INTRODUCTION

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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 so-called 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 at the modifications that occurred in a soil bacterial community and in various subcommunities (bacterial populations associated with different microenvironments) in response to a short exposure to inorganic mercury using RISA fingerprinting (36). This method is based on the length polymorphism of intergenic spacer (IGS) sequences between the small (16S) and the large (23S) subunit rRNA genes amplified with universal eubacterial primers directly on the DNA extracted from the community. RISA has also been used to contrast bacterial community structures associated with different soil microenvironments (37), in soils with different vegetation covers (6), or in the rhizosphere treated with different antibiotics (40).

The present study was done to determine the phylogenetic affiliations of bacterial populations responding to mercury spiking, as indicated by changes in the RISA band profiles of the soil community (36). Two major emerging bands (a 350-bp band and a 650-bp band) were excised from the gel derived from the RISA profile of the whole community, cloned, and further characterized by constructing and analyzing IGS clone libraries. Neither band was organism specific, since distinct and distantly related organisms had IGSs of different sequences but with similar sizes. Consequently, representative samples of clones (70 to 100) obtained from each RISA band were screened by amplified ribosomal DNA restriction analysis to determine pattern abundance profiles. Dominant clones in each RISA band were sequenced for phylogenetic affiliation and designing of oligonucleotide probes specific for the dominant responsive bacteria. Hybridization to the RISA profiles derived from the bacterial community before and after mercury spiking demonstrated the enrichment of these populations during adaptation of the community. Hybridization to the RISA profiles derived from the subcommunities associated with microenvironments highlighted the distribution and the dynamics of these responding populations at a microscale level.

	MATERIALS AND METHODS

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Soil origin and microcosm setup. A soil containing a background of 72.3 ng of Hg · g¹ (dry weight) was collected from a cultivated silt-loam soil at La Côte Saint André (LCSAc, France). Soil sampling, storage, and microcosm setup were as described by Ranjard et al. (34). Briefly, microcosms containing 10 g (dry weight) of soil received 1 ml of a mercuric chloride (HgCl₂) solution to obtain a final concentration of 10 µg of Hg(II) per g [e.g., 50 µM Hg(II)] and were incubated at 22°C for 30 days. Soil washing (34) and physical fractionation (35) were used to separate bacteria located outside aggregates and thus easily washed from the surface of aggregates (outer fraction) and to further separate the stable aggregates according to their size (size fractions). Details of the methods used to obtain various microenvironments, as well as the properties of the microenvironments have been published (36, 37).

RISA fingerprinting of bacterial communities. Bacterial DNA was extracted, purified, and quantified from soil samples (35). The IGS region between the small (16S) and the large (23S) subunits of ribosomal sequences was amplified by PCR using primers S-D-Bact-1522-b-S-20 (5'-TGCGGCTGGATCCCCTCCTT-3') and L-D-Bact-132-a-A-18 (5'-CCGGGTTTCCCCATTCGG-3'). PCR, and electrophoresis conditions were as previously described (36).

Purification of RISA products. Two major emerging bands, one of 350 bp (RISA band 1) and another of 650 bp (RISA band 2) were detected in the RISA profiles of the Hg(II)-spiked community (36). They were cut out of the gel and purified by electroelution with a Mini-Protean II (Bio-Rad, Ivry sur Seine, France) according to the manufacturer's instructions. The DNA was recovered in 20 µl of ultrapure water, and the concentrations were evaluated by comparing the fluorescent-band intensities on 2% (wt/vol) agarose gels to the fluorescent-band intensities of known concentrations of the standard Smart ladder (Eurogentec, Seraing, Belgium).

Clone library construction. Clone libraries from PCR products (the excised and purified DNA corresponding to RISA band 1 and RISA band 2) were constructed with the SureClone Ligation Kit (Pharmacia, Orsay, France) according to the manufacturer's instructions. PCR products were ligated to the vector pUC18 (Promega, Charbonnières, France). Ligation and transformation into Escherichia coli DH5 competent cells (Life Biotechnologies, Cergy Pontoise, France) were carried out according to the manufacturer's protocol. Cells were grown in Luria-Bertani medium at 37°C for 24 h. A total of 115 white clones were sampled for RISA band 1 and 200 for RISA band 2.

