Human saliva is clinically informative of both oral and general health. Since next generation shotgun sequencing (NGS) is now widely used to identify and quantify bacteria, we investigated the bacterial flora of saliva microbiomes of two healthy volunteers and five datasets from the Human Microbiome Project, along with a control dataset containing short NGS reads from bacterial species representative of the bacterial flora of human saliva. GENIUS, a system designed to identify and quantify bacterial species using unassembled short NGS reads was used to identify the bacterial species comprising the microbiomes of the saliva samples and datasets. Results, achieved within minutes and at greater than 90% accuracy, showed more than 175 bacterial species comprised the bacterial flora of human saliva, including bacteria known to be commensal human flora but also Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Gamma proteobacteria. Basic Local Alignment Search Tool (BLASTn) analysis in parallel, reported ca. five times more species than those actually comprising the in silico sample. Both GENIUSand BLAST analyses of saliva samples identified major genera comprising the bacterial flora of saliva, but GENIUS provided a more precise description of species composition, identifying to strain in most cases and delivered results at least 10,000 times faster. Therefore, GENIUS offers a facile and accurate system for identification and quantification of bacterial species and/or strains in metagenomic samples.

We recently determined the function of the gene product of Streptomyces sp. strain C5 doxA, a cytochrome P-450-like protein, to be daunorubicin C-14 hydroxylase (M. L. Dickens and W. R. Strohl, J. Bacteriol. 178: [3389][3390][3391][3392][3393][3394][3395] 1996). In the present study, we show that DoxA also catalyzes the hydroxylation of 13-deoxycarminomycin and 13-deoxydaunorubicin to 13-dihydrocarminomycin and 13-dihydrodaunorubicin, respectively, as well as oxidizing the 13-dihydro-anthracyclines to their respective 13-keto forms. The Streptomyces sp. strain C5 dauP gene product also was shown unequivocally to remove the carbomethoxy group of the -rhodomycinone-glycoside (rhodomycin D) to form 10-carboxy-13-deoxycarminomycin. Additionally, Streptomyces sp. strain C5 DauK was found to methylate the anthracyclines rhodomycin D, 10-carboxy-13-deoxycarminomycin, and 13-deoxy-carminomycin, at the 4-hydroxyl position, indicating a broader substrate specificity than was previously known. The products of Streptomyces sp. strain C5 doxA, dauK, and dauP were sufficient and necessary to confer on Streptomyces lividans TK24 the ability to convert rhodomycin D, the first glycoside in daunorubicin and doxorubicin biosynthesis, to doxorubicin. Daunorubicin (daunomycin [11,17]) and doxorubicin (14-hydroxydaunorubicin; adriamycin [2]) ( Fig. 1) are clinically important anthracycline chemotherapeutic agents (2, 11, 17), which are synthesized by a type II polyketide synthase (23,(42)(43)(44). The aglycone is formed by condensing nine extender units derived from malonyl coenzyme A onto a propionyl moiety to make a C 21 polyketide intermediate (21,23,(42)(43)(44)(45)50), which is reduced at C-9 from the carboxy terminus (43, 44), cyclized, and aromatized to form aklanonic acid (18,19,23,40,(42)(43)(44). Aklanonic acid is converted in four steps to ε-rhodomycinone (3,7,15,16,30,40,42,44), an intermediate that is accumulated in large quantities by most daunorubicin-producing strains (27,42,44,46). It is postulated that ε-rhodomycinone is glycosylated by condensation with TDP-daunosamine (3,8,23,38,42,44) to form the first glycoside intermediate, here named rhodomycin D, which is then converted by a series of reactions to daunorubicin and doxorubicin. Previously, however, the order of the reactions and the enzymes catalyzing some of the steps in the conversion of rhodomycin D to doxorubicin were not known. We recently showed that the doxA gene of Streptomyces sp. strain C5 encoded a cytochrome P-450 enzyme that was capable of conferring on Streptomyces lividans TK24 the ability to convert daunorubicin to doxorubicin (14). In the present work, we describe the enzymatic activities from Streptomyces sp. strain C5 that are responsible for the conversion of rhodomycin D to doxorubicin both in vivo and in vitro (Fig. 1). MATERIALS AND METHODSBacterial strains, plasmids, media, and genetic manipulations. Streptomyces sp. strain C5, originally obtained from the Frederick Cancer Research Center (33) and previously described in detail (3), was...

