High levels of endemicity of 3-chlorobenzoate-degrading soil bacteria

Abstract

Soils samples were obtained from pristine ecosystems in six regions on five continents. Two of the regions were boreal forests, and the other four were Mediterranean ecosystems. Twenty-four soil samples from each of four or five sites in each of the regions were enriched by using 3-chlorobenzoate (3CBA), and 3CBA mineralizers were isolated from most samples. These isolates were analyzed for the ability to mineralize 3CBA, and genotypes were determined with repetitive extragenic palindromic PCR genomic fingerprints and restriction digests of the 16S rRNA genes (amplified ribosomal DNA restriction analysis [ARDRA]). We found that our collection of 150 stable 3CBA-mineralizing isolates included 48 genotypes and 44 ARDRA types, which formed seven distinct clusters. The majority (91%) of the genotypes were unique to the sites from which they were isolated, and each genotype was found only in the region from which it was isolated. A total of 43 of the 44 ARDRA types were found in only one region. A few genotypes were repeatedly found in one region but not in any other continental region, suggesting that they are regionally endemic. A correlation between bacterial genotype and vegetative community was found for the South African samples. These results suggest that the ability to mineralize 3CBA is distributed among very diverse genotypes and that the genotypes are not globally dispersed.

FIG. 1

Composite photograph of 1.2% agarose gels showing the REP fingerprints of the unique genotypes. The genotypes are indicated at the top (each genotype designation includes the site or region and, in most cases, a number). From left to right the strains were isolated from sites in Australia, Saskatchewan, Chile, Russia, South Africa, and California. RC-2 is not included. Three of the genotypes, PMN1B, MBG3, and MUB4, lost the ability to degrade 3CBA. The left lane and the right lane of each gel are 1-kb marker lanes. Numbers at the left are marker fragment sizes in base pairs.

FIG. 2

Dendrogram showing the degrees of similarity of the ARDRA patterns of the genotypes. The analysis was performed by using the Wards method of neighbor joining of Jaccard similarity coefficients calculated from the presence or absence of restriction digestion fragments in each of the strains. R. eutrophus (GenBank accession no. M32021), B. cepacia¹ (GenBank accession no. X87275), and B. cepacia² (GenBank accession no. L28675) were included in the analysis.

FIG. 3

Diversity of 3CBA degraders found in each region calculated as ARDRA type cluster diversity (from the number of individuals in each ARDRA type cluster) and genotype diversity (from the number of individuals per REP genotype). Diversity was calculated by using n(1 − Σx_i²)/(n − 1), where x_i is the frequency of the ith taxon and n is the total number of individuals. The numbers of individuals in the regions are shown above the bars. The term n/(n − 1) corrects for sample size (30). Abbreviations: SASK, Saskatchewan; S.AFRICA, South Africa; AUS, Australia.

FIG. 4

Map of the Cape Region of South Africa showing the locations of the sampling sites and the designations of the genotypes found at each site. Note the widespread distribution of genotype RENOS, found at sites WG, MB, and MR, all of which are vegetated by renosterveld scrub.