New hydrocarbon degradation pathways in the microbial metagenome from Brazilian petroleum reservoirs

Abstract

Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs.

Figure 1. Functional assignment of the metagenomic fosmid clones using the COG database.

(C) Energy production and conversion; (E) Amino acid transport and metabolism; (R) General function prediction only. (T) Signal transduction mechanisms; (K) Transcription; (D) Cell cycle control, mitosis, and meiosis; (Q) Secondary metabolites biosynthesis, transport and catabolism; (S) Function unknown; (L) Replication, recombination, and repair; (M) Cell wall/membrane biogenesis; (G) Carbohydrate transport and metabolism; (I) Lipid transport and metabolism; (H) Coenzyme transport and metabolism; (O) Post-translational modification, protein turnover, chaperones; (P) Inorganic ion transport and metabolism; (J) Translation; (F) Nucleotide transport and metabolism.

Figure 2. Schematic representation of the sequence annotations of the entire insert in five metagenomic fosmid clones.

The identified open reading frames (ORFs) are shown in arrows, and the start and stop codons and the coding direction are indicated. The ORFs are color coded according to their functional category assigned by COG (Clusters of Orthologous Groups of proteins). Blue arrows: metabolism; green arrows: cellular processes and signaling; red arrows: information storage and processing; yellow arrows: poorly characterized; white arrows: not in COG. Further details on the ascribed putative function for each ORF are indicated in Supplementary tables S2 to S6.

Figure 3. Circular representation of the five annotated fosmid clones and synteny between them.

The outermost ring displays the different fosmids in different colors (dark green, purple, orange, dark blue and dark yellow). Annotated ORFs are shown in blocks (black is used to distinguish consecutive ORFs in the same fosmid). The connecting lines inside the circle join syntenic regions. The gray scale indicates the levels of synteny detected between the fosmids; the darkness is proportional to the number of genes in the two different fosmids (i.e., FOS 3B and FOS 2B showed more synteny, followed by FOS 3B and FOS 6A, and so on). Universal clusters of orthologous groups are shown in different colors (rings 2, 3, 4, 5); light blue: metabolism, light green: cellular processes and signaling, red: information storage and processing, light yellow: poorly characterized; white: not in COG. The innermost ring shows the G+C content (%). This figure was created using the Circos software (Krzywinski et al., 2009).

Figure 4. KEGG-based functional assignment of the sequences from the metagenomic fosmid clones.

Figure 5. Comprehensive map of biochemical pathways for aerobic and anaerobic bacterial degradation of hydrocarbon compounds.

Red bubbles show which genes of these routes are present in one or more fosmids. Two consecutive arrows represent more than one reaction. (UbiB) Ubiquinone biosynthesis protein UbiB; (UbiE) Ubiquinone biosynthesis protein C-methyltransferase UbiE; (ADH) Alcohol dehydrogenase; (ALDH) Aldehyde dehydrogenase: (DH) Dienelactone hydrolase; (UbiX) 3-octoprenyl-4-hydroxybenzoate; (RCH) Ring cleavage hydrolase; (GCD) Glutaryl-CoA dehydrogenase; (ACAT) Acetyl-CoA acyltransferase. Figure modified from .

Figure 6. Phylogenetic analysis based on the deduced amino acid sequences of selected proteins in the metagenomic fosmid clones.

(a) Primosomal protein N (COG L) of ORF 7 from fosmid clone FOS 1A, (b) ParB protein (COG K) of ORFs 18 from fosmid clone FOS 2B, ORF 33 from FOS 3B and ORF 2 from FOS 6A, and (c) arginine-tRNA ligase (COG J) of ORF 10 from fosmid clone FOS 10A. Bootstrap values (1000 replicate runs, shown as %) are listed. GenBank accession numbers are listed in parentheses after the species names.