Analysis of IGS clone libraries. The plasmid inserts were obtained from each clone by amplification with primers M13r and M13f, which are specifically designed to complement the polylinker of the vector pUC18 (Promega). The amplicons were run in 1.5% (wt/vol) agarose gel to determine the insert size. IGS diversity was investigated by digesting the amplified inserts from 5 µl of the PCR product with the restriction enzymes AluI, TaqI, and HaeIII (Boehringer Mannheim, Meylan, France) according to the manufacturer's instructions. The resulting fragments were separated by gel electrophoresis in 3.5% (wt/vol) Metaphor agarose (FMC Bioproducts, Le Perray en Yvelines, France). Individual clones were grouped into restriction groups or phylotypes based on a 100% identity threshold of the restriction patterns for the three enzymes used. The diversity of the phylotypes in the two RISA bands was compared by the Shannon diversity index (46).

Determination of nucleotide sequences and phylogenetic analysis. Sequencing was performed by Act Gene-Euro Sequence Gene Services (Grenoble, France) on an ABI 377 sequencer FS (Perkin-Elmer, Norwalk, Conn.) using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA Polymerase FS (Perkin-Elmer). The M13r and M13f primers were used to sequence both strands of the insert. Sequences from clones were aligned using CLUSTAL W (47) and were compared with databases available at the National Center for Biotechnology Information (NCBI) using the BLAST program (16).

Hybridization. Two 20-nucleotide probes were designed based on the alignments of the sequenced RISA bands. Alignment of the sequences obtained from RISA band 1 allowed to design a probe (5'-ACGCTCGGTAGCTCTTTGAC-3') located in the IGS zone that is specific for the dominant phylotypes 1, 2, 3, and 5. Alignment of the sequenced RISA band 2 allowed to design a probe (5'-GCATGCCTGATATACAAACG-3') located in the IGS zone between the two tRNAs that is specific for the dominant phylotypes 1 and 2. These probes were hybridized with RISA profiles of the spiked and control soil communities and subcommunities. Profiles were separated on 5% (wt/vol) polyacrylamide gels as described above, and the DNA was electrotransferred to nylon membranes (Genescreen Plus, Life Science Products, Boston, Mass.) using a TransBlot SD apparatus (Bio-Rad) according to the method of Muyzer et al. (27). Oligonucleotides were end labeled using T4 polynucleotide kinase (Boehringer Mannheim) as specified by the manufacturer, and hybridizations were performed at 57°C for the RISA 1 probe and at 53°C for the RISA 2 probe, according to the method of Sambrook et al. (44).

IGS length database. An overview of the size of the IGS within the main bacterial groups was obtained by comparing published data (5, 8, 15, 28, 45) and data from GenBank (NCBI) available as of September 2000. These data were from 99 bacterial genera and 332 species of bacteria (phylum -, -, -, and -subdivisions of the genera Proteobacteria, low G+C firmicutes, high G+C firmicutes, Cyanobacteria, Chlamydiae, Cytophagales, and Spirochetes). All multiple sequences were recorded for a given species or strain, whether or not their lengths were different.

Nucleotide sequence accession numbers. A total of 19 sequences obtained from RISA 1 and RISA 2 bands have been deposited in GenBank under the accession numbers listed in Table 1.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Changes in the RISA profiles of the whole community. Fingerprinting of bacterial community by electrophoretic separation of amplified IGS sequences between the rrs and rrl genes (RISA) allows the quick characterization of a community within various environmental contexts (6, 11, 40). Using this approach, changes in the structure of a bacterial community in soil spiked for 30 days with HgCl₂ were noted, but there was no change in a control community (36). These changes were likely to be the consequence of an Hg(II) addition due to the negligible concentration of chloride added in soil and the recognized toxicity of mercury for living cells. Shifts within RISA profiles were due to the emergence of four new bands and the loss of four preexisting bands and to variations in the relative intensity of eleven bands (36). The most noticeable changes were the increase in the intensity of the RISA band 1 at about 350 bp and the appearence of the new RISA band 2 at about 650 bp. These two bands were chosen for restriction analysis and sequencing to examine both the diversity associated with them and the community adaptation through the phylogenetic affiliation of responding bacterial populations.

