37Rv using MABA assay. The compound and its intermediates exhibited good in vitro anti-TB activity which can be taken up for future lead optimisation studies. Structure based virtual screening study was performed using a validated homology model against small molecules from two virtual compound libraries. Synthesised the lead compound 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl)benzamide obtained from virtual screening. In vitro activity against Mtb H37Rv has given a promising result.

The occurrence of nontuberculous mycobacteria in different hosts and their implication as obligate or opportunistic pathogens remain mainly unclear. Mycobacteriosis in pigs is usually associated with members of the Mycobacterium avium complex and, in particular, with ' Mycobacterium avium subsp. hominissuis '. Here we describe a novel slow-growing mycobacterial species isolated from lymph nodes obtained from two sows housed in different Swiss farms. The animals presented chronic inappetence and mild diarrhoea. Gross pathology revealed focal caseous lymphadenopathy of the mesenteric lymph nodes. Complete genome sequencing of the two isolates from the two sows was performed. The genomes comprised 5.76 Mb and an average nucleotide identity score of 99.97 %. Whole genome sequence, mycolic acid and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the two isolates were not related to any previously described Mycobacterium species. The closest related species was Mycobacterium parmense , a slow-growing scotochromogenic mycobacterium first isolated from a cervical lymph node of a 3-year-old child. The name proposed for the new species is Mycobacterium helveticum sp. nov. and 16-83 T (=DSM 109965 T = LMG 2019-02457 T ) is the type strain.

Cells of Mycobacterium alvei are known to contain a unique set of mycolic acids with a (ω-1)-methoxy group; although the various enzymes in the biosynthesis of other types of mycolic acid have been widely studied, the biosynthetic route to this substituent is unclear. We now define the stereochemistry of the (ω-1)-methoxy fragment as R, and describe the synthesis of a major R-(ω-1)-methoxy-mycolic acid and its sugar esters, and of two natural M. alvei diene mycolic acids., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Two Gram-stain-positive, strictly aerobic, non-spore-forming actinobacterial strains, designated YC2-7 T and YC5-17, were isolated from the Yongcheondonggul (larva cave) in Jeju, Republic of Korea and their taxonomic ranks were examined by a polyphasic approach. The 16S rRNA gene tree showed that the novel isolates occupied an independent position separated from recognized genera of the family Nocardiaceae . In the 92 core gene-based phylogenomic analysis, strain YC2-7 T was loosely associated with the type strain of Aldersonia kummingensis with 66.2 % average amino acid identity. The 16S rRNA gene sequence simairity between the isolate and members of the family Nocardiaceae was below 96.7 %. The cell-wall peptidoglycan was meso-diaminopimelic acid as a diagnostic diamino acid. Whole-cell sugars consisted of arabinose, galactose and glucose. The predominant menaquinone was MK-8(H4, ω-cycl). The major polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The cellular fatty acids consisted mainly of saturated and unsaturated components with small amounts of tuberculostearic acid. Mycolic acids of 52-58 carbon atoms were present. The DNA G+C content of the genome was 63.8 mol%. On the basis of combination of morphological and chemotaxonomic differences, in addition to phylogenetic distinctness, the novel isolates are considered to constitute members of a novel species of a new genus in the family Nocardiaceae , for which the name Antrihabitans stalactiti gen. nov., sp. nov. is proposed. The type strain is YC2-7 T (=KACC 19965 T =DSM 108733 T ).

