Application of Metagenomics for Identification of Novel Petroleum Hydrocarbon Degrading Enzymes in Natural Asphalts from the Rancho La Brea Tar Pits

Recent studies on the biodiversity of asphalt deposits at the Rancho La Brea Tar Pits in Los Angeles, California have revealed the existence of several hundred new species of bacteria and gene sequences encoding putative novel degradative enzymes (Kim and Crowley, 2007). The presence of fossilized extinct animal remains in the La Brea Tar Pits has led to estimations that these natural asphalt seeps have existed for at least 40,000 years (Akersten et al., 1983). These deposits consist of petroleum that has been degraded to the extent that the remaining material is comprised mainly of asphalts and heavy oils, which have saturated into the soil matrix. Since petroleum hydrocarbons are both a target and a product of microbial metabolism (Hamme et al., 2003), the role of microorganisms that inhabit this and other similar environments is directly relevant to development of technology for bioremediation, biotransformation of petroleum hydrocarbons, and microbial enhanced oil recovery for extracting and refining heavy oil.In this research, both culture-dependent and culture-independent methods were used to characterize microorganisms and consortia from the La Brea Tar Pits, which are able to degrade a variety of polycyclic aromatic hydrocarbons (PAHs) and BTEX (benzene, toluene, ethyl benzene and xylene). Initial studies applied PCR-DGGE methods to identify the microbial consortia that degrade selected petroleum hydrocarbons during enrichment cultures on pure compounds or mixtures of substances found in petroleum. This study revealed a single Pseudomonas sp. that may be able to degrade multiple PAHs and biphenyl. In addition, PCR based techniques identified a naphthalene dioxygenase from Pseudomonas stutzeri that appears to function as a major degradative enzyme in this system. DNA microarray analysis further revealed the presence of a wide variety of petroleum hydrocarbon degrading genes and has verified that naphthalene dioxygenase is the most commonly found degradative enzyme in this microbial community.To further investigate the true microbial diversity of the La Brea Tar Pits, the microbial community associated with the heavy oil at Rancho La Brea was studied used a metagenomics approach. A fosmid clone library was constructed using 38 Kb fragments of DNA extracted from the asphalt-soil samples of Pit 101. This library of about 3,000 clones was then screened for DNA inserts, which contained specific genes that could be targeted using PCR based methods. One selected clone contained the 16S rRNA gene of an unclassified Rhodospirallacaea and a putative 2-nitropropane dioxygenase, which suggest that this new organism degrades persistent nitroalkanes that are common in asphalt.The current investigation of the La Brea Tar pits concluded using Illumina technologies (Illumina, Inc.) to deep sequence the metagenomic library of over 3,000 clones using a high throughput sequencer. Over 75 MB of DNA has been sequenced, from which over 650 contigs with an average length of 500 bp were assembled. Bioinformatics analysis indicated the presence of genes encoding three types of dioxygenases, one of which encoded a naphthalene dioxygenase from Pseudomonas sp. along with two other genes that are most similar to previously reported genes encoding biphenyl and toluene dioxygenases. However, sequence analysis revealed that these genes were not significantly similar to these known dioxygenases and may thus be novel. The results of this research provide a foundation for further studies on the evolution and assembly of metabolic pathways in bacteria that have undergone long term adaptation to survival in natural asphalts

Culture-Independent Investigation of the Microbiome Associated with the Nematode <i>Acrobeloides maximus</i>

