Tuberculosis is a chronic infectious disease caused by bacteria of the Mycobacterium tuberculosis complex. One of the major contributors to virulence and intercellular spread of M. tuberculosis is the ESAT-6 secretion system 1 (ESX-1) that has been lost by the live vaccines Mycobacterium bovis BCG (Bacille Calmette Guérin) and Mycobacterium microti as a result of independent deletions. ESX-1 consists of at least 10 genes (Rv3868-Rv3877) encoding the T-cell antigens ESAT-6 and CFP-10 as well as AAA-ATPases, chaperones, and membrane proteins which probably form a novel export system. To better understand the mode of action of the ESX-1 proteins, as a prelude to drug development, we examined systematically the interactions between the various proteins using the two-hybrid system in Saccharomyces cerevisiae. Interestingly, ESAT-6 and CFP-10 formed both hetero- and homodimers. Moreover, Rv3866, Rv3868, and CFP-10 interacted with Rv3873 which also homodimerized. The data were summarized in a protein linkage map that is consistent with the model for the secretion apparatus and can be used as a basis to identify inhibitors of specific interactions.

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned from Streptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutant Streptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene. crtE alone was sufficient to induce zeaxanthin formation in an Escherichia coli clone containing the crt gene cluster from Erwinia herbicola deleted for crtE. The combination of crtE and crtB led to formation of phytoene in S. griseus. The putative crtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene into beta-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-type S. griseus strain.

In order to investigate mid and early biosynthetic steps of angucycline group antibiotics, approximately 400 mutants of the urdamycin producer Streptomyces fradiae (strain Tii 2717) were prepared, of which ca. 10 76 were selected for further investigations. The selection criterion, i.e., the consideration of only pale-colored metabolite-producing blocked mutants, yielded several mutants whose block was in close proximity to the known late-stage biosynthetic steps. The product patterns were characterized by TLC and HPLC methods, and the structures of five new and one known (but previously not detectable) metabolites were elucidated (3)(4)(5)(6)(7)(8). Their roles in the biosynthetic pathway leading to aquayamycin (1) and on to the urdamycins A (2) and B (9) are proposed. The glycosylation sequence of the urdamycin group and two additional earlier biosynthetic steps leading to aquayamycin (l), the most important angucyclinone, were established in this way.

The fermentation, isolation and structure elucidation of aklanonic acid are described. The compound was isolated from fermentations of Streptomyces strain ZIMET 43,717. Aklanonic acid is a yellow-orange crystalline substance, melting at 203-204'C (dec), having the molecular formula C:1H1sOs, and possessing UV maxima at 258, 282 (sh) and 438 nm (CHCI3). In dimethyl sulfoxide or pyridine aklanonic acid is unstable and a new compound (aklanone) is formed as a conversion product.The elucidation of the structures has shown that aklanonic acid and aklanone are derivatives of 1,8-dihydroxyanthraquinone.During our antibiotic screening program, a Streptomyces strain (designated as Streptomyces sp.ZIMET 43,717) was found to produce a new anthracycline-related pigment. The compound, which has been named aklanonic acid, is not active as an antibiotic. However, aklanonic acid is of particular interest because we have found that this compound was biotransformed to structurally different anthracyclinones in feeding experiments with several pigment-negative mutants"".Its role in the biosynthetic pathway of anthracyclines is now under study. The present paper is concerned with the physico-chemical properties of aklanonic acid and the results of the structural elucidation. The biological characteristics of the producing strain, which is unable to form aerial mycelia and spores, will be the subject of a separate communication. Results Isolation and Physico-chemical PropertiesAklanonic acid was obtained mainly from the mycelium by extraction with acetone. After purification by column chromatography and recrystallization from ethyl alcohol the compound was obtained as pure yellow-orange needles with a melting point of 203' 204°C (dec). Aklanonic acid is only slightly soluble in water, moderately soluble in ethyl alcohol, methanol, acetone and chloroform, and soluble in dimethyl sulfoxide and a solution of sodium bicarbonate in water. In concentrated sulfuric acid and sodium hydroxide the compound dissolves with a red color. When dissolved in dimethyl sulfoxide or pyridine aklanonic acid was rapidly converted to a new compound which we have named aklanone. The UV and visible spectrum of aklanonic acid in chloroform contains maxima at 258, 282 (sh), and 438 nm (s 23,300, 19,300, and 10,300). The IR spectrum in potassium bromide had characteristic absorptions at 1622, 1670 and 1700 cm-1.Aklanonic acid has the molecular formula C21H16O6. Attempts to derive the exact molecular formula based upon accurate molecular weight measurements by mass spectrometry and microanaly-

The close structural relationship of this compound (aklanonic acid, Ia, Fig. 1) to anthracyclinones induced us to attempt its biotransformation to anthracyclines by daunorubicin-negative mutants derived from different daunorubicin producing organisms. The results of our experiments provided evidence that