Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial

Abstract

The mcyABCDEFGHIJ gene cluster of Microcystis aeruginosa encodes the mixed polyketide synthase/nonribosomal peptide synthetase (microcystin synthetase) which is responsible for biosynthesis of the potent liver toxin microcystin. The sequence and orientation of the mcy genes have previously been reported, but no transcriptional analysis had been performed prior to this study. The mcyABCDEFGHIJ genes are transcribed as two polycistronic operons, mcyABC and mcyDEFGHIJ, from a central bidirectional promoter between mcyA and mcyD. Two transcription start sites were detected for both mcyA and mcyD when cells were exposed to light intensities of 68 and 16 mol of photons m ؊2 s ؊1 . The start sites, located 206 and 254 bp upstream of the translational start for mcyD under high and low light conditions, respectively, indicate long untranslated leader regions. Putative transcription start sites were also identified for mcyE, mcyF, mcyG, mcyH, mcyI, and mcyJ but not for mcyB and mcyC. A combination of reverse transcription-PCR and rapid amplification of cDNA ends was employed throughout this work, which may have been one of the first transcriptional analyses of a large nonribosomal polyketide gene cluster.Cyanobacteria are known to produce a wide range of bioactive compounds, including nonribosomally made peptides, polyketides, alkaloids, and lipopolysaccharides. Some of these compounds are neuro-or hepatotoxins, and their impact on human and animal health has resulted in concern worldwide. Microcystin is a hepatotoxic heptapeptide produced by several species of the genera Microcystis, Anabaena, Nostoc, and Oscillatoria. More than 65 structural isoforms of microcystins with various toxicities have been identified having the common structure cyclo(Adda-D-Glu-Mdha-D-Ala-L-X-D-MeAsp-L-Z), where X and Z are variable L amino acids, Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decadienoic acid, D-MeAsp is 3-methylaspartic acid, and Mdha is N-methyldehydroalanine (27, 31).Microcystin is synthesized nonribosomally via a mixed polyketide synthase/nonribosomal peptide synthetase system called microcystin synthetase (2,12,23). Recently, the gene cluster encoding the microcystin synthetase complex has been identified and sequenced (25,34). This 55-kb gene cluster consists of six open reading frames (ORFs) with a mixed nonribosomal peptide synthetase/polyketide synthase nature (mcyA to mcyE and mcyG) and four smaller ORFs with putative precursor and tailoring functions (mcyF and mcyH to mcyJ). Catalytic domains in mcyA to mcyE and mcyG are responsible for incorporation of the precursors phenylacetate, malonyl coenzyme A, S-adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The smaller ORFs encode monofunctional proteins which are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (McyI), and cellular localization (McyH) (24,34).

Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

“…The fact that McyI has two promoters, i.e. its own individual promoter as well as the central mcyD-J promoter, strongly supports this hypothesis (26,27). Table 2.…”

Cloning and characterization of a new hetero-gene cluster of nonribosomal peptide synthetase and polyketide synthase from the cyanobacterium Microcystis aeruginosa K-139

“…3). Microcystin synthetase and micropeptin synthetase genes have been reported to be dependent on light as well (Kaebernick et al, 2002;Nishizawa et al, 1999Nishizawa et al, , 2000. Although many peptide synthesis genes in cyanobacteria exhibit lightdependent expression, a precise conserved cis-element in the promoter region and trans-acting factor for 2007 A new hetero-gene cluster of NRPS and PKS from Microcystis 25 light-dependent expression has not been identified.…”

Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster

“…In E. coli, Plumbridge (1996) showed that in addition to being transcribed as part of the GlcNAc catabolic operon nagBACD, nagC expression is directed from two promoters located within the upstream nagA gene. It is possible that LA-PCR2 fragments may have been products of mRNA processing rather than individual transcripts, as acknowledged by Kaebernick et al (2002) who used LA-PCR for transcriptional analysis of the mcy gene cluster. Posttranscriptional processing of a primary transcript is possible, resulting in the production of mRNAs with differing stability and varying translational efficiency (Meinken et al, 2003).…”

“…The use of internal promoters enables bacteria to control protein production from an operon (Condon, 2003). Using LA-PCR, Kaebernick et al (2002) observed that the microcystin synthase, mcy, gene cluster of Microcystis aeruginosa is transcribed as two polycistronic operons from a central bi-directional promoter; the leader gene of each operon possesses two TSPs, the use of which depends on light intensity, and within operon mcyDEFGHIJ, each gene possesses a functional TSP. In E. coli, Plumbridge (1996) showed that in addition to being transcribed as part of the GlcNAc catabolic operon nagBACD, nagC expression is directed from two promoters located within the upstream nagA gene.…”