Novel forms of ring‐hydroxylating dioxygenases are widespread in pristine and contaminated soils

Abstract

Ring-hydroxylating dioxygenases (RHDs) are of central importance to bacterial recycling of aromatic hydrocarbons, including anthropogenic pollutants. The database of presently characterized RHDs is biased towards those from organisms readily isolated on anthropogenic substrates. To investigate the extent to which RHDs from extant organisms reflect the natural diversity of these enzymes, we developed a polymerase chain reaction (PCR) method for retrieval of RHD gene fragments from environmental samples. Gene libraries from two contaminated and two pristine soil samples were constructed. None of the inferred peptides from clones examined were identical to previously described RHDs; however, all showed significant sequence homology and contained key catalytic residues. On the basis of sequence identity, the environmental clones clustered into six distinct groups, only one of which included known RHDs. One of the new sequence groupings was particularly widespread, being recovered from all soil samples tested. Comparison of inferred peptide sequences of the environmental clones and known RHDs showed the former to have greater sequence variation at sites thought to influence accessibility of the active site than that seen between currently known RHDs. We conclude that presently characterized RHDs do not adequately represent the diversity of function found in in situ forms.

Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

“…DNA was isolated from 400 Ϯ 20 mg (500 ml for water samples) of material by a beadbeating procedure (28). Details of sample collection and processing have been previously described for most locations (13,29). The Yerranderie silver mine samples were collected at 1-m intervals along a linear transect crossing the mine drainage channel.…”

“…In anticipation of recovering useful genes from this unexplored gene pool, various research groups have designed methods for identifying genes in the yet to be cultured fraction of the microbiota. Two approaches are currently being used: PCR amplification of known gene families (14,23,29) and screening of shotgun libraries of large DNA fragments generated from environmental sources (often in bacterial artificial chromosomes) by using mass sequencing, hybridization, or activity assays (11,12,25). The shotgun approach is limited by the effort involved in identifying genes within the large sequence fragments.…”

Changes in Bacterial Populations and in Biphenyl Dioxygenase Gene Diversity in a Polychlorinated Biphenyl-Polluted Soil after Introduction of Willow Trees for Rhizoremediation

“…Recently, various culture-independent studies have shown that the ISP␣ sequences obtained from cultured strains (from which most available information on ISP␣ structure and mechanism derives) are not likely to represent the functional gene diversity in the environment, as distinct and numerically dominant (putative) functional genes were obtained from screening various different environments (43,44,51). The use of PCRbased genetic-profiling techniques for the study of ISP␣ diversity in the environment (23,43,52) can be employed for monitoring the predominant ISP␣'s structure and diversity and for detecting enzymes selected under the existing environmental conditions, which should prove highly valuable in future bioremediation strategies.…”

Diversity, Abundance, and Consistency of Microbial Oxygenase Expression and Biodegradation in a Shallow Contaminated Aquifer

“…The catabolic importance of archetypal NDO systems (encoded by nah genes) in many PAH-contaminated environments has been successfully demonstrated by functional-gene-targeted molecular approaches (6,12,14,18,23,57,63,65). Furthermore, broad-specificity PCR primers have enlarged the target range of molecular methods for environmental gene detection and uncovered previously unknown dioxygenase diversity in environmental samples (42,49,70).Previous work at a coal tar waste-contaminated aquifer (South Glens Falls, NY) has provided substantial insight on microbially mediated in situ degradation of contaminants (2,3,4,28,43,68,69). Over more than a decade of monitored natural attenuation at the site, naphthalene concentrations within the contamination plume have diminished from high levels of ϳ1.6 ppm (13 M) to persistent low ppb concentrations (48, 69a).…”

“…The catabolic importance of archetypal NDO systems (encoded by nah genes) in many PAH-contaminated environments has been successfully demonstrated by functional-gene-targeted molecular approaches (6,12,14,18,23,57,63,65). Furthermore, broad-specificity PCR primers have enlarged the target range of molecular methods for environmental gene detection and uncovered previously unknown dioxygenase diversity in environmental samples (42,49,70).…”