Register      Login
Marine and Freshwater Research Marine and Freshwater Research Society
Advances in the aquatic sciences
Shopping Cart: ( empty )
You are here: Home > Journals > MF > MF05083
RESEARCH ARTICLE
Contents Vol 56(8)

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 and G. J. Jones A E
+ Author Affiliations
- Author Affiliations

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

Marine and Freshwater Research 56(8) 1127-1135 https://doi.org/10.1071/MF05083
Submitted: 1 May 2005  Accepted: 6 September 2005   Published: 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.

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


Acknowledgments

The authors would like to acknowledge the assistance of Philip and Cheryl Orr in all aspects of this work.


References

Altschul, S. F. , Madden, T. L. , Schaeffer, A. A. , Zhang, J. , Zhang, Z. , Miller, W. , and Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402.
| Crossref | GoogleScholarGoogle Scholar | PubMed | Chorus I. (2001). ‘Cyanotoxins: Occurrence, Causes and Consequences.’ (Springer Verlag: Berlin.)

Chorus I., and Bartrim J. (Eds) (1999). ‘Toxic Cyanobacteria in Water: A Guide to their Public Health Consequences, Monitoring and Management.’ (World Health Organization, E. & F.N. Spon: London.)

Christoffersen, K. , Lyck, S. , and Winding, A. (2002). Microbial activity and bacterial community structure during degradation of microcystins. Aquatic Microbial Ecology 27, 125–136.
Giovannoni S. J. (1991). The polymerase chain reaction. In ‘Nucleic Acid Techniques in Bacterial Systematics’. (Eds E. Stackebrandt and M. Goodfellow.) pp. 177–203. (John Wiley & Sons: Chichester, UK.)

Hahn, D. , Amann, R. I. , Ludwig, W. , Akkermans, A. D. L. , and Schleifer, K. (1992). Detection of micro-organisms in soil after in situ hybridisation with rRNA-targeted, fluorescently labeled oligonucleotides. Journal of General Microbiology 138, 879–887.
| PubMed | Harrison J., Schalken A., Rankin T., and Croome R. (1990). Lake Mokoan and the Broken River: factors affecting water quality. Rural Water Commission of Victoria, Water materials and Environmental Science Branch, Report No. 105, Victoria, Australia.

Herrick, J. B. , Madsen, E. L. , Batt, C. A. , and Ghiorse, W. C. (1993). Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from Indigenous sediment bacteria. Applied and Environmental Microbiology 59, 687–694.
| PubMed | Lane D. J. (1991). 16S/23S rRNA sequencing. In ‘Nucleic Acid Techniques in Bacterial Systematics’. (Eds E. Stackebrandt and M. Goodfellow.) pp. 115–175. (John Wiley & Sons: Chichester, UK.)

Manz, W. , Szewzyk, U. , Ericsson, P. , Amann, R. , Schleifer, K. , and Stenstrom, T. (1993). In Situ identification of bacteria in drinking water and adjoining biofilms by hybridisation with 16S and 23S rRNA-directed fluorescent oligonucleotide probes. Applied and Environmental Microbiology 59, 2293–2298.
| PubMed | Sambrook J., Fritsch E. F., and Maniatis T. (1989). ‘Molecular Cloning: A Laboratory Manual.’ 2nd edn. (Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY.)

Sekar, R. , Pernthaler, A. , Pernthaler, J. , Warnecke, F. , Posch, T. , and Amann, R. (2003). An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization. Applied and Environmental Microbiology 69, 2928–2935.
| Crossref | GoogleScholarGoogle Scholar | PubMed |

Tsai, Y. , and Olson, B. H. (1991). Rapid method for direct extraction of DNA from soil and sediments. Applied and Environmental Microbiology 57, 1070–1074.
| PubMed |

Zurawell, R. W. , Chen, H. , Burke, J. M. , and Prepas, E. E. (2005). Hapatotoxic cyanobacteria: A review of the biological importance of; microcystins in freshwater environments. Journal of Toxicology and Environmental Health 8, 1–37.
| PubMed |