Methanocatella millerae SM9
Introduction
Methanocatella millerae SM9 (formerly Methanobrevibacter millerae) was isolated from the rumen of a sheep fed a fresh forage diet[1]. Although initially identified as Methanobrevibacter sp. or Methanobrevibacter smithii, SM9 shares 99% 16S rRNA gene sequence identity with the Methanocatella (Methanobrevibacter) millerae type strain ZA-10ᵀ (DSM 16643), supporting its assignment to this species[2]. Its robust growth and high plating efficiency make SM9 a strain of particular interest to 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
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Methylome
Methylation pattern
| Methylated motif | Percent methylated |
|---|---|
| C(5mC)TG | 92.68 |
| (5mC)AGG | 77.20 |
| GGN(5mC)C | 63.19 |
DefenseFinder Results
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Methylating E. coli
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Culturing Conditions
M. millerae SM9 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. millerae SM9 requires rumen fluid, yeast extract, and tryptone for optimal growth in MMSM. Omission of rumen fluid markedly reduces growth, whereas omission of either yeast extract or tryptone results in a moderate reduction in growth yield. Either cysteine (3 mM; autoclaved or filter-sterilized) or sodium sulfide (Na₂S; 0.4-1 mM) supports robust growth as a sulfur source, with no clear preference between the two. Under these conditions, cultures reach a maximum OD600 of approximately ~1.3. No growth was observed with formate as the sole energy substrate under the conditions tested.
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Antimicrobial MIC Tests
Solid Medium
Puromycin susceptibility of M. millerae SM9 was assessed on MMSM solid medium under anaerobic conditions, with the bottle preparation and incubation performed as described in the MMSM protocol. The absence of visible colonies was used as the endpoint. The apparent MIC was 10 µg/mL at an inoculum of 105 cells/plate and 20 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 initial inoculum densities of 8×105 and 2×107 cells/mL, using the absence of a detectable increase in OD600 throughout the observation period as the endpoint.
| Antibiotics | MIC at 8×105 cells/mL | MIC at 2×107 cells/mL | Observation period |
|---|---|---|---|
| Puromycin | 10 µg/mL | 20 µg/mL | ~700 h |
| nourseothricin | 40 µg/mL | 70 µg/mL | ~630 h |
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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. 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
- ↑ Joblin KN. (1999) Ruminal acetogens and their potential to lower ruminant methane emissions. Australian Journal of Agricultural Research 50, 1307–1314. https://doi.org/10.1071/AR99004
- ↑ Kelly WJ, Pacheco DM, Li D, Attwood GT, Altermann E, Leahy SC. The complete genome sequence of the rumen methanogen Methanobrevibacter millerae SM9. Stand Genomic Sci. 2016 Aug 17;11:49. doi: 10.1186/s40793-016-0171-9.