Mycolic acid methyl esters were extracted from Mycobacterium avium by a mild saponification protocol, designed to preserve labile components. The resulting mixture of α-, keto- and wax ester mycolates was accompanied by some degraded ω-carboxymycolic acid dimethyl esters, whose overall structures were found to support previous studies. Chromatography of the mono-carboxylic mycolates gave an inseparable mixture of keto- and wax ester mycolates and separate α-mycolates. Reduction of the ketomycolate components allowed isolation and characterisation of intact wax ester mycolates for the first time. Minor α- and ω-carboxymycolates were detected in which methyl branches were located on either the proximal or distal sides of trans-alkene groups., Competing Interests: Declaration of Competing Interest There are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel nocardioform strain, CICC 11023 T , was isolated from a tissue biopsy of neck lesions of a patient with primary cutaneous nocardiosis and characterized to establish its taxonomic position. The morphological, biochemical, physiological and chemotaxonomic properties of strain CICC 11023 T were consistent with classification in the genus Nocardia . Whole-cell hydrolysates were rich in meso-diaminopimelic acid, galactose, arabinose and fructose. Mycolic acids were present. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and two unidentified lipids, and the predominant menaquinone was cyclo MK-8 (H4, ω-cyclo). The main fatty acids (>5 %) were C 18 : 0 10-methyl (TBSA), C 16 : 0 , summed feature 4 (C 16 : 1 trans 9/C 15 : 0 iso 2OH), C 15 : 0 and C 17 : 0 10-methyl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the isolate is most closely related (>98 % similarity) to the type strains Nocardia ninae OFN 02.72 T , Nocardia iowensis UI 122540 T and Nocardia alba YIM 30243 T , and phylogenetic analysis of gyrB gene sequences showed similarity (89.1-92.2 %) to Nocardia vulneris NBRC 108936 T , Nocardia brasiliensis IFM 0236 T and Nocardia exalbida IFM 0803 T . DNA-DNA hybridization results for strain CICC 11023 T compared to Nocardia type strains ranged from 20.4 to 35.4 %. The genome of strain CICC 11023 T was 8.78 Mbp with a G+C content of 67.4 mol% overall. The average nucleotide identity (ANI) values between strain CICC 11023 T and N. alba YIM 30243 T were low (OrthoANIu=77.47 %), and the ANI values between strain CICC 11023 T and N. vulneris NBRC 108936 T were low (OrthoANIu=83.75 %). Consequently, strain CICC 11023 T represents a novel Nocardia species on the basis of this polyphasic study, for which the name Nocardia colli sp. nov. is proposed. The type strain is CICC 11023 T (=KCTC 39837 T ).

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis ( Mycobacterium tuberculosis ) and diphtheria ( Corynebacterium diphtheriae ). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 ( Rv0224c in M. tuberculosis ) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae., (© 2020 Rainczuk et al.)

A mycolic acid-containing actinobacterium designated strain MMS17-SY073 T was isolated from island soil. The isolate showed best growth at 25 °C, pH 6, and 0 % (w/v) NaCl. The phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MMS17-SY073 T belongs to the genus Gordonia , and is mostly related to the type strains of Gordonia soli (98.5 % sequence similarity), Gordonia polyisoprenivorans (98.1%), and Gordonia hankookensis (97.8%). The genome-based comparisons showed a clear distinction between the strain and the two neighbouring species, G. soli and G. polyisoprenivorans , with the average nucleotide identities (ANI) of 75.8 and 76.3 %, respectively. Notably, the genome of strain MMS17-SY073 T was the largest in total stretch and gene counts among the complete genomes of Gordonia , and contained a number of biosynthetic gene clusters for secondary metabolites, in particular those for non-ribosomal peptide synthetases. The major polar lipids were diphosphatidyl glycerol (DPG), phosphatidyl glycerol (PG), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl inositol mannoside (PIM). The isoprenoid quinone was MK-9(H 2 ), and the main fatty acids were C 16 : 0 (30.2%) and 10-methyl-C 18 : 0 (33.7%). The whole cell hydrolysates contained galactose, arabinose, and meso -diaminopimelic acid. The DNA G+C content was 67.4 mol%. Based on phenotypic, chemotaxonomic and genetic analysis, strain MMS17-SY073 T should be classified as a new species of the genus Gordonia , for which the name Gordonia insulae sp. nov. is proposed (type strain=MMS17-SY073 T =KCTC 49257 T =JCM 33277 T ).

In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis., Competing Interests: The authors declare no conflict of interest.

Three bacterial strains, HKU70 T , HKU71 T and HKU72 T , were isolated from the conjunctival swab, blood and sputum samples of three patients with conjunctivitis, bacteraemia and respiratory infection, respectively, in Hong Kong. The three strains were aerobic, Gram-stain positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from currently recognized Tsukamurella species. 16S rRNA, secA , rpoB and groEL gene sequence analyses revealed that the three strains shared 99.6-99.9, 94.5-96.8, 95.7-97.8 and 97.7-98.9 % nucleotide identities with their corresponding closest Tsukamurella species respectively. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella (26.2±2.4 to 36.8±1.2 % DNA-DNA relatedness), in line with results of in silico genome-to-genome comparison (32.2-40.9 % Genome-to-Genome Distance Calculator and 86.3-88.9 % average nucleotide identity values]. Fatty acids, mycolic acids, cell-wall sugars and peptidoglycan analyses showed that they were typical of members of Tsukamurella . The G+C content determined based on the genome sequence of strains HKU70 T , HKU71 T and HKU72 T were 69.9, 70.2 and 70.5 mol%, respectively. Taken together, our results supported the proposition and description of three new species, i.e. Tsukamurella sputi HKU70 T (=JCM 33387 T =DSM 109106 T ) sp. nov., Tsukamurella asaccharolytica HKU71 T (=JCM 33388 T =DSM 109107 T ) sp. nov. and Tsukamurella conjunctivitidis HKU72 T (=JCM 33389 T =DSM 109108 T ) sp. nov.

