Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197-222, in L. C. Vining and C. Stuttard, ed., Genetics and Biochemistry of Antibiotic Production, 1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced in Streptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnA mutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.The structurally identical antibiotics phosphinothricin tripeptide (PTT) and bialaphos are produced by Streptomyces viridochromogenes and by Streptomyces hygroscopicus (4, 18), respectively. They consist of two molecules, L-alanine and one molecule of the unusual amino acid phosphinothricin (PT). A biosynthetic pathway for bialaphos, consisting of at least 13 steps, was postulated following analysis of nonproducing S. hygroscopicus mutants (summarized in reference 35). Several enzymes were purified, and various genes of the PTT biosynthetic gene cluster were mapped in S. hygroscopicus (35), as well as in S. viridochromogenes (1,12,28,32). It was shown that the respective genes and enzymes were highly similar (up to 80%) on the DNA and amino acid levels (29,39,40). As the genetic organizations of the two clusters are basically identical, it has been concluded that the biosynthesis in both producing strains proceeds in the same way.The biosynthetic steps 6, 7, and 8 were found to be similar to the citrate synthase, aconitase, and isocitrate dehydrogenase reactions of the tricarboxylic acid (TCA) cycle, respectively (Fig. 1). In contrast to the step 6 reaction, for which a specific PTT biosynthetic gene and protein were identified (15), the subsequent steps, especially the isomerization of phosphinomethylmalate in step 7, were speculated to be catalyzed by the enzymes of the primary metabolism (35). Three facts supported this. First, inhibition of aconitase resulted i...

Previously, it was shown that inactivation of the tricarboxylic acid cycle aconitase gene acnA impairs the morphological and physiological differentiation of Streptomyces viridochromogenes Tü494, which produces the herbicide phosphinothricin tripeptide (PTT). In order to further characterize the role of the aconitase in the Streptomyces life cycle, aconitase activity was analyzed during growth of S. viridochromogenes in liquid culture. Two prominent maxima were measured in cell-free crude extracts. The first maximum was found at an early stage of growth, which is correlated with a decrease in pH when rapid glucose consumption is initiated. The second, lower maximum was detected at the beginning of the expression of the PTT-specific biosynthetic gene phsA,implying the onset of secondary metabolism. These results were confirmed by examining transcription of the acnA promoter in time-course experiments. The highest transcription rate was found during the early growth phases. In order to identify putative regulatory mechanisms, the transcriptional start site of the acnA transcript and subsequently the promoter were identified. Several putative, regulatory protein binding sites (e.g. regulators of oxygen stress or iron metabolism) were detected in the promoter region of acnA, which suggested complex regulation of acnA.

The tricarboxylic acid (TCA) cycle aconitase gene acnAfrom Streptomyces viridochromogenes Tü494 was cloned and analyzed. AcnA catalyzes the isomerization of citrate to isocitrate in the TCA cycle, as indicated by the ability of acnA to complement the aconitase-deficient Escherichia coli mutant JRG3259. An acnA mutant was unable to develop aerial mycelium and to sporulate, resulting in a bald phenotype. Furthermore, the mutant did not produce the antibiotic phosphinothricin tripeptide, demonstrating that AcnA also affects physiological differentiation.

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