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 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.

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: α-proteobacteria, Hyphomicrobium, Blastobacter, Rhodobacter, Rhodopseudomonas, Bartonella, Nitrobacter, Azospirillum, Agrobacterium, Rhizobium, Bradyrhizobium, Candidatus, Zymomonas, Gluconobacter, Acetobacter, Ochrobactrum, Brucella, Caulobacter, and Ehrlichia; β-proteobacteria, Acidithiobacillus, Thiobacillus, Ralstonia, Nitrosospira, Nitrosomonas, Burkholderia, Xylophilus, Nitrosolobus, Neisseria, and Microvirgula; γ-proteobacteria, Yersinia, Photorhabdus, Azotobacter, Haemophilus, Enterobacter, Citrobacter, Xanthomonas, Pseudomonas, Acinetobacter, Vibrio, Aeromonas, Klebsiella, Escherichia, Salmonella, Pasteurella, Actinobacillus, Thiobacillus, Dichelobacter, Piscirickettsia, Xylella, Erwinia, and Pectobacterium; ɛ-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.

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).

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.