A novel actinobacterial strain, designated NEAU-LL90 T , was isolated from cow dung collected from Shangzhi, Heilongjiang Province, north-east PR China and characterized by using a polyphasic approach. Morphological and chemotaxonomic characteristics were consistent with those members of the genus Nocardia . The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The predominant menaquinone detected was MK-8(H4, ω-cycl). Major fatty acids (>10 %) were identified as C 16:0 , C 18:1 ω9 c , C 18:0 and 10-methyl C 19:0 . Mycolic acids were present. The results of 16S rRNA gene sequence analysis showed that strain NEAU-LL90 T belongs to the genus Nocardia with high sequence similarity to Nocardia niigatensis JCM11894 T (98.1 %), similarities to other type strains of species of the genus Nocardia were found to be lower than 98.0 %. Furthermore, a combination of DNA-DNA hybridization results and some phenotypic characteristics demonstrated that strain NEAU-LL90 T could be distinguished from its closest relative. Therefore, it is proposed that strain NEAU-LL90 T represents a novel species of the genus Nocardia , for which the name Nocardia stercoris sp. nov. is proposed. The type strain is NEAU-LL90 T (=CGMCC 4.7500 T =JCM 32663 T ).

In this study, we report the first synthesis of an alkyne-based trehalose monomycolate probe containing a β-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.

Fragments of mycobacterial cell walls such as arabinoglycerol mycolate and dimycoloyl diarabinoglycerol, comprising complex mixtures of mycolic acids, have immunostimulatory and antigenic properties. A related di-mycoloyl tri-arabinofuranosyl glycerol fragment has been isolated from cell wall hydrolysates. An effective stereoselective synthesis of tri-arabinofuranosyl glycerol, followed by coupling with stereochemically defined mycolic acids of different structural classes, to provide unique di-mycoloyl tri-arabinofuranosyl glycerols is now described., Competing Interests: There are no conflicts of interest for any of the authors of this paper.

Mycolic acids are long chain alpha-alkyl beta-hydroxy fatty acids that are major constituents of the cell wall of Mycobacterium tuberculosis. M. tuberculosis produces three main types of mycolic acids, alpha mycolic acids and keto and methoxy mycolic acids. Cycloproponated mycolic acids make the cell wall less permeable, contribute to antibiotic resistance and host immunomodulation and protect from injury. Cyclopropanation is catalyzed by enzymes of the Cyclopropane Mycolic Acid Synthase (CMAS) family. In the current study, we addressed two CMAS enzymes, proximal alpha cyclopropane mycolic acid synthase (PcaA/CmaA3) and keto cyclopropane Mycolic acid synthase (CmaA2). All-atom Molecular Dynamics (MD) simulations were performed for these enzymes for a timeframe of 100ns each (in triplicate), using GROMACS. Based on the PDB structures of apo and holo states of related CMAS enzymes, we generated a framework which helped us correlate active or inactive states of the enzymes to different conformations sampled by the enzymes during MD simulations. Dynamics suggested that the free or unbound enzymes have intrinsic memory and sample different states of catalysis even in the absence of the substrate/cofactor. Additionally, we find that F200, P201 and W204 may have functional significance. MD simulation of CmaA2 was performed with the objective of gaining insights into the putative role of a loop insert. Analysis showed that acidic residues of this loop possibly play an important role during the active state by forming salt bridges. The insights gained in this study can potentially be utilized for design of effective inhibitors against CMAS enzymes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mycolic acids are fundamental cell wall components, found in the outer membrane barrier (mycomembrane) of Mycobacterium related genera, that have shown antigenic, murine innate immunity inducting and inflammatory activity triggering action. The mycolic acid derivatives, such as the lipid extractable trehalose monomycolates (TMM) and dimycolates (TDM), have been extensively investigated by several biochemical and biological methods and, more recently, we have performed the first neutron scattering measurements on these molecules in order to characterize their dynamical behavior as well as their rigidity properties. In the present paper, we show the first systematic FT-IR study on TMM, TDM and glucose monomycolate (GMM). It includes the analysis of individual lipids but also mixtures of TMM/TDM (ratio of 1:1) or TMM/GMM (ratio of 1:2). The present work is aimed to the first characterization of the vibrational behavior of mycolates and their mixtures enabling us to elucidate the molecular mechanisms responsible for the capability of mycolic acids to affect the flexibility and permeability properties of the mycomembrane. As a whole, the present FT-IR findings provide information that have relevant biological implications, allowing to demonstrate that the membrane fluidity is not only linked to the chain length, but also to the specific conformational behavior adopted by mycolates, which in the mixtures is strongly affected by their mutual interactions. In addition, the capability of trehalose to drive the mycolate conformational behavior and then the chain order and packing is emphasized; due to the TDM relevant evidences shown by our data, this trehalose effect could be related to the TDM toxicity and inflammation action., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Complex mixtures of natural dimycoloyl diarabinoglycerols isolated from mycobacteria have been shown to be both potent immune signalling agents and potentially valuable antigens in the serodiagnosis of mycobacterial infections. We now report the highly stereocontrolled synthesis of diacyl l-glycerol-(1'→1)-β-d-arabinofuranosyl-α-d-arabinofuranosides based on simple fatty acids and single defined synthetic mycolic acids. NMR analysis confirmed that the synthetic core was identical to that in natural mixtures., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The taxonomic position of an actinomycete designated AMA 120 T , isolated from mangrove sediment, was clarified by phenotypic, chemotaxonomic and phylogenetic studies. The 16S rRNA gene sequence revealed that strain AMA 120 T was most closely related to Gordonia rhizosphera NBRC 16068 T (98.9 %), Gordonia polyisoprenivorans NBRC 16320 T (98.1 %) and Gordonia bronchialis NBRC 16047 T (98.1 %). A fragment of the gyrB gene of strain AMA 120 T formed a distinct phyletic line with G. rhizosphera NBRC 16068 T (95.4 %). Strain AMA 120 T contained meso-diaminopimelic acid, arabinose and galactose as cell-wall components, and MK-9(H2) was the predominant menaquinone. The polar lipid profile for this strain consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and two unidentified phospholipids. Mycolic acid was present. The major fatty acids were C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The DNA-DNA relatedness values between AMA 120 T and close species were below 70 %. There was an obvious distinction in the average nucleotide identity distribution between strain AMA 120 T and its closely related strains at around 75-92%. The DNA G+C content of strain AMA 120 T was 66.6 mol%. These results, coupled with the phenotypic and chemotaxonomic data, indicated that strain AMA 120 T represents a novel species of the genus Gordonia, for which the name Gordoniasediminis sp. nov. is proposed. The type strain is AMA 120 T (=TBRC 7109 T =NBRC 113236 T ).

