Mycolic Acid-containing Bacterium Research Articles

A mycofactocin-associated dehydrogenase is essential for ethylene glycol metabolism by Rhodococcus jostii RHA1.

Ethylene glycol is an industrially important diol in many manufacturing processes and a building block of polymers, such as poly(ethylene terephthalate). In this study, we found that a mycolic acid-containing bacterium Rhodococcus jostii RHA1 can grow with ethylene glycol as a sole source of carbon and energy. Deletion of a putative glycolate dehydrogenase gene (RHA1_ro03227) abolished growth with ethylene glycol, indicating that ethylene glycol is assimilated via glycolate in R. jostii RHA1. Transcriptome sequencing and gene deletion analyses revealed that a gene homologous to mycofactocin (MFT)-associated dehydrogenase (RHA1_ro06057), hereafter referred to as EgaA, is essential for ethylene glycol assimilation. Furthermore, egaA deletion also negatively affected the utilization of ethanol, 1-propanol, propylene glycol, and 1-butanol, suggesting that EgaA is involved in the utilization of various alcohols in R. jostii RHA1. Deletion of MFT biosynthetic genes abolished growth with ethylene glycol, indicating that MFT is the physiological electron acceptor of EgaA. Further genetic studies revealed that a putative aldehyde dehydrogenase (RHA1_ro06081) is a major aldehyde dehydrogenase in ethylene glycol metabolism by R. jostii RHA1. KEY POINTS: • Rhodococcus jostii RHA1 can assimilate ethylene glycol via glycolate • A mycofactocin-associated dehydrogenase is involved in the oxidation of ethylene glycol • An aldehyde dehydrogenase gene is important for the ethylene glycol assimilation.

Mycobacterium smegmatis alters the production of secondary metabolites by marine-derived Aspergillus niger.

It is generally accepted that fungi have a number of dormant gene clusters for the synthesis of secondary metabolites, and the activation of these gene clusters can expand the diversity of secondary metabolites in culture. Recent studies have revealed that the mycolic acid-containing bacterium Tsukamurella pulmonis activates dormant gene clusters in the bacterial genus Streptomyces. However, it is not clear whether the mycolic acid-containing bacteria activate dormant gene clusters of fungi. We performed co-culture experiments using marine-derived Aspergillus niger with Mycobacterium smegmatis, a mycolic acid-containing bacteria. The co-cultivation resulted in the production of a pigment by A. niger and increased cytotoxic activity of the extract against human prostate cancer DU145 cells. An analysis of secondary metabolites in the extract of the co-culture broth revealed that the increase in cytotoxic activity was caused by the production of malformin C (1), and that TMC-256A1 (2), desmethylkotanin (3), and aurasperone C (4) were selectively produced under co-culture conditions. In addition, further study suggested that direct interaction between the two microorganisms was necessary for the production of the pigment and the cytotoxic compound malformin C (1) from A. niger. Given the biological activities of malformin C, including cytotoxic activity, our approach for increasing the production of bioactive secondary metabolites has important practical applications and may facilitate structural analyses of novel bioactive compounds.

Enhancement of saccharothriolide production and discovery of a new metabolite, saccharothriolide C2, by combined-culture of Saccharothrix sp. and Tsukamurella pulmonis

A combined-culture method was applied to saccharothriolide-producing strain Saccharothrix sp. A1506 with the mycolic acid-containing bacterium (MACB) Tsukamurella pulmonis TP-B0596. The combined-culture not only enhanced saccharothriolide production, but also produced saccharothriolide C2, a new C-2 epimer of saccharothriolide C, thereby indicating its potential for increasing the chemical diversity of microbial metabolites.

Catenulobactins A and B, Heterocyclic Peptides from Culturing Catenuloplanes sp. with a Mycolic Acid-Containing Bacterium

The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was significantly increased when it was cocultured with a mycolic acid-containing bacterium. The planar structures and absolute configurations of the catenulobactins were determined based on NMR/MS and chiral-phase GC-MS analyses. Catenulobactin B (2) displayed Fe(III)-chelating activity and moderate cytotoxicity against P388 murine leukemia cells.

Umezawamides, new bioactive polycyclic tetramate macrolactams isolated from a combined-culture of Umezawaea sp. and mycolic acid-containing bacterium.

New polycyclic tetramate macrolactams, Umezawamides A (1) and B (2) were isolated from a combined-culture of Umezawaea sp. RD066910 and mycolic-acid containing bacterium Tsukamurella pulmonis TP-B0596. Their planar structures and partial stereochemistries were determined based on the spectroscopic analysis, MMFF conformational search, and ECD calculations. Umezawamides are the first secondary metabolites isolated from the genus Umezawaea and they exhibited cytotoxicities to P388 murine leukemia cells. Furthermore, umezawamide A (1) showed growth inhibitory activity against Candida albicans.

Mycolic Acid Containing Bacterium Stimulates Tandem Cyclization of Polyene Macrolactam in a Lake Sediment Derived Rare Actinomycete.

Two novel macrolactams, dracolactams A and B, were identified from a combined-culture of Micromonospora species and a mycolic-acid containing bacterium (MACB). Their structures and stereochemistries were completely assigned, based on spectroscopic analyses and chemical derivatization. Both dracolactams were probably generated from a common macrolactam precursor produced by the Micromonospora species. In this combined-culture system, MACB is likely to activate cryptic oxidase genes in the Micromonospora species and induce the downstream polyene macrolactam cyclization.