Sequence variations in a one-size RISA band and phylogenetic affiliation. Of the 115 and 200 clones sampled from RISA band 1 and RISA band 2, 76 and 99, respectively, had an insert of the expected size. Restriction analysis with AluI, TaqI, and HaeIII resulted in 30 phylotypes for RISA band 1 and 54 phylotypes for RISA band 2. A dominant restriction pattern accounted for 54% of the RISA band 1 inserts analyzed. Restriction groups 2, 3, and 4 were represented by 4, 2, and 2 clones, respectively. The remaining patterns were each represented by a single clone. For RISA band 2, 2 major phylotypes were observed that accounted for 15 and 13% of all of the 99 clones; 7 phylotypes accounted for 2 to 7%, while the remaining 46 phylotypes were each represented by a single clone. Thus, RISA band 1 had a prominent rRNA dominance structure of the first phylotype (54%), while RISA band 2 showed no phylotype frequency greater than 15%. The Shannon diversity index was calculated for the two RISA bands; it was 0.66 for RISA band 1 and 0.87 for band 2. Such diversity in a single-size RISA band was expected, since several authors who have studied variations between and within genera or species (14, 15, 18, 28) have reported spacer length heterogeneity. Similarly, published data (Fig. 1) stresses the extent of IGS length and the large overlap between eubacterial phyla.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Length distribution of 428 IGSs between the rrs and rrl genes among the groups of eubacterial domains represented by approximately 99 genera and 332 species. The data were compiled from the literature and the GenBank database. The numbers in parentheses indicate the numbers of genera and species from each phylum. The vertical lines within the boxes indicate the median-size IGS for that phylum. The following genera were used to build the database: -proteobacteria, Hyphomicrobium, Blastobacter, Rhodobacter, Rhodopseudomonas, Bartonella, Nitrobacter, Azospirillum, Agrobacterium, Rhizobium, Bradyrhizobium, Candidatus, Zymomonas, Gluconobacter, Acetobacter, Ochrobactrum, Brucella, Caulobacter, and Ehrlichia; -proteobacteria, Acidithiobacillus, Thiobacillus, Ralstonia, Nitrosospira, Nitrosomonas, Burkholderia, Xylophilus, Nitrosolobus, Neisseria, and Microvirgula; -proteobacteria, Yersinia, Photorhabdus, Azotobacter, Haemophilus, Enterobacter, Citrobacter, Xanthomonas, Pseudomonas, Acinetobacter, Vibrio, Aeromonas, Klebsiella, Escherichia, Salmonella, Pasteurella, Actinobacillus, Thiobacillus, Dichelobacter, Piscirickettsia, Xylella, Erwinia, and Pectobacterium; -proteobacteria, Campylobacter; high-G+C-content gram-positive bacteria, Streptomyces, Rhodococcus, Frankia, Arthrobacter, Brevibacterium, Microbispora, Bifidobacterium, Corynebacterium, Staphylococcus, Mycobacterium, Renibacterium, Tropheryma, Thermonospora, Spirillospora, Excellospora, Actinocorallia, and Actinomadura; low-G+C-content gram-positive bacteria, Bacillus, Lactobacillus, Clostridium, Leuconostoc, Streptococcus, Acholeplasma, Anaeroplasma, Listeria, Enterococcus, Mycoplasma, Ureaplasma, Phytoplasma, Lactococcus, Pectinatus, Zymophilus, and Planococcus; chlamydiae, Chlamydia, Chlamydophila, Simkania, Parachlamydia, and Waddlia; cyanobacteria, Microcystis, Spirulina, Trichodesmium, Arthrospira, and Anacystis; spirochetes, Leptonema and Treponema; and cytophagales, Prevotella, Rhodothermus, and Flavobacterium.

