Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR

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Contents Vol 56(8)

doi:10.1071/MF05083

Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR

D. G. Bourne ^A ^B ^C ^D ^E ^G, R. L. Blakeley ^C, P. Riddles ^D ^F, G. J. Jones ^A ^E

^A CSIRO Land and Water, PMB 3, Griffith, NSW 2680, Australia.
^B Australian Institute of Marine Science, PMB 3, Townsville, Qld 4810, Australia.
^C Department of Biochemistry and Molecular Sciences, The University of Queensland, St Lucia, Qld 4072, Australia.
^D CSIRO Tropical Agriculture, Indooroopilly, Qld 4068, Australia.
^E Cooperative Research Centre for Freshwater Ecology, University of Canberra, Canberra, ACT 2600, Australia.
^F Present address: Institute for Molecular Biosciences, University of Queensland, Brisbane, Qld 4072, Australia.
^G Corresponding author. Email: d.bourne@aims.gov.au





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Marine and Freshwater Research 56(8) 1127–1135 http://dx.doi.org/10.1071/MF05083
Submitted: 1 May 2005 Accepted: 6 September 2005 Published online: 22 November 2005

Abstract

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) techniques were developed for the detection of a Sphingomonas bacterium (strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA (rDNA) of strain MJ-PV. A 16S ribosomal RNA (rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.

Keywords: microcystin LR degradation, mlrA, 16S rDNA PCR, Sphingomonas strain MJ-PV.