Effective Production of Aromatic Polyketides in Streptomyces using a Combined-Culture Method

Combinedculture is a fermentation method which efficiently induces secondary metabolite production in Streptomyces by co-culturing them with mycolic acid-containing bacteria. As a result of combined-culture screening -of our terrestrial Streptomyces collection using UV-HPLC, one of the tested strains, Streptoinyces. sp. TAKO-2, produced two known aromatic polyketides, julichrome Q6 (1) and julichrome Q₈.₈ (2), when co-cultured with the mycolic acid- containing bacterium Tsukamurella pulmonis TP-B0596. The structures of 1 and 2 were confirmed by spectroscopic analysis and literature data.

Niizalactams A-C, Multicyclic Macrolactams Isolated from Combined Culture of Streptomyces with Mycolic Acid-Containing Bacterium.

A terrestrial bacterium, Streptomyces sp. NZ-6, produced niizalactams A-C (1-3), unprecedented di- and tricyclic macrolactams, by coculturing with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. Their complete structures, including absolute configurations, were elucidated on the basis of spectroscopic data and chemical derivatization. Their unique skeletons are proposed to be biosynthesized from a common 26-membered macrolactam intermediate by SN2 cyclization or an intramolecular Diels-Alder reaction.

UNLOCKING “SILENT” GENES VIA COMBINE CULTURE–AN ALTERNATIVE GATEWAY TO NATURAL PRODUCTS DISCOVERY

Streptomyces sp. has been known to produce antibiotics and other bioactive natural products. However, the production of these secondary metabolites depends on the culture conditions, where in most cases the secondary-metabolite genes are not expressed in fermentation culture. Recently, a novel fermentation method known as combined-culture has been introduced to unlock these “silent” genes, hence induces the production of cryptic metabolites. We report herein, our preliminary work on combined-culture using two soil-borne bacterial strains; Streptomyces and Tsukamurella. From the results, it is shown that the presence of Tsukamurella, a mycolic acid-containing bacterium induces the production of new metabolites in Streptomyces. Moreover, the production of compounds associated with Streptomyces was enhanced via combination-culture as compared to culture of Streptomyces strain alone. These findings may promote the feasibility of combined-culture in unlocking the “silent” genes of microorganisms which could lead to the discovery of novel metabolites.

Mycolic acid-containing bacteria induce the production of novel secondary metabolites in streptomyces strains

It was previously reported that several kinds of mycolic acid-containing bacteria interact with various kinds of Streptomyces strains and induce the secondary metabolites production (Onaka, H. et al. Appl. Environ. Microbiol. 2011, 77, 400 – 406). We applied this fermentation method to our collection of Streptomyces strains, employing Tsukamurella pulmonis as a mycolic acid-containing bacterium. As a result, novel cytotoxic alkaloid arcyriaflavin E (1) was isolated from Streptomyces cinnamoneus NBRC 13823 and novel cytotoxic butanolides chojalactones A-C (2-4) were obtained from soil-derived Streptomyces CJ-5 respectively. The stereochemistries (including absolute configurations) of 2-4 were determined based on careful spectroscopic analysis and the total synthesis of 2 and 3, together with all of their stereoisomers.

Chojalactones A-C, cytotoxic butanolides isolated from Streptomyces sp. cultivated with mycolic acid containing bacterium.

The soil-derived bacterium, Streptomyces sp. CJ-5, was cocultured with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. The combined culture method significantly enhanced the production of the secondary metabolites in Streptomyces sp. CJ-5, leading to the isolation of three novel butanolide chojalactones A-C (1-3), with unusual γ-butyrolactone scaffolds. The complete structures, including the absolute configurations of 1-3, were determined based on spectroscopic data and total syntheses. In methylthiazole tetrazolium (MTT) assays, 1 and 2 showed moderate cytotoxicity against P388 cells.

Mycolic Acid-Containing Bacteria Induce Natural-Product Biosynthesis in Streptomyces Species

Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.

Rapid detection of Nocardia amarae in the activated sludge process using enzyme-linked immunosorbent assay (ELISA)

Nocardia amarae, a mycolic acid-containing bacterium, has often been reported to cause foaming of activated sludge in wastewater treatment plants. In this study, the number of N. amarae cells in the activated sludge process was estimated by enzyme-linked immunosorbent assay (ELISA) with anti- N. amarae polyclonal antibody. Use of the antibody enabled N. amarae to be detected at levels of 10 4 to 10 7 colony forming units. On the other hand, the antibody reacted with only a small portion of activated sludge, in which no N. amarae cells were detected by the plate count method. Competitive ELISA was employed to estimate the N. amarae cells in samples taken from a municipal wastewater treatment plant, including raw wastewater and activated sludge foam. The cell numbers estimated by competitive ELISA corresponded well with those obtained by plate counts. Hence, the antibody produced in this study was shown to be effective for the rapid monitoring of N. amarae in the activated sludge process.

Identification of a Lipoarabinomannan-like Lipoglycan in Gordonia rubropertincta

Lipoarabinomannans are well characterised lipoglycans present in the cell envelopes of mycobacteria and closely related bacteria. To define further the distribution of these lipoglycans we have investigated the mycolic acid-containing bacterium Gordonia rubropertincta and, by analysis of carbohydrate composition and antigenic cross-reactivity, demonstrated the presence of a lipoarabinomannan-like lipoglycan. A fraction that appeared to contain higher phosphatidylinositolmannosides was also isolated.