Subtle structural features in bacterial lipids such as unsaturation elements can have vast biological implications. Cyclopropane rings have been correlated with tolerance to a number of adverse conditions in bacterial phospholipids. They have also been shown to play a major role in Mycobacterium tuberculosis ( M. tuberculosis or Mtb) pathogenesis as they occur in mycolic acids (MAs) in the mycobacterial cell. Traditional collisional activation methods allow elucidation of basic structural features of lipids but fail to reveal the presence and position of cyclopropane rings. Here, we employ 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for structural characterization of cyclopropane rings in bacterial phospholipids and MAs. Upon UVPD, dual cross-ring C-C cleavages on both sides of the cyclopropane ring are observed for cyclopropyl lipids, resulting in diagnostic pairs of fragment ions spaced 14 Da apart, thus enabling cyclopropane localization. These diagnostic pairs of ions corresponding to dual cross-ring cleavage are observed in both negative and positive ion modes and afford localization of multiple cyclopropane rings within a single lipid. This method was integrated with liquid chromatography (LC) for LC/UVPD-MS analysis of cyclopropyl glycerophospholipids in Escherichia coli ( E. coli) and for analysis of MAs in Mycobacterium bovis ( M. bovis) and M. tuberculosis lipid extracts.

Bacterial secondary metabolites (SM) are rich sources of drug leads, and in particular, numerous metabolites have been isolated from actinomycetes. It was revealed by recent genome sequence projects that actinomycetes harbor much more secondary metabolite-biosynthetic gene clusters (SM-BGCs) than previously expected. Nevertheless, large parts of SM-BGCs in actinomycetes are dormant and cryptic under the standard culture conditions. Therefore, a widely applicable methodology for cryptic SM-BGC activation is required to obtain novel SM. Recently, it was discovered that co-culturing with mycolic-acid-containing bacteria (MACB) widely activated cryptic SM-BGCs in actinomycetes. This "combined-culture" methodology (co-culture methodology using MACB as the partner of actinomycetes) is easily applicable for a broad range of actinomycetes, and indeed, 33 novel SM have been successfully obtained from 12 actinomycetes so far. In this review, the development, application, and mechanistic analysis of the combined-culture method were summarized.

