Methanobrevibacter olleyae YLM1
Introduction
Methanobrevibacter olleyae YLM1 is a hydrogenotrophic methanogen isolated from the rumen of a lamb[1]. Unlike the type species of the genus Methanobrevibacter ruminantium M1[2], YLM1 possesses the comADE genes, supporting the prediction that it can biosynthesize coenzyme M. It also possesses the mrtBDGA operon encoding methyl-coenzyme M reductase II, which is absent from M1 and may contribute to differences in growth under varying hydrogen gas concentrations[3]. These genomic features make YLM1 a useful strain for comparative studies of rumen methanogen physiology and the development of methane mitigation strategies. Its robust growth and high plating efficiency, observed in the Whitman Lab, make it particularly valuable for research supported by the Global Methane Hub.
Global Methane Hub Metadata
| Origin: | Lucidome Bio, NZ |
| Sequenced by: | Plasmidsaurus, ONT + Illumina |
| Assembled by: | Nana Shao |
| Methylome called by: | William G. Alexander |
| Methyltransferases cloned by: | Roy Rodriguez Carrero |
| MIC and Culturing conditions by: | Nana Shao |
| Transformation by: |
Genome
The draft genome of Methanobrevibacter olleyae YLM1 is 2,201,192 bp, with a GC content of 26.9% and 1,834 predicted protein-coding genes[3]. It contains one approximately 40-kb prophage, two CRISPR regions, and no identified plasmids. Notable features include the hdrABC genes encoding CoB-CoM heterodisulfide reductase subunits, the comADE genes involved in coenzyme M biosynthesis, the mrtBDGA operon encoding methyl-coenzyme M reductase II, and 64 large adhesin-like protein genes. The GenBank accession number is CP014265.
Methylome
Methylation pattern
Pending Roy's input.
- Searching dataset for following motifs: GSNGA, GNSNGA
- Motifs explain the following about the (6mA) modified dataset:
- Low-quality methylated sites: 7170/6856 explained (104.57992998833139%)
- Mid-quality methylated sites: 7158/6524 explained (109.71796443899449%)
- High-quality methylated sites: 300/244 explained (122.95081967213115%)
- No 5mC motifs present in output file, skipping...
- No 4mC motifs present in output file, skipping...
- Only one replicon present, skipping differential genomic coverage
DefenseFinder Results
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Methylating E. coli
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Culturing Conditions
M. olleyae YLM1 is cultivated in Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM) at 37°C. To ensure good gas transfer to the media, tubes are incubated on their sides (or at a shallow angle so that the media does not contact the stoppers) with gentle shaking at 50 rpm and 207 kPa of H2/CO2 (80:20, v/v) as an energy substrate for 2-4 days. This methanogen can also be cultivated on MMSM plates using 1.5% agar as a gelling agent with the same pressure of H2/CO2 as an energy substrate for 3-7 days.
Growth requirements
M. olleyae YLM1 requires rumen fluid for robust growth in MMSM, whereas yeast extract and tryptone are individually dispensable under the conditions tested. Omission of rumen fluid markedly reduces growth, while omission of either yeast extract or tryptone still allows growth to OD600 values of ~1.4. The inability of filter-sterilized cysteine to support good growth indicates that is not used as a sulfur source and that sulfide from the breakdown of cysteine serves as a sulfur source for this strain.
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Antimicrobial MIC Tests
Solid Medium
Puromycin susceptibility of M. olleyae YLM1 was assessed on solid medium under anaerobic conditions, using the absence of visible colonies as the endpoint. The apparent MIC was 10 µg/mL at an inoculum of 105 cells/plate and 15 µg/mL at 107 cells/plate. Below the MIC, confluent growth or numerous small colonies are observed on the agar surface. Large colonies are observed when fewer than 100 cells survive. When growth is inhibited but not completely suppressed, small colonies are observed.
Liquid Medium
Puromycin and nourseothricin susceptibility was assessed in liquid MMSM at an initial inoculum density of 2×107 cells/mL, using the absence of a detectable increase in OD600 throughout the observation period as the endpoint.
| Antibiotics | MIC at 2×107 cells/mL | Observation period |
|---|---|---|
| Puromycin | 30 µg/mL | ~650 h |
| nourseothricin | >80 µg/mL | ~550 h |
Overall, the results demonstrated inoculum-dependent inhibition under the tested conditions, with higher apparent MICs at higher inoculum densities in both solid and liquid media.
Overall, puromycin showed inoculum-dependent inhibition on solid medium and prevented detectable growth in liquid medium, whereas nourseothricin delayed but did not prevent growth at the tested concentrations. For the lower inoculum of about 105 cells, the MIC represents the lowest concentration that completely inhibits growth of wild-type cells. For the higher inoculum of about 107 cells, the MIC represents the lowest concentration that completely inhibits growth of spontaneous resistance mutants.
Protocols
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Transformation Results
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External Links
- ↑ Skillman LC, Evans PN, Naylor GE, Morvan B, Jarvis GN, Joblin KN. 16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs. Anaerobe. 2004 Oct;10(5):277-85. doi: 10.1016/j.anaerobe.2004.05.003.
- ↑ Leahy SC, Kelly WJ, Altermann E, Ronimus RS, Yeoman CJ, Pacheco DM, Li D, Kong Z, McTavish S, Sang C, Lambie SC, Janssen PH, Dey D, Attwood GT. The genome sequence of the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions. PLoS One. 2010 Jan 28;5(1):e8926. doi: 10.1371/journal.pone.0008926.
- ↑ 3.0 3.1 Kelly WJ, Li D, Lambie SC, Cox F, Attwood GT, Altermann E, Leahy SC. Draft Genome Sequence of the Rumen Methanogen Methanobrevibacter olleyae YLM1. Genome Announc. 2016 Apr 7;4(2):e00232-16. doi: 10.1128/genomeA.00232-16.
