Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment

Abstract

Microorganisms maintain the biosphere by catalyzing biogeochemical processes, including biodegradation of organic chemical pollutants. Yet seldom have the responsible agents and their respective genes been identified. Here we used field-based stable isotopic probing (SIP) to discover a group of bacteria responsible for in situ metabolism of an environmental pollutant, naphthalene. We released 13C-labeled naphthalene in a contaminated study site to trace the flow of pollutant carbon into the naturally occurring microbial community. Using GC/MS, molecular biology, and classical microbiological techniques we documented 13CO2 evolution (2.3% of the dose in 8 h), created a library of 16S rRNA gene clones from 13C labeled sediment DNA, identified a taxonomic cluster (92 of 95 clones) from the microbial community involved in metabolism of the added naphthalene, and isolated a previously undescribed bacterium (strain CJ2) from site sediment whose 16S rRNA gene matched that of the dominant member (48%) of the clone library. Strain CJ2 is a beta proteobacterium closely related to Polaromonas vacuolata. Moreover, strain CJ2 hosts the sequence of a naphthalene dioxygenase gene, prevalent in site sediment, detected before only in environmental DNA. This investigative strategy may have general application for elucidating the bases of many biogeochemical processes, hence for advancing knowledge and management of ecological and industrial systems that rely on microorganisms.

Fig. 1.

Net respiration of ¹³CO₂ beneath glass chambers enclosing 28 cm² of sediment in the contaminated field site that was dosed with aqueous ¹³C-naphthalene. Data reflect averages of four replicate chambers. Background ¹³CO₂ respired was inferred from evolved ¹³CO₂ (19). Error bars show standard deviations. Values at successive sampling times were significantly different (Student's t test, P < 0.008).

Fig. 2.

Microbial community composition reflected by T-RFLP profiles of 16S rRNA genes digested with HhaI, in bulk coal tar-contaminated sediment (A) and CsCl gradient-ultracentrifuged preparations from the sediment ¹²C-DNA (B) and ¹³C-DNA (C) fractions.

Fig. 3.

Phylogenetic analysis of 95 cloned bacterial 16S rRNA genes from the sediment-derived ¹³C-DNA fraction and isolated strain CJ2. Sequences found in this study (bold) are contrasted with those from reference strains (italic). The clones were screened by their enzyme restriction patterns, and 22 full 16S rRNA genes representing these patterns were fully sequenced. Numbers in parentheses indicate frequencies of clones exhibiting the same restriction pattern. Sequence alignment and phylogenic relationships were completed with clustal w and phylip software packages (Kimura model; Department of Genomics, University of Washington). Values at each node are the percentage of 1,000 boot-strap trees. (Bar = 0.1 changes per nucleotide position.) T, type strain.

Fig. 4.

Photomicrographs of strain CJ2. Note coccoid shape of two dividing cells, rough surface texture from extracellular polysaccharide, absence of flagella, and size (1-2 μm) visible in scanning electron microscopy image (Left). Alcian-blue staining at low pH reveals acidic nature of extracellular polysaccharide (Right). (Inset) India ink staining of capsular material.

Fig. 5.

Cluster analysis examining the percent dissimilarity of 446-bp naphthalene dioxygenase (nahAc)-like sequences from cultured microorganisms and amplicons from site sediment. Shown are partial nahAc sequences for eight reference strains, new isolate CJ2 (bold), 20 clones from site sediment-derived DNA extracts (bold), and four GenBank sequences previously cloned from well-water mRNA taken from this study site. Dendrograms were constructed with GenBank DNA sequences by using the clustal procedure in dnastar with the default parameters. The horizontal scale denotes the percentage of divergence of the sequences. In parentheses, numbers indicate frequencies of clones found representing the same sequence; GenBank accession nos. are also given. Roman numerals indicate three clades mentioned in the text; note that the sequence labeled “strain CJ2” is the only one in Clade I whose host bacterium has been cultured.