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

Abstract

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 brpA 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 brpApl-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 proffle 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.Bialaphos, a linear tripeptide produced as a secondary metabolite by Streptomyces hygroscopicus, is composed of two L-alanine residues and the glutamate analog phosphinothricin (PT) (4, 29). After cleavage of bialaphos by intracellular nonspecific peptidases, PT acts as an inhibitor of glutamine synthetase (4, 29). For this reason, bialaphos has herbicide and antibiotic activities. The biosynthetic pathway has been well characterized by analyzing intermediates accumulated in cultures of blocked mutants (19). Primary metabolic substrates are converted to the demethylated form of PT, which is then condensed with two alanine residues. Althoug...

Biosynthetic Gene Cluster of the Herbicide Phosphinothricin Tripeptide from Streptomyces viridochromogenes Tü494

“…5), which would lead to proteins of 301 and 261 amino acids. The proteins have high sequence similarities to the gene product of the orthologous gene brpA from the bialaphos producer S. hygroscopicus (28). According to its amino acid sequence, PrpA can be identified as a response regulator of a two-component transcriptional control system, similar to those employed by diverse bacteria to transduce metabolic signals and activate gene expression.…”

“…(36). The sequence of the deduced gene product of trs was shown to have similarities to citrate-proton symporters (Table 2) and to the corresponding protein of bialaphos biosynthesis, which is proposed to be involved in the secretion of PTT (28).…”

An Acyl-CoA Synthase (acoas) Gene Adjacent to the Mycocerosic Acid Synthase (mas) Locus Is Necessary for Mycocerosyl Lipid Synthesis in Mycobacterium tuberculosisvar. bovis BCG

“…A coupling of multifunctional synthase and transferase activities is believed to occur during gramicidin biosynthesis; a thioesterase-like domain, located at the 5Ј end of the gramicidin synthase gene and displaying homology to fatty acid thioesterases, has been suggested to function as a transacylase in the chain termination step (26,27). Similar mechanisms also appear to be involved in the synthesis of nonribosomal peptide synthases such as ␦-(L-␣-aminoadipyl)-Lcysteinyl-D-valine synthase, which is involved in penicillin and cephalosporin biosynthesis (28,29) and the bialaphos antibiotic synthesizing gene cluster (30). In mycobacteria each acoas-like gene, located adjacent to each of the many multifunctional polyketide synthases, probably functions at the terminal step to channel the product of the synthase to the appropriate acceptor in the cell wall as demonstrated in this paper for mycocerosyl lipid synthesis.…”

Accumulation of bldA-specified tRNA is temporally regulated in Streptomyces coelicolor A3(2)

“…Indeed, when the sequences of 100 known Streptomyces genes were scanned, the occurrence of TTA codons was shown to be nonrandom with respect to the class of genes concerned (36). Most of the TTA-containing genes specify antibiotic-resistance proteins or antibiotic-pathwayspecific regulators associated with the production of antibiotics, including actinorhodin (15), bialaphos (40), streptomycin (14), and spiramycin (19). Such products would be expected to be required only under conditions associated with secondary metabolism, such as the cessation of rapid growth and the absence of repressing conditions such as excessive phosphate, nitrogen, or carbon (12).…”