The antibiotic valanimycin is a naturally occurring azoxy compound produced by Streptomyces viridifaciens MG456-hF10. Precursor incorporation experiments showed that valanimycin is derived from L-valine and L-serine via the intermediacy of isobutylamine and isobutylhydroxylamine. Enzymatic and genetic investigations led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes. A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene (vlmL) that encodes a class II seryl-tRNA synthetase. Previous studies suggested that the role of this enzyme is to provide seryl-tRNA for the valanimycin biosynthetic pathway. Here, we report the results of investigations to elucidate the role of seryltRNA in valanimycin biosynthesis. A combination of enzymatic and chemical studies has revealed that the VlmA protein encoded by the valanimycin biosynthetic gene cluster catalyzes the transfer of the seryl residue from seryl-tRNA to the hydroxyl group of isobutylhydroxylamine to produce the ester O-seryl-isobutylhydroxylamine. These findings provide an example of the involvement of an aminoacyl-tRNA in an antibiotic biosynthetic pathway.Streptomyces ͉ aminoacyl-tRNA ͉ antibiotics

SummaryStreptomyces viridifaciens MG456-hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing S. viridifaciens DNA was isolated that conferred valanimycin production upon Streptomyces lividans TK24. Subcloning of DNA from the valanimycin-producing cosmid has led to the identification of a 22 kb segment of DNA sufficient to allow valanimycin production in S. lividans TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decarboxylase gene ( vlmD ), a valanimycin resistance gene ( vlmF ), at least two regulatory genes ( vlmE , vlmI ), two genes encoding a flavin monooxygenase ( vlmH , vlmR ), a seryl tRNA synthetase gene ( vlmL ) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of L -valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5 ¢ ¢ ¢ ¢ end.

Obtaining information on the genetic capabilities and phylogenetic affinities of individual prokaryotic cells within natural communities is a high priority in the fields of microbial ecology, microbial biogeochemistry, and applied microbiology, among others. A method for prokaryotic in situ PCR (PI-PCR), a technique which will allow single cells within complex mixtures to be identified and characterized genetically, is presented here. The method involves amplification of specific nucleic acid sequences inside intact prokaryotic cells followed by color or fluorescence detection of the localized PCR product via bright-field or epifluorescence microscopy. Prokaryotic DNA and mRNA were both used successfully as targets for PI-PCR. We demonstrate the use of PI-PCR to identify nahA-positive cells in mixtures of bacterial isolates and in model marine bacterial communities.

An open reading frame located upstream of the bacterioferritin gene in Escherichia coli encodes a hypothetical 64-residue protein [Andrews, S.C., Harrison, P.C., & Guest, J.R. (1989) J. Bacteriol. 171, 3940-3947)]. The spacing of the four cysteine residues in this hypothetical protein is identical to that in a region of NIFU, a [2Fe-2S] protein found in nitrogen-fixing bacteria [Fu, W., Jack, R.F., Morgan, T.V., Dean, D.R., & Johnson, M.K. (1994) Biochemistry 33, 13455-13463)]. The NIFU-like E. coli gene was cloned and overexpressed with a C-terminal "His tag" in E. coli using the T7 RNA polymerase/promoter system, and the protein was purified by metal-chelate affinity chromatography. UV-vis absorption and EPR spectra together with iron and amino acid analyses conclusively established that this NIFU-like E. coli protein contains one [2Fe-2S] cluster which can exist in at least two oxidation levels: +2 for the as-purified protein, and +1 for dithionite-reduced protein. Size-exclusion chromatography established that this His-tagged [2Fe-2S] protein is monomeric in solution. Affinity chromatography demonstrated specific complex formation between bacterioferritin (Bfr) and this NIFU-like [2Fe-2S] protein, which is dubbed Bfd. An open reading frame encoding a homologous Bfd is located near a Bfr gene in at least one other bacterium. Bfd may, therefore, constitute a general redox and/or regulatory component participating in the iron storage or mobilization functions of Bfr.

The antibiotic valanimycin is a naturally occurring azoxy compound isolated from Streptomyces viridifaciens. Detailed investigations have shown that valanimycin is derived from L-valine and L-serine via the intermediacy of O-(L-seryl)-isobutylhydroxylamine. Sequence analysis of the valanimycin biosynthetic genes provides relatively few clues to the nature of the later stages of the pathway. Two exceptions are provided by the vlmJ and vlmK genes. The translation product of vlmJ exhibits similarity to diacylglycerol kinases, while the translation product of vlmK exhibits low similarity to the MmgE/PrpD superfamily of proteins. This superfamily includes 2-methylcitrate dehydratase. This communication describes the isolation and structure elucidation of valanimycin hydrate from vlmJ and vlmK mutants of S. viridifaciens. Additional studies show that the conversion of valanimycin hydrate into valanimycin by S. viridifaciens requires both the vlmJ and vlmK genes, and that VlmJ catalyzes the ATP-dependent phosphorylation of the hydroxyl group of valanimycin hydrate prior to a VlmK-catalyzed dehydration.