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Applied and Environmental Microbiology, November 2004, p. 6370-6378, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6370-6378.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Inactivation of an ABC Transporter Gene, mcyH, Results in Loss of Microcystin Production in the Cyanobacterium Microcystis aeruginosa PCC 7806

Leanne A. Pearson,1 Michael Hisbergues,2,{dagger} Thomas Börner,2 Elke Dittmann,2 and Brett A. Neilan1*

School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia,1 Institute for Biology, Humboldt University, Berlin, Germany2

Received 13 April 2004/ Accepted 22 June 2004

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. Microcystin is synthesized nonribosomally by the thiotemplate function of a large, modular enzyme complex encoded within the 55-kb microcystin synthetase (mcy) gene cluster. Also encoded within the mcy gene cluster is a putative ATP binding cassette (ABC) transporter, McyH. This study details the bioinformatic and mutational analyses of McyH and offers functional predictions for the hypothetical protein. The transporter is putatively comprised of two homodimers, each with an N-terminal hydrophobic domain and a C-terminal ATPase. Phylogenetically, McyH was found to cluster with members of the ABC-A1 subgroup of ABC ATPases, suggesting an export function for the protein. Two mcyH null mutant ({Delta}mcyH) strains were constructed by partial deletion of the mcyH gene. Microcystin production was completely absent in these strains. While the mcyH deletion had no apparent effect on the transcription of other mcy genes, the complete microcystin biosynthesis enzyme complex could not be detected in {Delta}mcyH mutant strains. Finally, expression levels of McyH in the wild type and in {Delta}mcyA, {Delta}mcyB, and {Delta}mcyH mutants were investigated by using immunoblotting with an anti-McyH antibody. Expression of McyH was found to be reduced in {Delta}mcyA and {Delta}mcyB mutants and completely absent in the {Delta}mcyH mutant. By virtue of its association with the mcy gene cluster and the bioinformatic and experimental data presented in this study, we predict that McyH functions as a microcystin exporter and is, in addition, intimately associated with the microcystin biosynthesis pathway.

* Corresponding author. Mailing address: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia. Phone: (61) 2 9385 3235. Fax: (61) 2 9385 1591. E-mail: b.neilan{at}unsw.edu.au.

{dagger} Present address: Institute of Biology of Lille, Department of Microbiology of Ecosystems, 59019 Lille, France.

Applied and Environmental Microbiology, November 2004, p. 6370-6378, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6370-6378.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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