Heterologous expression of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes from a novel pyrene-degrading betaproteobacterium

Abstract

A betaproteobacterium within the family Rhodocyclaceae previously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed in Escherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of the E. coli constructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.

Fig 1

DGGE showing the diversity of bacterial 16S rRNA genes in the simplified enrichment culture from which RHD genes were amplified for expression studies. All lanes were run on a single gel, but the image was cropped to remove intervening lanes and areas without obvious bands. The vertical thin black lines indicate where lanes were digitally removed. In the two leftmost lanes, the simplified enrichment culture was grown with either amended phenanthrene or pyrene. In the middle two lanes, genomic DNA from isolated bacteria is shown. In lane PG1 clone PYR10d2, a PG1 clonal sequence from the earlier SIP experiment is shown. A no-DNA control is shown in the rightmost lane.

Fig 2

Neighbor-joining tree of predicted RHD α-subunit protein sequences. Sequences from this experiment are shown in boldface type. The open and closed circles at the nodes indicate ≥95% and ≥50% bootstrap support, respectively. GenBank accession numbers are indicated in parentheses. A biphenyl dioxygenase was used as an outgroup.

Fig 3

Gene neighborhoods of RHD genes on contigs putatively associated with the PG1 organism. Diagrams are derived from information provided by the IMG/MER system. Predicted RHD α- and β-subunits are shown with a black background. Ferredoxin and reductase genes are shown on a gray background. The highest BLASTP hits to the predicted amino acid sequence of each open reading frame (ORF) can be found in supplemental information (see Table S3 in the supplemental material).

Fig 4

Decrease in PAH parent compound concentration by E. coli cells expressing all four PG1 RHD genes relative to E. coli cells expressing only the RHD ferredoxin and reductase genes. Values are averages plus standard deviations (error bars) of triplicate incubations. Experiments for which an expressed RHD displayed at least 25% removal of the PAH and significantly lower PAH concentration than the control without expressed RHD α- and β-subunits are indicated by asterisks. Abbreviations: NAP, naphthalene; FLU, fluorene; ANT, anthracene; BAA, benz[a]anthracene; PHN, phenanthrene; PYR, pyrene.