Identification of Genes and Pathways Related to Phenol Degradation in Metagenomic Libraries from Petroleum Refinery Wastewater

<div><p>Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, <i>Escherichia coli</i> EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.</p></div

Metabolic profile based on metagenomic pyrosequencing data from phenol degrading clones by MG-RAST platform.

<p>(N = 129,635 reads).</p

Predicted ORFs in the fosmid insert sequence.

<p>Sequence annotation was based on Blastx searches.</p

Network of predicted functional associations between proteins from contig 20 of metagenomic pyrosequencing data using STRING database.

<p>The circles represent phenol hydroxylase subunits and catechol 2,3- dioxygenase enzyme and the links between circles represent a putative interaction of these enzymes.</p

Profile of peripheral degradation pathways of aromatic compounds of metagenomic pyrosequencing data from phenol degrading clones by MG-RAST platform.

<p>(N = 129,635 reads).</p

Phylogenetic classification based on metagenomic pyrosequencing data from phenol degrading clones by MG-RAST platform.

<p>(N = 129,635 reads).</p

Neighbour-joining phylogenetic tree based on amino acid sequences of subunits of phenol hydroxylase and catechol 2,3- dioxygenase enzymes from metagenomic pyrosequencing data.

<p>(<b>a</b>) Phenol hydroxylase positive regulator, (<b>b</b>) Phenol hydroxylase sub. 1 and (<b>c</b>) Catechol 2,3-dioxygenase. All reference protein sequences used were obtained from <i>SwissProt protein</i> and <i>Refseq</i>_<i>protein</i>. The bootstrap values greater than 70% are listed.</p

Schematic map of the gene cluster found in the fosmid insert.

<p>The squares indicate the predicted ORFs in positive (white) and negative (gray) strands, and marks indicate the coding sequence (CDS) start site. The annotation of ORFs is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061811#pone-0061811-t002" target="_blank">Table 2</a>.</p