Hybridization and the role of the identified populations in the adaptation of the whole community to mercury stress. The 23S partial sequences indicated the phylogenetic affiliations of the bacterial population. The IGS, because of its variability, permits an analysis with a finer resolution and is particularly powerful for designing very specific probes (5). Two probes, RISA probe 1 and RISA probe 2, were designed from the IGS of the dominant clone sequences derived from RISA band 1 and RISA band 2. RISA probe 1 was designed to be specific for the IGS sequences similar to Clostridium (phylotypes 1, 2, 3, and 5 of RISA band 1), and RISA probe 2 was intended to be specific for the IGS sequences similar to Ralstonia (phylotypes 1 and 2 of RISA band 2). This specificity was confirmed by hybridization with the various Clostridium-like phylotypes and the five Ralstonia-like phylotypes (data not shown). The RISA profile hybridization with the oligonucleotide RISA probes 1 and 2 yielded a strong positive signal on the profiles of the spiked community at the expected positions of 350 and 650 bp, whereas little or no signal was found with the control community profiles (Fig. 2).

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Autoradiogram of a Southern blot of RISA profile from soil DNA, hybridized at 58°C with RISA probe 1 (A) and at 53°C with RISA probe 2 (B). Lanes 1, RISA profile of DNA extracted from soil before adding Hg(II); lanes 2, RISA profile of DNA extracted from soil 30 days after adding Hg(II).

Microscale distribution of responsive bacteria. It was shown previously (36) that mercury spiking causes major changes in the RISA profiles of subcommunities associated with the outer and dispersible clay fractions but that there are only minor shifts in those of the macroaggregate and microaggregate fractions. The heterogeneous mercury impact at a microscale level was explained in part by differences in the bioaccessibility and bioavailability of metal to bacteria according to their location. We concluded that all microenvironments did not contribute similarly to the overall community response. In the present study, it was found that the two selected RISA bands could be easily detected on the profiles of the outer and dispersible clay fractions after contamination with Hg(II). Whether or not the major phylotypes of the whole soil community are also involved in the response of the subcommunities was investigated by performing hybridizations with the designed oligonucleotide probes on RISA profiles derived from each microenvironment before and after mercury contamination. Strong positive signals of the expected size were obtained with the subcommunities associated with the outer and dispersible clay fractions, but little or no signal was obtained for other subcommunities after hybridizations with RISA probe 1 (Fig. 3) and RISA probe 2 (data not shown). Little or no signal was detected for the outer and dispersible clay fraction before Hg(II) contamination. These results show the nonuniform distribution of these populations as well as the preferential enrichment in the outer and dispersible clay fractions. Therefore, populations of subcommunities associated with the outer and dispersible clay fractions are more involved in the whole soil community response and adaptation, as previously suggested by multivariate analysis of RISA profiles (36).

View larger version (99K): [in this window] [in a new window]   FIG. 3.   Autoradiograms of a Southern blot of RISA profiles from soil microenvironments before and after adding Hg(II) hybridized at 58°C with RISA probe 1. Lanes [before and after addition of Hg(II), respectively]: 1 and 2, outer fraction; 3 and 4, 250 to 2,000 µM; 5 and 6, 50 to 250 µM; 7 and 8, 20 to 50 µM; 9 and 10, 2 to 20 µM; 11 and 12, dispersible clay fraction.

Conclusion. As previously shown (40), our study confirms that RISA is a valuable tool for monitoring the structure and dynamics of complex bacterial communities under stress and that excising, cloning, and sequencing shifted RISA bands can be used to identify the populations involved in the community adaptation. The variations within a single RISA band indicate that this method can be used to examine the diversity in soil communities and to focus on diversity within them, perhaps within even a single species, because of the large variability of the IGS. Responding bacterial populations were found to belong to distantly related genera and to be located in similar microenvironments. Isolating these populations and understanding the way they manage their adaptation to mercury and compete together will be our next challenge.