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

Abstract

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 ␤-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.Recent major advances in the field of molecular biology have made it possible to develop community DNA fingerprinting methods with which to monitor complex bacterial communities in their natural environment without the need for isolation and cultivation steps. These methods involve in situ extraction of DNA from the microbial community, the PCR amplification of sequences of interest, and analysis of part of the genetic information. The sequences most commonly used as markers of communities are the genes of the ribosomal operon. The diversity of the amplified sequences is then resolved by differential electrophoretic migration on agarose or polyacrylamide gels, based on their size (terminal-restriction fragment length polymorphism, ribosomal intergenic spacer analysis [RISA]) or sequence (denaturing gel gradient electrophoresis, thermal gel gradient electrophoresis). These socalled fingerprinting methods provide band profiles that are representative of the genetic structure of the community as a whole or of a section of it, as defined by selected primers (for a review, see reference 26). These methods are valuable tools for characterizing complex bacterial communities and detecting shifts following environmental perturbations and are less time-consuming and labor-intensive than strategies such as small-subunit rRNA gene clone library construction (for a review, see reference 38). Individual bands can also be excised from a fingerprint profile, cloned and sequenced, or challenged with a range of probes providing precise information on the phylogenetic groups constituting a community (25,17).We previously looked...

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