Directed culturing of microorganisms using metatranscriptomics

Abstract

The vast majority of bacterial species remain uncultured, and this severely limits the investigation of their physiology, metabolic capabilities, and role in the environment. High-throughput sequencing of RNA transcripts (RNA-seq) allows the investigation of the diverse physiologies from uncultured microorganisms in their natural habitat. Here, we report the use of RNA-seq for characterizing the metatranscriptome of the simple gut microbiome from the medicinal leech Hirudo verbana and for utilizing this information to design a medium for cultivating members of the microbiome. Expression data suggested that a Rikenella-like bacterium, the most abundant but uncultured symbiont, forages on sulfated- and sialated-mucin glycans that are fermented, leading to the secretion of acetate. Histological stains were consistent with the presence of sulfated and sialated mucins along the crop epithelium. The second dominant symbiont, Aeromonas veronii, grows in two different microenvironments and is predicted to utilize either acetate or carbohydrates. Based on the metatranscriptome, a medium containing mucin was designed, which enabled the cultivation of the Rikenella-like bacterium. Metatranscriptomes shed light on microbial metabolism in situ and provide critical clues for directing the culturing of uncultured microorganisms. By choosing a condition under which the desired organism is rapidly proliferating and focusing on highly expressed genes encoding hydrolytic enzymes, binding proteins, and transporters, one can identify an organism's nutritional preferences and design a culture medium.

Importance: The number of prokaryotes on the planet has been estimated to exceed 10(30) cells, and the overwhelming majority of them have evaded cultivation, making it difficult to investigate their ecological, medical, and industrial relevance. The application of transcriptomics based on high-throughput sequencing of RNA transcripts (RNA-seq) to microorganisms in their natural environment can provide investigators with insight into their physiologies under optimal growth conditions. We utilized RNA-seq to learn more about the uncultured and cultured symbionts that comprise the relatively simple digestive-tract microbiome of the medicinal leech. The expression data revealed highly expressed hydrolytic enzymes and transporters that provided critical clues for the design of a culture medium enabling the isolation of the previously uncultured Rikenella-like symbiont. This directed culturing method will greatly aid efforts aimed at understanding uncultured microorganisms, including beneficial symbionts, pathogens, and ecologically relevant microorganisms, by facilitating genome sequencing, physiological characterization, and genetic manipulation of the previously uncultured microbes.

FIG 1

Glycan utilization operon. The numbers below the arrows represent the expression values.

FIG 2

Expression of endoG and smdS1 inside the leech was confirmed using qRT-PCR. Relative expression of endoG and smdS1 was measured at 8, 24, 42, and 96 h after feeding. The results are expressed as fold changes in expression relative to the level for rpoD and normalized to the level obtained 8 h after feeding. Each target was quantified in duplicate. Error bars represent standard deviations.

FIG 3

Histochemical staining of the leech crop epithelium tissue detected sulfated and sialated carbohydrates. Results for HID/AB staining of leech tissue obtained at the below-indicated sampling time points after feeding are shown. Staining patterns consistent with the presence of both sulfoglycans (black-gray) and sialoglycans (blue) were present at all sampling time points, 8 h (A), 24 h (B), 42 h (C), 96 h (D), and 7 days (E). Sulfoglycans (black-gray) appear in the epithelial tissue with an increased signal seen at the lumen interface (black arrows) over time. Sulfoglycans can also be found in areas near the cuticle (red arrow). Sialoglycans (blue) are also present. LU, lumen of the crop. The scale bar represents 50 µm. (F) HID-AB staining of the epithelial flap (indicated by black arrows) extending into the lumen. LU, lumen. The scale bar represents 100 µm.

FIG 4

Evidence supporting the cultivation of Rikenella-like bacteria. (A) Diagnostic PCR identification of Rikenella-like bacteria. Lanes: R, Rikenella clone M3 (genomic DNA template); 4, Rikenella clone M4 (colony template); 5, Rikenella clone M5 (colony template); 6, Rikenella clone M6 (colony template); C, crop contents (genomic DNA template); N, no template control; A, A. veronii (genomic DNA template); M, DNA marker (100, 200, 300, 400, 500, 600, 700, and 800 bp, from bottom to top). (B) Phase contrast image of Rikenella-like bacteria. The scale bar represents 10 µm.

FIG 5

Maximum-likelihood tree constructed from the alignment of partial 16S rRNA gene sequences. Partial 16S rRNA gene sequences were aligned using MUSCLE, and a PhyML tree was constructed from the alignment. Bootstrap support values from 1,000 resamplings and GenBank accession numbers are shown.

FIG 6

Schematic displaying the proposed physiological processes carried out by the Rikenella-like bacteria (red) and A. veronii (green) in the crop. The circled numbers correspond to the steps as follows: 1, Rikenella-like bacteria remove sulfate from mucin glycans that are shed from or adjacent to the host tissue and are more concentrated near the endothelium (SmdS1 and SmdS2); 2, glycans are liberated from the protein (EndoG and SusC- and SusD-like proteins); 3, liberated glycans are fermented to short chain fatty acids (SCFAs) and secreted (phosphofructokinase, phosphate acetyltransferase, and acetate kinase); 4, A. veronii converts SCFAs to malate and succinate with the glyoxylate shunt of the TCA cycle (isocitrate lyase and malate synthase); 5, pelagic A. veronii catabolizes sugars to CO₂ (phosphofructokinase, 2-oxoglutarate dehydrogenase, and cytochrome c oxidase).