Mycobacterium tuberculosis remains a persistent pathogen, partly due to its lipid rich cell wall, of which mycolic acids (MAs) are a major component. The fluidity and conformational flexibilities of different MAs in the bacterial cell wall significantly influence its properties, function, and observed pathogenicity; thus, a proper conformational description of different MAs in different environments (e.g., in vacuum, in solution, in monolayers) can inform about their potential role in the complex setup of the bacterial cell wall. Previously, we have shown that molecular dynamics (MD) simulations of MA folding in vacuo can be used to characterize MA conformers in seven groupings relating to bending at the functional groups (W, U and Z-conformations). Providing a new OPLS-based forcefield parameterization for the critical cyclopropyl group of MAs and extensive simulations in explicit solvents (TIP4P water, hexane), we now present a more complete picture of MA folding properties together with improved simulation analysis techniques. We show that the 'WUZ' distance-based analysis can be used to pinpoint conformers with hairpin bends at the functional groups, with these conformers constituting only a fraction of accessible conformations. Applying principle component analysis (PCA) and refinement using free energy landscapes (FELs), we are able to discriminate a complete and unique set of conformational preferences for representative alpha-, methoxy- and keto-MAs, with overall preference for folded conformations. A control backbone-MA without any mero-chain functional groups showed significantly less folding in the mero-chain, confirming the role of functionalization in directing folding. Keto-MA showed the highest percentage of WUZ-type conformations and, in particular, a tendency to fold at its alpha-methyl trans-cyclopropane group, in agreement with results from Villeneuve et al. MAs demonstrate similar folding in vacuum and water, with a majority of folded conformations around the W-conformation, although the molecules are more flexible in vacuum than in water. Exchange between conformations, with a disperse distribution that includes unfolded conformers, is common in hexane for all MAs, although with more organization for Keto-MA. Globular, folded conformations are newly defined and may be specifically relevant in biofilms. Graphical abstract Through advanced simulation analysis, including principle component analysis and free energy landscapes, we reveal detailed physical insights into the solvent-dependant folding behavior of mycolic acids from M. tb.

Mixed Langmuir films of type 1 alpha-(α-) and keto-mycolic acids (MAs) were investigated to understand the roles of α-methyl trans-cyclopropane containing keto-MA in determining the physical and chemical properties of the monolayers. Surface pressure (π) vs. mean molecular area (A) isotherms were measured at constant mole fractions defined as the ratio of the keto-MA molarity to the total molarity of α-MA and keto-MA (X keto ) at 25 °C and 37 °C. A and the elastic modulus (E) of the mixed monolayer were compared for different X keto at fixed π values. In keto-MA rich monolayers, A values were much larger than values of the combined areas of α-MA and keto-MA, while the E values were close to those of solid keto-MA monolayers. A and E were also plotted against the mole fraction of α-methyl trans-cyclopropane containing keto-MA, which showed that the α-methyl trans-cyclopropane group stabilized the W-form conformation of mycolic acids in monolayers, and rendered them solid state. Furthermore, a comparison of the experimental results and the α-methyl trans-cyclopropane content in cell-wall MAs from various strains indicated that the ratio of trans-cyclopropane content was important in determining the nature of the mixed MA layer., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human's, there have been some early drug discovery efforts towards developing potent and selective inhibitors., Objective: Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors., Conclusion: Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

A novel actinobacterium, designated strain SYSU K10002 T , was isolated from a soil sample collected from a karst cave in Xingyi county, Guizhou province, south-western China. The taxonomic position of the strain was investigated using a polyphasic approach. Cells of the strain were aerobic and Gram-stain-positive. On the basis of 16S rRNA gene sequence similarities and phylogenetic analysis, strain SYSU K10002 T was most closely related to the type strains of Nocardiaaltamirensis NBRC 108246 T (99.0 % sequence similarity) and Nocardiatenerifensis NBRC 101015 T (98.8 %) and is therefore considered to represent a member of the genus Nocardia. DNA-DNA hybridization values between strain SYSU K10002 T and the closely related type strains of the genus Nocardia were less than 70 %. In addition, meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan. The whole-cell sugars were arabinose, ribose and galactose. The major isoprenoid quinone was MK-8(H4,ω-cycl), while the major fatty acids (>10 %) were C16 : 0, C18 : 1ω9c and C18 : 0 10-methyl. The polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified glycolipid. Mycolic acids were present. The genomic DNA G+C content of strain SYSU K10002 T was 67.4 mol%. On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU K10002 T represents a novel species of the genus Nocardia, for which the name Nocardiaaurea sp. nov. is proposed. The type strain is SYSU K10002 T (=KCTC 39849 T =DSM 103986 T ).

