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1.
Appl Environ Microbiol ; 73(20): 6436-43, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17720843

ABSTRACT

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of

Subject(s)
Anti-Bacterial Agents/pharmacology , Bacillus subtilis/cytology , Bacillus subtilis/drug effects , Biosensing Techniques/methods , Bacillus subtilis/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/metabolism , Fatty Acids/antagonists & inhibitors , Fatty Acids/metabolism , Microbial Sensitivity Tests/methods , Promoter Regions, Genetic , RNA, Bacterial/antagonists & inhibitors , RNA, Bacterial/metabolism
2.
Phytochemistry ; 62(3): 271-86, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12620338

ABSTRACT

Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.


Subject(s)
Acyltransferases/metabolism , Benzopyrans , Coumarins/metabolism , Salicylates/metabolism , Stilbenes/metabolism , Acyltransferases/chemistry , Acyltransferases/genetics , Amino Acid Sequence , Amino Acid Substitution , Chalcone/metabolism , Cloning, Molecular , Coenzyme A/chemistry , Coenzyme A/metabolism , Coumarins/chemistry , Decarboxylation , Electrophoresis, Polyacrylamide Gel , Hydrangea/enzymology , Hydrangea/genetics , Hydrangea/metabolism , Isocoumarins , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Stilbenes/chemistry , Substrate Specificity
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