The design and development of an integrated natural products screening database.

We designed and developed NEXUS--a new natural products screening database and related suite of software applications--to utilize the spectacular increases in assay capacity of the modern high throughput screening (HTS) environment. NEXUS not only supports seamless integration with separate HTS systems, but supports user-customized integration with external laboratory automation, particularly sample preparation systems. Designed and developed based on a detailed process model for natural products drug discovery, NEXUS comprises two integrated parts: (1) a single schema of Oracle tables and callable procedures and functions, and (2) software "front-ends" to the database developed using Microsoft Excel and Oracle Discovery/2000. Many of the back-end processing functions were written in Programming Language/Structured Query Language (PL/SQL) to provide an Application Programmer's Interface, which allows end users to create custom applications with little input from information technology professionals.

Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus.

Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.

Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus.

Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C.J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brp A within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpAp1-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity profile of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411.

Bioactive compounds from aquatic and terrestrial sources.

The world of nature provides a never-ending set of fascinating problems for the chemist. Many of the most intriguing problems, however, concern compounds available in only truly minute quantities. One solution is to focus on bioassay-guided separations. In so doing one can isolate compounds with novel structures or unsuspected activities from almost any phylum, including tunicates, sponges, insects, or even the much-studied terrestrial plants, as exemplified in several recent studies in our laboratory involving activities ranging from antiviral and antimicrobial activity to cytotoxicity and immunomodulation. Moreover, newer spectroscopic techniques, especially fast atom bombardment mass spectrometry and tandem mass spectrometry, enhance one's ability to study compounds present in minute quantities, including those of importance to the host organism, such as neuropeptides in insects or marine invertebrates.

Thiostrepton-induced gene expression in Streptomyces lividans.

Thiostrepton induced the expression of four proteins (17, 19, 30, and 56 kilodaltons) of unknown function in Streptomyces lividans. The chromosomal gene which encoded the 19-kilodalton protein (tipA) was cloned and sequenced. Transcription of the tipA promoter was induced at least 200-fold by thiostrepton. The tipA 200-fold by thiostrepton. The tipA transcriptional start site (located by S1 mapping and primer extension experiments) was preceded by a 45-base-pair imperfect inverted-repeat sequence which included the -10 and -35 regions of the promoter. Under noninducing conditions in vivo, this might form a cruciform structure which is not recognized by RNA polymerase. A 143-base-pair fragment including this region was cloned into a promoter probe vector, pIJ486. In this plasmid, pAK114, the thiostrepton-inducible tipA promoter controlled the expression of a kanamycin resistance gene encoding an aminoglycoside phosphotransferase. As little as 1 ng of thiostrepton spotted on a lawn of S. lividans(pAK114) induced kanamycin-resistant growth. Other thiostreptonlike antibiotics also induced tipA, but structurally unrelated antibiotics which inhibit translation had no effect. In S. lividans, the promoter could be induced by thiostrepton during either growth or stationary phase. The tipA promoter should be a valuable tool for expression studies in streptomycetes.