Two mycobacterial strains with close similarity to the Mycobacterium tuberculosis complex (MTBC) were isolated from cutaneous lesions of patients in the USA and Italy. At the phenotypic level, similarities to the MTBC included slow growth rate, rough morphotype of the unpigmented colonies and nearly identical high-performance liquid chromatography profiles of mycolic acids. In contrast to the MTBC, the strains were niacin- and nitrate-negative, and catalase-positive both at 68 °C and in semi-quantitative tests. The clinical isolates were more closely related to M. tuberculosis than to any other known mycobacterium and scored positive with commercial DNA probes (Hologic AccuProbe M. tuberculosis). Both average nucleotide identity and genome-to-genome distance suggested the strains are different from the MTBC. Therefore, given the distinguishing phenotypic and genomic-scale differences, we submit that the strains belong to a new species we have named Mycobacteriumdecipiens with type strain TBL 1200985 T (=ATCC TSD-117 T =DSM 105360 T ).

Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In Mycobacterium tuberculosis, it is made up of alpha-, methoxy- and keto-MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single-chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first recognized all three classes of chemically synthetic MAs, the second only the two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated after grafting each of the three scFv types onto two configurations of constant chain domains-CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only with CH2-4 versions, notably those two that were also able to recognize the trans-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the trans-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies., Competing Interests: The authors have declared that no competing interests exist.

An electrogenic bacterium was isolated from a marine coral, designated as strain JC435 T and its taxonomic status examined by using a polyphasic approach. Results from the 16S rRNA gene sequence study showed that the isolate belonged to the genus Rhodococcus and formed a cluster with Rhodococcus ruber KCTC 9806 T (99.5 % 16S rRNA gene sequence similarity) and Rhodococcus aetherivorans JCM 14343 T (99.3 %), respectively. Genome relatedness based on DNA-DNA hybridization to the type strains of closest-related species was less than 30 % and the ΔTm of >7 °C, suggesting that strain represents a new species of the genus Rhodococcus. The major fatty acids were C16 : 0, C18 : 1ω9c, C18 : 010-methyl and C16 : 1ω6c and/or C16 : 1ω7c. The polar lipids of strain JC435 T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, phosphatidylinositol, three unknown phospholipids and an unknown amino lipid. The major isoprenoid quinone was MK-8(H2), with 8 % of MK-7(H2) and 2 % of MK-9(H2) as minor components. Whole-cell hydrolysates contained meso-diaminopimelic acid, arabinose and galactose as the diagnostic diamino acid and sugars. Mycolic acids were detected. The genomic DNA G+C content of strain JC435 T was 69.8 mol%. On the basis of phylogenetic genotypic, physiological and chemotaxonomic analysis, strain JC435 T is considered to represent a novel species of the genus Rhodococcus for which the name Rhodococcuselectrodiphilus sp. nov. is proposed. The type strain is JC435 T (=KCTC 39856 T =LMG 29881 T =MCC 3659 T ).

Among non-tuberculous mycobacteria (NTM), the Mycobacterium simiae complex is one of the largest groups, consisting of 18 species of slow-growing mycobacteria. In 2009, a case of NTM-associated infectious skin disease was reported in Shiga Prefecture, Japan. The patient presented with scattered nodules on the chest, back and extremities, and an M. simiae-like organism was isolated from skin biopsy specimens obtained from one of these lesions. Based on several assessments, including multiple-gene analyses, biochemical characterization and drug susceptibility testing, we concluded that this isolate represented a novel species of NTM, and proposed the name 'Mycobacterium shigaense'. Since 2009, five more cases of NTM-associated infectious disease in which there was a suspected involvement of 'M. shigaense' have been reported. Interestingly, four of these six cases occurred in Shiga Prefecture. Here we performed multiple-gene phylogenetic analyses, physiological and biochemical characterization tests, drug susceptibility tests, and profiling of proteins, fatty acids and mycolic acids of eight clinical isolates from the six suspected 'M. shigaense' cases. The results confirmed that all of the clinical isolates were 'M. shigaense', a slow-growing, scotochromogenic species. Here M. shigaense is validly proposed as a new member of the M. simiae complex, with the type strain being UN-152 T (=JCM 32072 T =DSM 46748 T ).