(23,25). Only in the cases of the tetracenomycin (40) and actinorhodin (5,19,34,55) PKSs, however, have biochemical characterizations also been carried out. Thus, most of the information on type II PKSs is derived from the strong conservation of gene structure among the PKS genes (23,25) and cross-functionality of the components (5,26,34,41). We describe here the structure of a gene region from Streptomyces sp. strain C5 that putatively encodes daunomycin biosynthesis and show that it has a significantly different overall structure from other type II PKS gene regions. MATERUILS AND METHODSBacterial strains and media used. Streptomyces sp. strain C5 and mutants derived from it have been described previously (3,4). Streptomyces lividans TK24 (22), used as a recombinant host strain, was obtained from D. A. Hopwood. Streptomyces coelicolor mutants, described by Rudd and Hopwood (37), were obtained from H. G. Floss. Streptomyces galilaeus ATCC 31671, which lacks a functional polyketide reductase (PKR), has been described previously (5, 50).

(4,5,8,9,33), a pathway in which two methyltransferase reactions have been proposed (8,9,33). Aklanonic acid methyltransferase (AAMT) catalyzes the methyl esterification of aklanonic acid (Fig. 1A) (8). Aklanonic acid methyl ester (AAME) cyclase catalyzes the formation of aklaviketone from AAME, and in wild-type anthracycline producers, aklaviketone is reduced to form aklavinone (8). We have generated mutants specifically blocked in AAMT activity (dauC mutants) and AAME cyclase activity (dauD) (4, 5).Carminomycin 4-O-methyltransferase (CMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 4-O-position of carminomycin and 13-dihydrocarminomycin (Fig. 1B) to form daunomycin and 13-dihydrodaunomycin, respectively (9, 10). CMT has been purified to near homogeneity and is an apparent homotetramer with an M r of ca. 161,000 (10). A gene from S. peucetius ATCC 29050 encoding CMT has been isolated and sequenced (25). In this article, we show the isolation and sequence analyses of AAMT, AAME cyclase, and CMT of Streptomyces sp. strain C5. On the basis of a comparison of the sequence similarities and differences, the two methyltransferases apparently belong to two different subgroups. The AAME cyclase appears to be unusual with respect to both its activity and proteins in the databases. We also show the sequence of dauP, encoding an esterase-like function which we hypothesize also to be involved in daunomycin biosynthesis. MATERIALS AND METHODSBacterial strains, plasmids, and media. Streptomyces sp. strain C5 and mutants derived from it have been described previously (4, 5). Streptomyces lividans TK24 (18) was obtained from D. A. Hopwood. Streptomyces strains normally were grown in YEME (18) supplemented with 20% sucrose. R2YE medium, also used for growth as well as for preparation of streptomycete protoplasts, was prepared as described by Hopwood et al. (18). Nitrate-defined-plus-yeast-extract (NDYE) medium, used for growth of Streptomyces sp. strain C5 and its mutants, has been described previously (9). Strains carrying streptomycete plasmids pIJ486 (38), pIJ702 (18), and pWHM3 (36) or derivatives of them were grown and stored on plates containing 40 g of thiostrepton per ml.Escherichia coli JM83 was used to propagate plasmids for sequencing and restriction analyses. E. coli was grown in Luria-Bertani medium, and plasmids were introduced into E. coli by transformation done by standard procedures (26). For E. coli strains harboring plasmids, ampicillin was added at a final concentration of 100 g/ml.General genetic manipulations. The procedures for protoplast formation, transformation, and regeneration of protoplasts for Streptomyces sp. strain C5 have been described elsewhere (24). The procedures used for the preparation of Streptomyces plasmid and chromosomal DNA have been described by Hopwood et al. (18). The digestion of DNA with restriction endonucleases was carried out according to the manufacturers' directions. Restriction mapping and other routine molecular methods used in th...

DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located between the daunomycin polyketide biosynthesis gene cluster and a dnrI (transcriptional activator) homolog, revealed the presence of a gene encoding a P-450-like enzyme with a deduced M r of 46,096. Expression of this gene, named herein doxA, in Streptomyces lividans TK24 resulted in in vivo bioconversion of daunomycin to doxorubicin. DoxA showed specificity for only daunomycin and 13-dihydrodaunomycin, both of which were converted to doxorubicin. Daunomycinone (daunomycin aglycone), carminomycin, 13-dihydrocarminomycin, idarubicin, and aklavin were not apparent substrates for DoxA. In vector controls or in vectors in which doxA was poorly expressed, S. lividans catalyzed the reduction of daunomycin and other 13-oxo-anthracyclines and -anthracyclinones to their 13-dihydro homologs.