Global Methane Hub Wikipedia - User contributions [en]

Methanobrevibacter olleyae YLM1

2026-10-28T18:47:22Z

Nanashao:

== Introduction ==
''Methanobrevibacter olleyae'' YLM1 is a hydrogenotrophic methanogen isolated from the rumen of a lamb<ref>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: [https://www.sciencedirect.com/science/article/pii/S1075996404000496?via%3Dihub 10.1016/j.anaerobe.2004.05.003]. </ref>. Unlike the type species of the genus ''Methanobrevibacter ruminantium'' M1<ref>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: [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008926 10.1371/journal.pone.0008926].</ref>, YLM1 possesses the ''com''ADE genes, supporting the prediction that it can biosynthesize coenzyme M. It also possesses the ''mrt''BDGA operon encoding methyl-coenzyme M reductase II, which is absent from M1 and may contribute to differences in growth under varying hydrogen gas concentrations<ref name=":0">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: [https://journals.asm.org/doi/10.1128/genomea.00232-16 10.1128/genomeA.00232-16].</ref>. 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 ==
{| class="wikitable"
|'''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<ref name=":0" />. It contains one approximately 40-kb prophage, two CRISPR regions, and no identified plasmids. Notable features include the ''hdr''ABC genes encoding CoB-CoM heterodisulfide reductase subunits, the ''com''ADE genes involved in coenzyme M biosynthesis, the ''mrt''BDGA operon encoding methyl-coenzyme M reductase II, and 64 large adhesin-like protein genes. The GenBank accession number is [https://www.ncbi.nlm.nih.gov/nuccore/CP014265.1/ 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 ===
add here

=== Methylating ''E. coli'' ===
add here

== Culturing Conditions ==
''M. olleyae'' YLM1 is cultivated in [[Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM)|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.
{| class="wikitable"
|+
|[[File:Rumen fluid is required for M. olleyae YLM1 growth.jpg|thumb|Rumen fluid is required for ''M. olleyae'' YLM1 growth. Source: Dr. Nana Shao|center|333x333px]]
|[[File:Sulfide serves as a sulfur source for M. olleyae YLM1 .jpg|thumb|524x524px|Sulfide serves as a sulfur source for ''M. olleyae'' YLM1. Source: Nana Shao|center]]
|}

== Antimicrobial MIC Tests ==
[[File:Puromycin MIC determination in M. olleyae YLM1. Source- Dr. Nana Shao.jpg|thumb|Puromycin MIC determination in ''M. olleyae'' YLM1. Source: Dr. Nana Shao|left|290x290px]]'''Solid Medium'''

Puromycin susceptibility of ''M.'' ''olleyae'' YLM1 was assessed on MMSM solid medium under anaerobic conditions, with the bottle preparation and incubation performed as described in the [[Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM)|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 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'''
[[File:Antimicrobial MIC Determination in M. olleyae YLM1 in Liquid Medium.jpg|left|thumb|290x290px|Antimicrobial MIC determination in ''M. olleyae'' YLM1 in Liquid MMSM]]
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.
{| class="wikitable"
|+
!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 ==
add here

== Transformation Results ==
add here

== External Links ==
<references />

Methanocatella millerae SM9

2026-10-28T18:46:23Z

Nanashao:

== Introduction ==
''Methanocatella millerae'' SM9 (formerly ''Methanobrevibacter millerae'') was isolated from the rumen of a sheep fed a fresh forage diet<ref>Joblin KN. (1999) Ruminal acetogens and their potential to lower ruminant methane emissions. ''Australian Journal of Agricultural Research'' 50, 1307–1314. [https://connectsci.au/cp/article/50/8/1307/33473/Ruminal-acetogens-and-their-potential-to-lower https://doi.org/10.1071/AR99004]</ref>. 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<ref>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: [https://link.springer.com/article/10.1186/s40793-016-0171-9 10.1186/s40793-016-0171-9].</ref>. Its robust growth and high plating efficiency make SM9 a strain of particular interest to the Global Methane Hub.

== Global Methane Hub Metadata ==
{| class="wikitable"
|'''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 ==
add here

== Methylome ==

=== Methylation pattern ===
Pending Roy's input.

