Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, inAnabaena strain 90

Anabaena strain 90 produces three hepatotoxic heptapeptides (microcystins), two seven-residue depsipeptides called anabaenopeptilide 90A and 90B, and three six-residue peptides called anabaenopeptins. The anabaenopeptilides b elong to a group of cyanobacterial depsipeptides that share the structure o f a six-amino-acid ring with a side-chain. Despite their similarity to know n cyclic peptide toxins, no function has been assigned to the anabaenopepti lides. Degenerate oligonucleotide primers based on the conserved amino acid sequences of other peptide synthetases were used to amplify DNA from Anaba ena 90, and the resulting polymerase chain reaction (PCR) products were use d to identify a peptide synthetase gene cluster. Four genes encoding putati ve anabaenopeptilide synthetase domains were characterized. Three genes, ap dA, apdB and apdD, contain two, four and one module, respectively, encoding a total of seven modules for activation and peptide bond formation of seve n l-amino acids. Modules five and six also carry methyltransferase-like dom ains. Before the first module, there is a region similar in amino acid sequ ence to formyltransferases. A fourth gene (apdC), between modules six and s even, is similar in sequence to halogenase genes. Thus, the order of domain s is co-linear with the positions of amino acid residues in the finished pe ptide. A mutant of Anabaena 90 was made by inserting a chloramphenicol resi stance gene into the apdA gene. DNA amplification by PCR confirmed the inse rtion. Mass spectrometry analysis showed that anabaenopeptilides are not ma de in the mutant strain, but other peptides, such as microcystins and anaba enopeptins, are still produced by the mutant.