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Applied and Environmental Microbiology, December 2000, p. 5334-5339, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Sequencing Bands of Ribosomal Intergenic Spacer Analysis Fingerprints for Characterization and Microscale Distribution of Soil Bacterium Populations Responding to Mercury Spiking

Lionel Ranjard, Elisabeth Brothier, and Sylvie Nazaret*

Laboratoire d'Ecologie Microbienne, UMR CNRS 5557, Université Claude Bernard, Lyon I, F-69622 Villeurbanne Cedex, France

Received 25 May 2000/Accepted 22 September 2000

Two major emerging bands (a 350-bp band and a 650-bp band) within the RISA (ribosomal intergenic spacer analysis) profile of a soil bacterial community spiked with Hg(II) were selected for further identification of the populations involved in the response of the community to the added metal. The bands were cut out from polyacrylamide gels, cloned, characterized by restriction analysis, and sequenced for phylogenetic affiliation of dominant clones. The sequences were the intergenic spacer between the rrs and rrl genes and the first 130 nucleotides of the rrl gene. Comparison of sequences derived from the 350-bp band to The GenBank database permitted us to identify the bacteria as being mostly close relatives to low G+C firmicutes (Clostridium-like genera), while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to beta -proteobacteria (Ralstonia-like genera). Oligonucleotide probes specific for the identified dominant bacteria were designed and hybridized with the RISA profiles derived from the control and spiked communities. These studies confirmed the contribution of these populations to the community response to the metal. Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different soil microenvironments) also permitted to characterize the distribution and the dynamics of these populations at a microscale level following mercury spiking.

* Corresponding author. Mailing address: UMR-CNRS 5557-Ecologie Microbienne, Université Claude Bernard Lyon I, Bât 741, 4ème étage, 43 Bd du 11 Novembre 1918, F-69622 Villeurbanne cedex, France. Phone: 33 (0) 472431324. Fax: 33 (0) 472431223. E-mail: nazaret{at}biomserv.univ-lyon1.fr.

Applied and Environmental Microbiology, December 2000, p. 5334-5339, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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