* No 6mA motifs present in output file, skipping...
* Searching dataset for following motifs: CCTG, CAGG, GGNCC
* Motifs explain the following about the (5mC) modified dataset:
* Low-quality methylated sites: 3603/4435 explained (81.2401352874859%)
* Mid-quality methylated sites: 12352/13030 explained (94.7966231772832%)
* High-quality methylated sites: 8337/8480 explained (98.31367924528301%)
* No 4mC motifs present in output file, skipping...
* Only one replicon present, skipping differential genomic coverage
=== DefenseFinder Results ===
add here

=== Methylating ''E. coli'' ===
add here

== Culturing Conditions ==
''M. millerae'' SM9 is cultivated in [[Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM)|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.
{| class="wikitable"
|+
|[[File:Rumen fluid, yeast extract and tryptone are all required for optimal growth of M. millerae SM9 .jpg|thumb|Rumen fluid, yeast extract and tryptone are all required for optimal growth of ''M. millerae'' SM9. Source: Dr. Nana Shao]]
|[[File:Sulfur source of M. millerae SM9.jpg|thumb|Sulfur source of ''M. millerae'' SM9. Source: Dr. Nana Shao]]
|[[File:Influence of Na2S concentration on M. millerae SM9 growth.jpg|thumb|Influence of Na2S concentration on ''M. millerae'' SM9 growth. Source: Dr. Nana Shao]]
|}

== Antimicrobial MIC Tests ==
[[File:Puromycin MIC determination in M. millerae SM9. Source- Dr. Nana Shao..jpg|thumb|246x246px|Puromycin MIC determination in ''M. millerae'' SM9. Source: Dr. Nana Shao|left]]'''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 [[Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM)|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.

{| class="wikitable"
|+
|-
!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
|}
{| class="wikitable"
|+
|[[File:Puromycin MIC determination in M. millerae SM9 in liquid MMSM.jpg|left|thumb|408x408px|Puromycin MIC determination in ''M. millerae'' SM9 in liquid MMSM. Source: Dr. Nana Shao]]
|[[File:Nourseothricin MIC determination in M. millerae SM9 in liquid MMSM.jpg|center|thumb|411x411px|Nourseothricin MIC determination in ''M. millerae'' SM9 in liquid MMSM. Source: Dr. Nana Shao]]
|}
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 ==
add here

== Transformation Results ==
add here

== External Links ==
<references />

Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM)

2026-10-28T18:42:21Z

Nanashao: /* Sources */

== Description ==
Modified ''Methanobrevibacter smithii'' and ''Methanosphaera stadtmanae'' medium (MMSM) is a rich, undefined medium commonly used for the anaerobic cultivation of ''Methanobrevibacter'' species.

== Medium components ==
Table 1 MMSM medium composition
{| class="wikitable"
|'''Composition per 100 mL'''
|'''Amount'''
|-
|''Modified Rich medium for Methanobrevibacter (MMSM)''
|
|-
|Mineral 1
|3.3 mL
|-
|Mineral 2
|3.3 mL
|-
|Yeast extract
|0.2 g
|-
|Tryptone
|0.2 g
|-
|Na Acetate·3H2O (150 g/L)
|2 mL
|-
|Fe(NH4)2(SO4)2·6H2O (2 g/L)
|1 mL
|-
|Rezasurin (1 g/L)
|0.1 mL
|-
|Trace vitamins
|1 mL
|-
|Trace minerals
|1 mL
|-
|NaHCO3
|0.5 g
|-
|Rumen fluid
|3 mL
|-
|NH4Cl
|0.1 g
|-
|L-Cysteine hydrochloride·H2O
|0.05 g
|-
|Na2S·9H2O
|0.1 mL
|-
|DTT
|0.05 g
|-
|ddH2O
|85 mL
|-
|''Modification of MMSM for growth on formate''
|
|-
|Add sodium formate
|1.5 g
|-
|Add glycylglycine buffer, 1 M
|20 mL
|-
|ddH2O
|65 mL
|}
Table 2 Composition of stock solutions for MMSM medium
{| class="wikitable"
|'''Composition per 1 L'''
|'''Amount'''
|-
|''Mineral 1''
|
|-
|K2HPO4
|6 g
|-
|''Mineral 2''
|
|-
|KH2PO4
|6 g
|-
|(NH4)2SO4
|6 g
|-
|NaCl
|12 g
|-
|MgSO4·7H2O
|2.6 g
|-
|CaCl2·2H2O
|0.16 g
|-
|''Iron stock solution''
|
|-
|Fe(NH4)2(SO4)2·6H2O
|2 g
|-
|37% HCl
|100 μL
|-
|''Na2S·9H2O''
|
|-
|2.5 %
|25 g
|-
|0.5 %
|5 g
|-
|''Trace minerals''
|
|-
|Nitriloacetic acid
|1.5 g
|-
|MnSO4·H2O / MnSO4·2H2O
|0.45 / 0.5 g
|-
|Fe(NH4)2(SO4)2·6H2O
|0.2 g
|-
|CoCl2·6H2O
|0.1 g
|-
|ZnSO4·7H2O
|0.1 g
|-
|CuSO4·5H2O
|0.01 g
|-
|H3BO3
|0.01 g
|-
|Na2MoO4·2H2O
|0.01 g
|-
|NiCl2·6H2O
|0.025 g
|-
|''Trace vitamins''
|
|-
|Biotin
|2 mg
|-
|Folic acid
|2 mg
|-
|Pyridoxine hydrochloride
|10 mg
|-
|Thiamine hydrochloride
|5 mg
|-
|Riboflavin
|5 mg
|-
|Nicotinic acid
|5 mg
|-
|DL-Pantolactone
|5 mg
|-
|Vitamin B12
|0.1 mg
|-
|p-aminobenzoic acid
|5 mg
|-
|Lipoic acid
|5 mg
|}
== Preparation of stock solutions ==
''Preparation of Mineral 1''. Dissolve 6 g of K2HPO4 in 1 liter of glass-distilled water. Use 3.3 mL per 100 mL of medium.

