Biosynthetic gene cluster of the herbicide phosphinothricin tripeptide from Streptomyces viridochromogenes Tü494

Abstract

The antibiotic phosphinothricin tripeptide (PTT) consists of two molecules of L-alanine and one molecule of the unusual amino acid phosphinothricin (PT) which are nonribosomally combined. The bioactive compound PT has bactericidal, fungicidal, and herbicidal properties and possesses a C-P-C bond, which is very rare in natural compounds. Previously uncharacterized flanking and middle regions of the PTT biosynthetic gene cluster from Streptomyces viridochromogenes Tü494 were isolated and sequenced. The boundaries of the gene cluster were identified by gene inactivation studies. Sequence analysis and homology searches led to the completion of the gene cluster, which consists of 24 genes. Four of these were identified as undescribed genes coding for proteins that are probably involved in uncharacterized early steps of antibiotic biosynthesis or in providing precursors of PTT biosynthesis (phosphoenolpyruvate, acetyl-coenzyme A, or L-alanine). The involvement of the genes orfM and trs and of the regulatory gene prpA in PTT biosynthesis was analyzed by gene inactivation and overexpression, respectively. Insight into the regulation of PTT was gained by determining the transcriptional start sites of the pmi and prpA genes. A previously undescribed regulatory gene involved in morphological differentiation in streptomycetes was identified outside of the left boundary of the PTT biosynthetic gene cluster.

FIG. 1.

Chemical structure of PTT and related compounds.

FIG. 2.

Postulated PTT biosynthetic pathway (according to reference 41).

FIG. 3.

Genetic organization of the complete PTT biosynthetic gene cluster. (A to D) Newly characterized DNA regions described in this paper. The deduced ORFs are drawn to scale. phsA, phosphinothricin tripeptide synthetase A gene; phsB, phosphinothricin tripeptide synthetase B gene; phsC, phosphinothricin tripeptide synthetase C gene; pmi, phosphinomethyl malate isomerase gene; ppm, PEP phosphomutase gene; ppd, phosphonopyruvate decarboxylase gene; cppm, carboxy-PEP phosphonomutase gene; cpps, carboxy-PEP synthase gene; pmet, P-methylase gene; pms, phosphinomethylmalic acid synthase gene; dea, deacetylase gene; pat, phosphinothricin N-acetyltransferase gene; the1 and the2, thioesterase genes; trs, putative transporter gene; prpA, phosphinothricin tripeptide biosynthesis regulatory gene; orf1, mbtH-like gene; adhP, putative alcohol dehydrogenase gene; orf3, gene of unknown function; pgdP, putative d-3-phosphoglycerate dehydrogenase gene; pgmP, putative phosphoglycerate mutase gene; orfM, putative membrane protein gene; orfx, putative nucleotidyltransferase gene; and aldP, putative aldehyde dehydrogenase gene. The positions of the overlapping inserts of cosmids pPtcos1 and p4L5 (approximately 12 kb of the cosmid insert is shown) and the phage clone λ-WT8 are shown. The insertion site of the aprP resistance cassette in the orfM gene is indicated. Genes that were inactivated by disruption are indicated by cross marks. The locations of promoter regions within the cluster are indicated by arrows. The numbers of the biosynthetic steps carried out by the encoded proteins are indicated below the respective genes; hypothetical step numbers are shown in parentheses. The new PTT biosynthetic genes analyzed in this work are boxed.

FIG. 4.

Expression of phsA in wild-type (WT) S. viridochromogenes and a prpA overexpression strain (OV) (A) and in a prpA null mutant (PA) (B). In immunoblotting experiments using polyclonal antibodies raised against PhsA, the expression of the PTT biosynthetic gene phsA was examined at 24, 48, 72, 96, and 120 h. The location of the PhsA protein band is marked by an arrow.

FIG. 5.

Mapping of pmi and prpA transcriptional start sites by a reverse transcription assay. RNAs were isolated from cells in different growth phases. Ppmi and PrpA1 were used as primers. The DNA transcript was characterized by running the samples alongside prpA (A) and pmi (B) sequencing reactions generated with the corresponding ³²P-labeled primers. The sequences of the beginning of the transcripts and the transcriptional start sites (*) are presented. For the prpA transcripts, the positions of the minor (*) and major (**) signals are indicated. Lanes: 1, 24 h; 2, 48 h; and 3, 72 h. The corresponding promoter regions in prpA (C) and pmi (D) are shown. The −10 and −35 regions are marked. pmi, phosphinomethylmalate isomerase gene; phsC, phosphinothricin tripeptide synthetase C gene; trs, putative transporter gene; prpA1, phosphinothricin tripeptide biosynthesis regulatory gene (first start codon).

FIG. 6.

Postulated reaction for the formation of phosphonoformiate from phosphonoacetaldehyde in the PTT biosynthetic pathway.