Mycolic acid (MA) plays important role in Corynebacterium glutamicum, but the key enzymes in the biosynthetic pathway of MA in C. glutamicum ATCC13869 have not been characterized. Since the locus BBD29_RS14045 in C. glutamicum ATCC13869 shows high similarity to the gene Cgl2871, which encodes Pks13, the key enzyme for synthesizing MA in C. glutamicum ATCC13032, it was deleted, resulting in the mutant WG001. Compared with the wild-type ATCC13869, MA was not synthesized in WG001, but more phosphatidylglycerol and phosphatidylinositol containing longer unsaturated fatty acids were produced. WG001 cells also show hindered cell growth and defective cell separation when compared with ATCC13869 cells. Transcriptomic analysis shows that many genes relevant to the pathways of fatty acids, inositol, phospholipids, cell wall, and cell division were significantly regulated in WG001 cells when compared with ATCC13869 cells. This study demonstrates that the locus BBD29_RS14045 encodes a key enzyme that plays important role for synthesizing MA in C. glutamicum ATCC13869., (© 2017 International Union of Biochemistry and Molecular Biology, Inc.)

Within the Actinobacteria, the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus form the so-called CMNR group, also designated as mycolic acid-containing actinomycetes. Almost all members of this group are characterized by a mycolic acid layer, the mycomembrane, which covers the cell wall and is responsible for a high resistance of these bacteria against chemical and antibiotic stress. Furthermore, components of the mycomembrane are crucial for the interaction of bacteria with host cells. This review summarizes the current knowledge of mycolic acid synthesis and interaction with components of the immune system for the genus Corynebacterium with an emphasis on the pathogenic species Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis and Corynebacterium ulcerans as well as the biotechnology workhorse Corynebacterium glutamicum.

Mycobacterium smegmatis is often used to study the different components of mycobacterial cell wall. Mycolic acids are important components of mycobacterial cell wall that have been associated with virulence. Recently, a novel lipid containing mycolic acids has been described in M. smegmatis. However, some uncertainties regarding the structure of this molecule named mycolate ester wax have been reported. The objective of this work was to perform an in depth structural study of this molecule for its precise characterization. Using 1 H and 13 C NMR spectroscopy, the molecular structure of mycolate ester wax found in M. smegmatis has been elucidated. The characterization was complemented with MS analyses. This molecule is formed by a carbon chain with three methyl substituted olefinic units and a mycolate structure with trans double bonds and cis cyclopropane rings. The present molecular study will facilitate the detection and identification of pentatriacontatrienyl mycolate in future studies by the performance of a simple 1D 1 H NMR experiment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis antigen 85 (Ag85) enzymes catalyze the transfer of mycolic acid (MA) from trehalose monomycolate to produce the mycolyl arabinogalactan (mAG) or trehalose dimycolate (TDM). These lipids define the protective mycomembrane of mycobacteria. The current model of substrate binding within the active sites of Ag85s for the production of TDM is not sterically and geometrically feasible; additionally, this model does not account for the production of mAG. Furthermore, this model does not address how Ag85s limit the hydrolysis of the acyl-enzyme intermediate while catalyzing acyl transfer. To inform an updated model, we obtained an Ag85 acyl-enzyme intermediate structure that resembles the mycolated form. Here, we present a 1.45-Å X-ray crystal structure of M. tuberculosis Ag85C covalently modified by tetrahydrolipstatin (THL), an esterase inhibitor that suppresses M. tuberculosis growth and mimics structural attributes of MAs. The mode of covalent inhibition differs from that observed in the reversible inhibition of the human fatty-acid synthase by THL. Similarities between the Ag85-THL structure and previously determined Ag85C structures suggest that the enzyme undergoes structural changes upon acylation, and positioning of the peptidyl arm of THL limits hydrolysis of the acyl-enzyme adduct. Molecular dynamics simulations of the modeled mycolated-enzyme form corroborate the structural analysis. From these findings, we propose an alternative arrangement of substrates that rectifies issues with the previous model and suggest a direct role for the β-hydroxy of MA in the second half-reaction of Ag85 catalysis. This information affords the visualization of a complete mycolyltransferase catalytic cycle.