''Preparation of Mineral 2''. Use 3.3 mL per 100 mL of medium (see Table 2 for details).

''Preparation of Iron stock solution''. Add 0.2 g of Fe(NH4)2(SO4)2·6H2O into a small screw top bottle. Add 100 μL of concentrated HCl, then bring the volume to 100 mL with glass-distilled water.

''Preparation of L-cysteine hydrochloride hydrate solution.'' Use 0.05 g per 100 mL of medium. L-cysteine hydrochloride hydrate can be added directly to the medium before autoclaving. When preparing a filter-sterilized cysteine stock, proceed as follows:

1. Sparge 20 mL of ddH₂O in an acid-washed serum bottle with N2/CO2 (80:20, v/v) for 1 h. Seal the bottle and transfer it into the anaerobic chamber.

2. Add 1 g of L-cysteine hydrochloride hydrate in an acid-washed Balch tube, transfer the tube into the chamber, and leave it overnight.

3. Add 20 mL anaerobic water to the tube containing cysteine and mix well.

4. Filter-sterilize the cysteine solution by passage through a 0.22 µm filter. Dispense 5 mL aliquots into sterile 28 mL serum tubes and seal the tubes with sterile blue stoppers and aluminum seals.

5. For medium preparation, add 0.05 mL of the cysteine stock solution to each 5 mL of medium prior to inoculation.

''Preparation of Trace mineral solution''. Use 1 mL per 100 mL of medium (see Table 2 for details). Nitriloacetic acid was first dissolved in ~800 mL of ddH2O and the pH was adjusted to 6.5 with KOH. Add minerals in order, allowing each one to dissolve before adding the next mineral. At the end, bring the final volume to 1 L and adjust pH to 7.0.

''Preparation of Trace vitamin mix solution''. Use 0.05 mL per 5 mL of medium (see Table 2 for details). Dissolve the 10 ingredients listed in Table 2 in 1 liter of ddH2O with stirring under room temperature (about 10 min). Then transfer the solution to a serum bottle and sparge with N2 for 1 h. Inside the anaerobic chamber, the solution is filter-sterilized by passage through a 0.22 µm filter. Aliquots of 20 mL are transferred to sterile serum bottles and stored inside the chamber.

''Preparation of Glycylglycine solution''. Use 20 mL per 100 mL of formate medium (see Table 1 for details). Dissolve 132 g glycylglycine with 800 mL ddH2O. Adjust the pH to 8.0 by adding about 5 g NaOH. Bring the final volume to 1 L with ddH2O.

''Preparation of 2.5% and 0.5% sodium sulfide solutions''.

1. Add 100 mL ddH2O to an acid-washed 500 mL round-bottom flask. Add one pellet of NaOH (approximately 0.1 g).

2. Sparge the solution with N2 for 90 minutes, then add either 2.5 g or 0.5 g Na2S·9H2O. Loosely stopper the flask and continue sparging for an additional 10 min. Transfer the flask to the anaerobic chamber and dispense the solution into 5 mL aliquots in 28 mL aluminum-sealed tubes.

