Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill

Abstract

The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

Figure 1

Methods schematic. Each type of molecular approach—metagenomics, metatranscriptomics and single cell genomics—is shown, as are the subsequent, novel bioinformatics approaches that were used to analyze the various data sets.

Figure 2

Relative abundance of bacteria and archaea in the proximal and distal plume samples and in the uncontaminated sample collected from plume depth. (a) Relative OTU abundance of rarified 16S rRNA gene 454-pyrotag data. Universal primers for archaea and bacteria were used. Taxonomy was assigned using the Greengenes (DeSantis et al., 2006) 16S rRNA gene database. (b) Raw, unassembled metagenomic and metatranscriptomic reads were compared with the Greengenes (DeSantis et al., 2006) database. Less-abundant bacteria and archaea are grouped under the category ‘other.' The complete list of bacteria and archaea observed in these analyses are presented in Supplementary Tables S2, S3 and S4.

Figure 3

Analysis of genes involved in hydrocarbon degradation in the metagenome data. Blue bars denote the distal station metagenome, black bars denote the uncontaminated sample metagenome and red bars denote the proximal station metagenome. Raw, unassembled metagenomic reads were compared with proteins involved in hydrocarbon degradation, using a custom database using the tblastn algorithm. A bit score cutoff of ⩾40 was used. Genes were grouped according to function. ^AIndicates that a corrected P-value was not significant. Gene categories denoted with an ‘‡' indicate a similar substrate degradation pathway in which the different substrates are degraded by the same enzyme (simple ring oxygenases). A complete list of all gene categories is provided in Supplementary Table S6.

Figure 4

Analysis of genes involved in hydrocarbon degradation in the metatranscriptome data. Blue bars denote the distal station metatranscriptome and red bars denote the proximal station metatranscriptome. Raw, unassembled metatranscriptome reads were compared with proteins involved in hydrocarbon degradation, using a custom database using the tblastn algorithm. A bit score cutoff of ⩾40 was used. Genes were grouped according to function. An asterisk indicates that the difference in relative abundance of a particular gene group in the proximal station metatranscriptome compared with the distal station metatranscriptome was statistically significant. Gene categories denoted with an ‘‡' indicate a similar substrate degradation pathway in which the different substrates are degraded by the same enzyme (simple ring oxygenases). Within this category, ring cleavage/hydroxylating enzymes were observed at very low abundance and only in the proximal plume station. Simple ring oxygenases that are involved in the degradation of benzene, toluene and PAHs were not observed in the metatranscriptome data. A complete list of all gene categories is provided in Supplementary Table S6.

Figure 5

Oceanospirillales single-cell metabolic reconstruction using COG annotations of assembled sequence data and the blast comparison of unassembled single-cell reads to genes involved in hydrocarbon degradation. All genes in the single-cell metabolic reconstruction were present in the metagenomes and most were expressed in the metatranscriptome, except for those with an asterisk following the gene name.