<div><p>Background</p><p>Symbioses between metazoans and microbes are widespread and vital to many ecosystems. Recent work with several nematode species has suggested that strong associations with microbial symbionts may also be common among members of this phylu. In this work we explore possible symbiosis between bacteria and the free living soil bacteriovorous nematode <i>Acrobeloides maximus</i>.</p><p>Methodology</p><p>We used a soil microcosm approach to expose <i>A. maximus</i> populations grown monoxenically on RFP labeled <i>Escherichia coli</i> in a soil slurry. Worms were recovered by density gradient separation and examined using both culture-independent and isolation methods. A 16S rRNA gene survey of the worm-associated bacteria was compared to the soil and to a similar analysis using <i>Caenorhabditis elegans</i> N2. Recovered <i>A. maximus</i> populations were maintained on cholesterol agar and sampled to examine the population dynamics of the microbiome.</p><p>Results</p><p>A consistent core microbiome was extracted from <i>A. maximus</i> that differed from those in the bulk soil or the <i>C. elegans</i> associated set. Three genera, <i>Ochrobactrum</i>, <i>Pedobacter</i>, and <i>Chitinophaga</i>, were identified at high levels only in the <i>A. maximus</i> populations, which were less diverse than the assemblage associated with <i>C. elegans</i>. Putative symbiont populations were maintained for at least 4 months post inoculation, although the levels decreased as the culture aged. Fluorescence <i>in situ</i> hybridization (FISH) using probes specific for <i>Ochrobactrum</i> and <i>Pedobacter</i> stained bacterial cells in formaldehyde fixed nematode guts.</p><p>Conclusions</p><p>Three microorganisms were repeatedly observed in association with <i>Acrobeloides maximus</i> when recovered from soil microcosms. We isolated several <i>Ochrobactrum sp.</i> and <i>Pedobacter sp.</i>, and demonstrated that they inhabit the nematode gut by FISH. Although their role in <i>A. maximus</i> is not resolved, we propose possible mutualistic roles for these bacteria in protection of the host against pathogens and facilitating enzymatic digestion of other ingested bacteria.</p></div

Identified Isolates from <i>A. maximus</i> microcosm and long term culture.

a<p>OTU assigned using RDP phylogeny. Isolates assigned to the same OTU were verified to be distinct using direct sequence comparison (bl2seq).</p

Phylogenetic tree created using Bayesian inference on the Ochrobactrum 16S rDNA sequences obtained from sample Acro2, the isolated <i>Ochrobactrum</i> sp., and several related sequences from the literature (see Materials and Methods).

<p><i>Rhizobium leguminosarum</i> bv. trifolii WSM597 was used as the outgroup for this analysis.</p

Phylogenetic tree created using Bayesian inference on the <i>Chitinophaga</i> 16S rDNA sequences obtained from sample Acro2, and several related sequences from the literature (see Materials and Methods).

<p>Clone A Heterodera and Clone B Heterodera were previously identified microbial sequence tags recovered from the Soybean Cyst Nematode <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067425#pone.0067425-Nour1" target="_blank">[21]</a>. <i>Flavobacterium johnsoniae</i> UW101 was used as the outgroup for this analysis.</p

A consistent bacterial population is associated with soil exposed <i>A.</i><i>maximus</i> nematodes.

<p>Samples isolated from <i>A. maximus</i> (N = 72, 93, 92 for Acro1-3) are dominated by Sphingobacteria and α-Proteobacteria after 24 h soil exposure. The bacteria from the identically treated soil sample (N = 94) contain a much higher percentage of Bacilli, and those associated with <i>C. elegans</i> after 24 h soil exposure (N = 94) are more broadly distributed across taxa.</p

Phylogenetic tree created using Bayesian inference on the <i>Pedobacter</i> 16S rDNA sequences obtained from sample Acro2, the isolated <i>Pedobacter</i> sp., and several related sequences from the literature (see Materials and Methods).

<p>Clone A (Heterodera) and Clone B (Heterodera) are partial sequences from Nour et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067425#pone.0067425-Nour1" target="_blank">[21]</a>. <i>Chitinophaga terrae</i> was used as the outgroup for this analysis.</p

Within the dominant subgroups the <i>Acrobeloides maximus</i> populations were consistent and limited compared to <i>C.</i>

<p><b>elegans<i> </i></b><b> associated bacteria.</b><b> </b><b>A</b>) The alpha-proteobacteria within <i>A. maximus</i> were predominantly Ochrobactrum. <b>B</b>) The Sphingobacteria within <i>A. maximus</i> were predominantly <i>Pedobacter</i>, with a consistent representation of <i>Chitinophaga</i> at a lower level. The <i>C. elegans samples</i> were more diverse at the genus level (both panels).</p

Shannon-Wiener Population Statistics for Microcosm Samples.

<p>Shannon-Wiener Population Statistics for Microcosm Samples.</p

Fluorescence <i>in situ</i> hybridization to identify <i>Ochrobactrum</i> (green) and <i>Pedobacter</i> (red) in the gut of formaldehyde fixed <i>A.</i><i>maximus</i> after recovery from soil microcosm.

<p>Samples from soil microcosms (<b>A–D</b>) and monoxenic culture on <i>E. coli</i> DH5μ (<b>E, F</b>) were imaged by DIC (<b>A,C,E</b>) or epifluorescence (<b>B,D,F</b>). Scale bar = 10 microns.</p