3. Remove the tubes from chamber and pressurize with N2 to 103 kPa.

4. Autoclave the tubes using gravity cycle 121 °C, 20 min, D: 5 min.

5. Store the sodium sulfide tubes inside the anaerobic chamber. They remain stable for up to two months. Discard if a precipitate forms.

== Medium preparation ==
'''Preparation of 100 mL of MMSM liquid medium.''' Prior to medium preparation, glassware was cleaned with 5% HCl overnight and dried at 150 °C for five hours to ensure that it was free of organic matter before use. MMSM liquid medium was prepared by combining the components indicated in Table 1 within an acid-washed 500 mL round-bottom flask. Sparge the medium with N2/CO2 (80:20, v/v) for 1 hour. Add 0.05 g DL-dithiothreitol (DTT) and 0.05 g L-cysteine hydrochloride hydrate to reduce the medium and continue sparging for 0.5 hour. Tightly stopper and transfer the flask into the anaerobic chamber. Dispense the medium into Balch tubes or serum bottles. Seal the tubes with 20 mM blue stoppers (Bellco Glass, Inc.) and secure each stopper with an aluminum seal. Remove the tubes from the chamber and sterilize the tubes by autoclaving at 121 °C for 20 min. Prior to inoculation, 0.05 mL of sterile vitamin mix and 0.1 mL of sterile 0.5% sodium sulfide are added. For cultivation of ''Methanobrevibacter'' species, the cultures are repressurized with 207 kPa of H2/CO2 (80:20, v/v) once per day.

For growth on formate, modify the MMSM preparation by adding sodium formate and glycylglycine as listed in Table 1. Replace the H2/CO2 headspace with 103 kPa N2/CO2.

'''Preparation of MMSM solid medium.''' MMSM solid medium containing 1.5% agar (w/v) was prepared in 70 mL serum bottles for plating ''Methanobrevibacter'' strains using a protocol modified from Long et al. <ref>Long F, Wang L, Lupa B, Whitman WB. A Flexible System for Cultivation of ''Methanococcus'' and Other Formate-Utilizing Methanogens. Archaea. 2017 Nov 19;2017:7046026. doi: [https://onlinelibrary.wiley.com/doi/10.1155/2017/7046026 10.1155/2017/7046026].</ref>. Before medium preparation, 0.15 g of agar was added to each serum bottle. The remaining medium components were prepared as described above for MMSM liquid medium. The liquid medium and agar-containing bottles were transferred into an anaerobic chamber, and 10 mL of medium was dispensed into each bottle. The bottles were sealed with blue rubber stoppers (Bellco Glass, Inc.), and secured with aluminum crimp seals. The sealed bottles were removed from the chamber, pressurized to 103 kPa of H2/CO2 (80:20, v/v), and autoclave at 121 °C for 20 min.

To obtain individual colonies of ''Methanobrevibacter'', cultures were serially diluted in MMSM broth. Undiluted and diluted culture were plated by transferring 1 mL of each suspension onto agar bottle surface using a 1 mL syringe, with each bottle resting on its side. The bottles were gently swirled to distribute the suspension evenly across the agar surface, then left on their sides overnight (~20 h), with the agar surface level and horizontal, to allow the suspension to be fully absorbed. The inoculated bottles were subsequently incubated upright outside of the anaerobic chamber and repressurized to 207 kPa with H₂/CO₂ (80:20, v/v) every 2 days. Colonies appeared after ~7 days of incubation.

== Sources ==

*External Links
<references />
* Used by Metcalf and Whitman Lab

2026-10-23T19:53:53Z

Nanashao: /* Antimicrobial MIC Tests */

== Introduction ==
''Methanobrevibacter olleyae'' YLM1 was a hydrogenotrophic methanogen isolated from the rumen of a lamb<ref>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: [https://www.sciencedirect.com/science/article/pii/S1075996404000496?via%3Dihub 10.1016/j.anaerobe.2004.05.003]. </ref>. Unlike ''Methanobrevibacter ruminantium'' M1<ref>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: [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008926 10.1371/journal.pone.0008926].</ref>, YLM1 carries ''com''ADE genes supporting its predicted ability to synthesize coenzyme M. It also possesses the ''mrt''BDGA operon encoding methyl-coenzyme M reductase II, which is absent from M1 and may contribute to differences in growth under varying hydrogen concentrations<ref name=":0">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: [https://journals.asm.org/doi/10.1128/genomea.00232-16 10.1128/genomeA.00232-16].</ref>. 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 ==
{| class="wikitable"
|'''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<ref name=":0" />. It contains one approximately 40-kb prophage, two CRISPR regions, and no identified plasmids. Notable features include the ''hdr''ABC gene encoding CoB-CoM heterodisulfide reductase subunits, the ''com''ADE genes involved in coenzyme M biosynthesis, the ''mrt''BDGA operon encoding methyl-coenzyme M reductase II, and 64 large adhesin-like protein genes. The GenBank accession number is [https://www.ncbi.nlm.nih.gov/nuccore/CP014265.1/ CP014265].

