Single-cell genome and metatranscriptome sequencing reveal metabolic interactions of an alkane-degrading methanogenic community

Abstract

Microbial interactions have a key role in global geochemical cycles. Although we possess significant knowledge about the general biochemical processes occurring in microbial communities, we are often unable to decipher key functions of individual microorganisms within the environment in part owing to the inability to cultivate or study them in isolation. Here, we circumvent this shortcoming through the use of single-cell genome sequencing and a novel low-input metatranscriptomics protocol to reveal the intricate metabolic capabilities and microbial interactions of an alkane-degrading methanogenic community. This methanogenic consortium oxidizes saturated hydrocarbons under anoxic conditions through a thus-far-uncharacterized biochemical process. The genome sequence of a dominant bacterial member of this community, belonging to the genus Smithella, was sequenced and served as the basis for subsequent analysis through metabolic reconstruction. Metatranscriptomic data generated from less than 500 pg of mRNA highlighted metabolically active genes during anaerobic alkane oxidation in comparison with growth on fatty acids. These data sets suggest that Smithella is not activating hexadecane by fumarate addition. Differential expression assisted in the identification of hypothetical proteins with no known homology that may be involved in hexadecane activation. Additionally, the combination of 16S rDNA sequence and metatranscriptomic data enabled the study of other prevalent organisms within the consortium and their interactions with Smithella, thus yielding a comprehensive characterization of individual constituents at the genome scale during methanogenic alkane oxidation.

Figure 1

(a) Formation of gas (methane) in an anaerobic enrichment culture growing in 300 ml mineral medium containing hexadecane (•). The methanogenic consortium was propagated in the laboratory and formed a total of 809 ml of gas consisting of 10.9 mM methane (8.81 mmol total formed) after 1141 days of incubation. A control without hexadecane (▪) showed no gas formation. The previous enrichment described in Zengler et al., 1999 (open symbols, overlaid for comparison) formed a total of 370 ml of gas after 1051 days of incubation. The community with hexadecane is represented by open circles (∘), and the control by open squares (□). (b) The consortium, although highly metabolically active as seen by gas formation (bubbles), grows to very low biomass densities as can be seen in this 3-year-old culture. Teflon boiling stones were coated with hexadecane to increase the contact between cells and the hydrophobic substrate.

Figure 2

Schematic of the integrated workflow applied to study the methanogenic community. A single-cell genome sequencing approach established a working-draft genome of Smithella. Then, low-input metatranscriptomics was used in order to determine which genes were active during alkane degradation. After determining the major role of Smithella within the community, the metatranscriptomics data sets were extended to analyze the activity of other microbial community members. A genome-scale metabolic model was used to facilitate the integration of both the genomic and transcriptomic data in order to extract functional information about the organisms.

Figure 3

Example of the coverage obtained from the metatranscriptome (hexadecane-degrading community) when mapped to the Smithella draft genome (teal tracks) and the published genome of M. concilii (orange tracks). This snapshot is representative of the level of coverage observed across the entire genome of these organisms. Ninety-four percent of the genes from Smithella were represented by the metatranscriptomic data set.

Figure 4

(a) Overview of the metabolism of Smithella and two representative methanogens during hexadecane degradation. Active pathways were determined from metatranscriptomic data mapped to the Smithella draft genome and the published M. concilii genome. The red arrow indicates that the mechanism driving this step is currently unknown. In this paper, we present a list (Supplementary Tables S7 and S8) of highly expressed, hypothetical proteins and radical-activating enzymes that may be facilitating hexadecane activation. (b) 16S rDNA gene analysis of the bacterial and archaeal community under hexadecane-, caprylic acid- and butyric acid-degrading conditions.