Sequencing bands of ribosomal intergenic spacer analysis fingerprints for characterization and microscale distribution of soil bacterium populations responding to mercury spiking

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 communit y spiked with Hg(II) were selected for further identification of the popula tions involved in the response of the community to the added metal, The ban ds were cut out from polyacrylamide gels, cloned, characterized by restrict ion analysis, and sequenced for phylogenetic affiliation of dominant clones . The sequences were the intergenic spacer between the rrs and rrl genes an d the first 130 nucleotides of the rrl gene. Comparison of sequences derive d 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 (Clostridiu m-like general, while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to P-proteobacteria (Ralstonia-like genera l, Oligonucleotide probes specific for the identified dominant bacteria wer e designed and hybridized with the RISA profiles derived from the control a nd spiked communities. These studies confirmed the contribution of these po pulations to the community response to the metal. Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different so il microenvironments) also permitted to characterize the distribution and t he dynamics of these populations at a microscale level following mercury sp iking.