Fluorescent Pseudomonas strains were isolated from 38 undisturbed pristine soil samples from 10 sites on four continents. A total of 248 isolates were confirmed as Pseudomonas sensu stricto by fluorescent pigment production and group-specific 16S ribosomal DNA (rDNA) primers. These isolates were analyzed by three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA), 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, and repetitive extragenic palindromic PCR genomic fingerprinting with a BOX primer set (BOX-PCR). All isolates showed very similar ARDRA patterns, as expected. Some ITS-RFLP types were also found at every geographic scale, although some ITS-RFLP types were unique to the site of origin, indicating weak endemicity at this level of resolution. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 85 unique fluorescent Pseudomonas genotypes in our collection. There were no overlapping genotypes between sites as well as continental regions, indicating strict site endemism. The genetic distance between isolates as determined by degree of dissimilarity in BOX-PCR patterns was meaningfully correlated to the geographic distance between the isolates' sites of origin. Also, a significant positive spatial autocorrelation of the distribution of the genotypes was observed among distances of <197 km, and significant negative autocorrelation was observed between regions. Hence, strong endemicity of fluorescent Pseudomonas genotypes was observed, suggesting that these heterotrophic soil bacteria are not globally mixed."Everything is everywhere, the environment selects" (1, 3) has been a fundamental paradigm in microbial ecology for nearly a century. However, little emphasis has been given to the study of bacterial biogeography, and everything and everywhere were never defined. Improved resolution of this principle has important implications for a broad range of topics, ranging from evolution to the search for new pharmaceuticals to quarantine.There have been a large number of studies on the population genetics of human and animal pathogenic or commensal bacteria, such as Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus and Streptococcus pneumoniae, to elucidate epidemiological patterns (6,25,28,29,31,39,40,48). The results from those studies, obtained mainly by multilocus enzyme electrophoresis (MLEE) (38), suggest that bacterial populations are clonal and in linkage disequilibrium; i.e., genetic recombination occurs too infrequently to destroy genetic linkage, so that these bacterial taxa are comprised of a limited number of clones with worldwide distribution. This clonal paradigm has reinforced the centuryold principle of global distribution. Although those studies provide important insights into bacterial population genetics, the distribution patterns of those host-associated bacteria were inevitably a...

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.Bacterial identification methods currently used include analysis of morphological, physiological, biochemical, and genetic data. In the last two decades, molecular methods, especially 16S rRNA gene sequencing, have been a reliable aid to the identification of diverse bacteria. Although the 16S rRNA method has served as a powerful tool for finding phylogenetic relationships among bacteria (24), because of its molecular clock properties and the large database for sequence comparison, the molecule is too conserved to provide good resolution at the species and subspecies levels (2,4,9,20,23). The relationship between 16S rRNA gene similarity and percent DNA-DNA reassociation is a logarithmic function in which the sequence similarity within a species (Ͼ70% DNA relatedness) is expected to be Ͼ98% (3), and the similarity among different species in a genus, e.g., fluorescent Pseudmonas spp., is 93.3 to ϳ99.9% (11). Considering the high sequence conservation and relative standard errors at 98 and 90% sequence similarities of 19 and 8%, respectively (5), 16S rDNA analysis results on closely related strains could be inaccurate and inconsistent with the results obtained by other methods. Incongruity between genome structure and 16S rDNA sequence similarity was also reported (8). Since many important ecological and clinical characteristics of bacteria, such as pathogenicity, competitiveness, substrate range, and bioactive molecule production, vary below the species level, methods with higher resolution than that of 16S rDNA sequencing are needed.DNA-DNA hybridization is one method that provides more resolution than 16S rDNA sequencing, and the 70% criterion (22) has been a cornerstone for describing a bacterial species. In spite of these values, the method is not popular. Major disadvantages are the laborious nature of pairwise cross-hyb...

A culture-independent survey was performed to search for 16S rRNA gene sequences representing dominant and metabolically active bacteria in rhizosphere soil. PCR- and reverse transcription-PCR-derived clone libraries were constructed from DNA and RNA directly extracted from the soil sample. Acidobacteria-related sequences occupied an unusually large proportion (>50%) of both rDNA- and rRNA-derived clone libraries. This study suggested that the bacteria belonging to the phylum Acidobacteria might be numerically dominant as well as metabolically active in the soil sample, implying that the phylum Acidobacteria might be highly involved in the biogeochemical cycles of the rhizosphere soil.

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