Three bacterial strains, HKU63 T , HKU64 and HKU65 T , were isolated from the conjunctival swabs of three patients with conjunctivitis in Hong Kong. The three strains were aerobic, Gram-stain-positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from closely related Tsukamurella species. 16S rRNA gene sequence analysis revealed that the three strains shared identical sequences with each other, being most closely related to Tsukamurella tyrosinosolvens and Tsukamurella pulmonis, sharing 99.9 % sequence identity. Sequence analysis of three additional housekeeping genes, groEL, secA and rpoB, revealed 100 % nucleotide sequence identity between HKU63 T and HKU64, 94.2-97.0 % nucleotide sequence identities between HKU63 T /HKU64 and HKU65 T and the three strains shared 82.9-98.9 % sequence identities with other currently recognized Tsukamurella species. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella(23.0±4.2 to 50.7±3.7 % DNA-DNA relatedness), of which HKU63 T and HKU64 represented the same species (≥95.2±4.8 % DNA-DNA relatedness) while HKU65 T represented another species. Fatty acid, mycolic acid, cell-wall sugar and peptidoglycan analyses showed that they were typical of members of Tsukamurella. The G+C content of strains HKU63 T , HKU64 and HKU65 T were 71.3±1.9, 71.3±2.0 and 71.2±2.3 mol% (mean±sd; n=3), respectively. A novel species, Tsukamurella ocularis sp. nov. is proposed to accommodate strains HKU63 T and HKU64, with HKU63 T (=JCM 31969 T =DSM 105034 T ) designated as the type strain whilst another novel species, Tsukamurella hominis sp. nov., is proposed to accommodate the third strain, HKU65 T , which is designated as the type strain (=JCM 31971 T =DSM 105036 T ).

In the cell surface display, the choice of host cell and anchoring motif are the most crucial for the efficient display of passenger proteins. Corynebacterium glutamicum has mycolic acid layer in outer membrane and the use of protein in the mycolic acid layer as an anchoring motif can provide a potential platform for surface display in C. glutamicum. All 19 mycolic acid layer proteins of C. glutamicum are analyzed, and two proteins, NCgl0535 and NCgl1337, which have a signal peptide and predicted O-mycoloylation site, are selected as anchoring motifs candidates. Among them, NCgl1337, which shows better expression with higher display efficiency, is chosen as a potential anchoring motif. Two forms of the NCgl1337 anchoring motif, a full-length (1-324 amino acids) and a short-length (1-50 amino acids) containing only signal peptide and O-mycoloylation site, are constructed and their abilities for surface display are examined using two protein models, endoxylanase from Streptomyces coelicolor and α-amylase from Streptococcus bovis. For both model proteins, the short-length NCgl1337 anchoring motif exhibits higher yield of protein display on the surface of C. glutamicum than the full-length NCgl1337. Finally, with C. glutamicum displaying α-amylase, a batch fermentation is performed for the production of l-lysine from starch degradation, and a production of l-lysine as high as 10.8 ± 0.92 g L -1 was achieved after 18 h of culture., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mycolic acid-containing bacteria (MACB) are known to activate cryptic natural product biosynthesis in co-cultures with actinobacteria. We cultured Actinosynnema mirum NBRC 14064, a producer of the mono-cyclic polyene macrolactam mirilactam A (6), with the MACB Tsukamurella pulmonis TP-B0596. As a result, three novel compounds (mirilactams C-E, 1-3) were produced in the co-culture conditions. Compounds 1-3 were likely derived from 6 by epoxidation and subsequent spontaneous cyclization. The chemical structures and stereochemistries of 1-3 were determined by spectroscopic analyses (NMR and MS), conformational searches in the optimized potentials for liquid simulations-3 (OPLS3) force field, and calculations of electronic circular dichroism (ECD).

Tuberculosis (TB), caused by Mycobacterium tuberculosis , remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in TB granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells., Competing Interests: The authors declare no conflict of interest.

Two rapidly growing mycobacteria with identical 16S rRNA gene sequences were the subject of a polyphasic taxonomic study. The strains formed a well-supported subclade in the mycobacterial 16S rRNA gene tree and were most closely associated with the type strain of Mycobacterium novocastrense. Single and multilocus sequence analyses based on hsp65, rpoB and 16S rRNA gene sequences showed that strains SN 1900 T and SN 1904 T are phylogenetically distinct but share several chemotaxonomic and phenotypic features that are are consistent with their classification in the genus Mycobacterium. The two strains were distinguished by their different fatty acid and mycolic acid profiles, and by a combination of phenotypic features. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values for strains SN 1900 T and SN 1904 T were 61.0 % and 94.7 %, respectively; in turn, the corresponding dDDH and ANI values with M. novocastrense DSM 44203 T were 41.4 % and 42.8 % and 89.3 % and 89.5 %, respectively. These results show that strains SN1900 T and SN 1904 T form new centres of taxonomic variation within the genus Mycobacterium. Consequently, strains SN 1900 T (40 T =CECT 8763 T =DSM 43219 T ) and SN 1904 T (2409 T =CECT 8766 T =DSM 43532 T ) are considered to represent novel species, for which the names Mycobacteriumlehmannii sp. nov. and Mycobacteriumneumannii sp. nov. are proposed. A strain designated as 'Mycobacteriumacapulsensis' was shown to be a bona fide member of the putative novel species, M. lehmannii.