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. 2002 Feb;68(2):449-55.
doi: 10.1128/aem.68.2.449-455.2002.

Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial Nonribosomal Peptide

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Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial Nonribosomal Peptide

Melanie Kaebernick et al. Appl Environ Microbiol. .
Free PMC article

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 micromol 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.

Figures

Fig. 1.
Fig. 1.
RACE products identified for mcyA and mcyD from RNA extracted from cells grown with 31 μmol of photons m−2 s−1 (intermediate light conditions) and exposed prior to sampling to high light conditions (68 μmol of photons m−2 s−1) and low light conditions (16 μmol of photons m−2 s−1). φX174 DNA/HaeIII (Promega) was used as a size marker (lanes M). The bands represent 1,353, 1,078, 872, 603, 310, 281, 271, 234, and 194 bp (from top to bottom).
Fig. 2.
Fig. 2.
Nucleotide sequence of the region separating mcyA and mcyD, including portions of each ORF. The site of transcription initiation for each gene is indicated by +1, and the translation start codon is indicated by boldface type. Putative promoter sequences at −10 and −35 are underlined.
Fig. 3.
Fig. 3.
RT-PCR analysis showing the presence of transcripts between mcyA and mcyB, between mcyB and mcyC, between mcyD and mcyE, between mcyE and mcyF, between mcyF and mcyG, between mcyG and mcyH, between mcyH and mcyI, and between mcyI and mcyJ. For each gene, negative controls in which RNA was used for the PCR (lanes −) and positive controls in which DNA was used for the PCR (lanes +) were included. φX174 DNA/HaeIII (Promega) was used as a size marker (lanes M). The bands represent 1,353, 1,078, 872, 603, 310, 281, 271, 234, and 194 bp (from top to bottom).
Fig. 4.
Fig. 4.
RACE products detected for mcy genes in the polycistronic mcyDEFGHIJ transcript (mcyEa, mcyEb, mcyF, mcyG, mcyH, mcyI, and mcyJ). Lanes Ea and Eb show results obtained with two different RNA samples. mcyE RACE products 1, 2, 3, and 4 were purified and sequenced (Table 2). φX174 DNA/HaeIII (Promega) was used as a size marker (lanes M). The bands represent 1,353, 1,078, 872, 603, 310, 281, 271, 234, and 194 bp (from top to bottom).
Fig. 5.
Fig. 5.
Organization of the microcystin synthetase gene cluster, mcyABCDEFGHIJ, showing putative promoters for mcyEFGHIJ (PmcyE to PmcyJ) and the alternate promoters identified for mcyA and mcyD (PmcyA and PmcyD) under high light conditions (AH and DH) and low light conditions (AL and DL).

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