== DefenseFinder Results ==
add here

== Methylome ==
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

== Methylating ''E. coli'' ==
add here

== Culturing Conditions ==
''M. olleyae'' YLM1 is cultivated in [[Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM)|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.

== Antimicrobial MIC Tests ==
[[File:Puromycin MIC determination in M. olleyae YLM1. Source- Dr. Nana Shao.jpg|thumb|Puromycin MIC determination in ''M. olleyae'' YLM1. Source: Dr. Nana Shao]]'''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.

'''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.
{| class="wikitable"
|+
!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
|}
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.
{| class="wikitable"
|+
! rowspan="2" |Antibiotics
! colspan="2" |Concentration
|-
|'''Liquid'''
|'''Solid'''
|-
|Puromycin
|
|
|-
|nourseothricin
|
|μM
|}

== Protocols ==
add here

== Transformation Results ==
add here

== External Links ==
<references />

Methanocatella millerae SM9

2026-10-23T19:53:23Z

Nanashao:

Methanobrevibacter olleyae YLM1

2026-10-23T19:36:35Z

Nanashao:

== Introduction ==
''Methanobrevibacter olleyae'' YLM1 was a hydrogenotrophic methanogen isolated from the rumen of a lamb<ref>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: [https://www.sciencedirect.com/science/article/pii/S1075996404000496?via%3Dihub 10.1016/j.anaerobe.2004.05.003]. </ref>. Unlike ''Methanobrevibacter ruminantium'' M1<ref>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: [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008926 10.1371/journal.pone.0008926].</ref>, YLM1 carries ''com''ADE genes supporting its predicted ability to synthesize coenzyme M. It also possesses the ''mrt''BDGA operon encoding methyl-coenzyme M reductase II, which is absent from M1 and may contribute to differences in growth under varying hydrogen concentrations<ref name=":0">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: [https://journals.asm.org/doi/10.1128/genomea.00232-16 10.1128/genomeA.00232-16].</ref>. 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 ==
{| class="wikitable"
|'''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<ref name=":0" />. It contains one approximately 40-kb prophage, two CRISPR regions, and no identified plasmids. Notable features include the ''hdr''ABC gene encoding CoB-CoM heterodisulfide reductase subunits, the ''com''ADE genes involved in coenzyme M biosynthesis, the ''mrt''BDGA operon encoding methyl-coenzyme M reductase II, and 64 large adhesin-like protein genes. The GenBank accession number is [https://www.ncbi.nlm.nih.gov/nuccore/CP014265.1/ CP014265].

== DefenseFinder Results ==
add here

== Methylome ==
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

== Methylating ''E. coli'' ==
add here

== Culturing Conditions ==
''M. olleyae'' YLM1 is cultivated in [[Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM)|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.

== Antimicrobial MIC Tests ==
[[File:Puromycin MIC determination in M. olleyae YLM1. Source- Dr. Nana Shao.jpg|thumb|Puromycin MIC determination in ''M. olleyae'' YLM1. Source: Dr. Nana Shao]]'''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.

'''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.
{| class="wikitable"
|+
!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
|}
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.
{| class="wikitable"
|+
! rowspan="2" |Antibiotics
! colspan="2" |Concentration
|-
|'''Liquid'''
|'''Solid'''
|-
|Puromycin
|
|
|-
|nourseothricin
|
|μM
|}

== Protocols ==
add here

== Transformation Results ==
add here

== External Links ==
<references />

Methanocatella millerae SM9

2026-10-23T19:32:00Z

Nanashao: /* Antimicrobial MIC Tests */

Methanocatella millerae SM9

2026-10-23T17:54:18Z

Nanashao: /* Antimicrobial MIC Tests */

Methanocatella millerae SM9

2026-10-23T17:53:24Z

Nanashao: /* Antimicrobial MIC Tests */

Methanocatella millerae SM9

2026-10-23T17:52:15Z

Nanashao: /* Antimicrobial MIC Tests */

Methanocatella millerae SM9

2026-10-21T17:10:37Z

Nanashao: