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Applied and Environmental Microbiology, October 2003, p. 5875-5883, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.5875-5883.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Rapid Screening for Freshwater Bacterial Groups by Using Reverse Line Blot Hybridization{dagger}

Gabriel Zwart,1* Erik J. van Hannen,1,{ddagger} Miranda P. Kamst-van Agterveld,1 Katleen Van der Gucht,2 Eva S. Lindström,3 Jeroen Van Wichelen,2 Torben Lauridsen,4 Byron C. Crump,5 Suk-Kyun Han,6 and Steven Declerck7

Centre for Limnology, NIOO-KNAW, 3631 AC Nieuwersluis, The Netherlands,1 Laboratory of Protistology and Aquatic Ecology, Department of Biology, 9000 Ghent,2 Laboratory of Aquatic Ecology, University of Leuven, 3000 Leuven, Belgium,7 Department of Limnology, Evolutionary Biology Centre, Uppsala University, SE-752 36 Uppsala, Sweden,3 Department of Lake and Estuarine Ecology, The National Environmental Research Institute, DK-8600 Silkeborg, Denmark,4 The Ecosystem Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543,5 Department of Environmental Sciences, University of California, Riverside, California 925216

Received 28 April 2003/ Accepted 19 July 2003

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.

* Corresponding author. Mailing address: NIOO-KNAW, Centre for Limnology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands. Phone: 31-294-239315. Fax: 31-294-232224. E-mail: g.zwart{at}nioo.knaw.nl.

{dagger} Publication no. 3194 of NIOO-KNAW (Netherlands Institute of Ecology).

{ddagger} Present address: Medical Microbiology and Immunology Department, St. Antonius Hospital, 3430 EM Nieuwegein, The Netherlands.

Applied and Environmental Microbiology, October 2003, p. 5875-5883, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.5875-5883.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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