Methanobacterium formicicum BRM9
Introduction
Methanobacterium formicicum BRM9 is isolated from the rumen enrichment of non-lactating, rumen-fistulated, 6-year-old New Zealand Friesian cows grazing ryegrass/clover pastures[1]. Although Methanobacterium species are relatively low in abundance in the rumen environment[2], they have been found as endosymbionts of anaerobic amoebae and ciliate protozoa[3]. This uniqueness makes M. formicicum BRM9 a methanogen of interest for GMH, as understanding these methanogenic endosymbiont interactions might open a venue for novel strategies in livestock methane mitigation.
Global Methane Hub Metadata
| Origin: | BSI, New Zealand |
|---|---|
| Sequenced by: | Plasmidsaurus, ONT sequencing |
| Assembled by: | Christian Heryakusuma |
| Methylome called by: | William G. Alexander |
| Methyltransferases cloned by: | Guss Lab, ORNL |
| MIC and Culturing Conditions by: | Christian Heryakusuma |
| Transformation by: |
Abbreviation: BSI, Bioeconomy Science Institute; ONT, Oxford Nanopore Technologies; ORNL, Oak Ridge National Laboratory.
Genome
| Assembly Summary | |
|---|---|
| Completeness | 99.94% |
| Contamination | 0.35% |
| Total contigs | 1 |
| Max contig length | 2,456,253 bp |
| GC content | 41.3% |
| Annotation Summary | |||
|---|---|---|---|
| Coding density | 84.8% | CDSs | 2397 |
| tRNAs | 44 | Pseudogenes | 0 |
| tmRNAs | 0 | Hypotheticals | 2061 |
| rRNAs | 7 | sORFs | 0 |
| ncRNAs | 1 | Gaps | 0 |
| ncRNAs regions | 2 | oriCs | 0 |
| CRISPR arrays | 3 | oriVs | 0 |
| oriTs | 0 | ||
Genomic DNA (gDNA) of M. formicicum BRM9 was extracted using NEB Monarch® Spin gDNA Extraction Kit following the manufacturer's protocols for gram-positive bacteria and archaea, except that lysozyme was replaced with the phage ΨM2 pseudomurein endoisopeptidase of Methanothermobacter marburgensis (PeiP)[4].
The extracted gDNA was sequenced with Oxford Nanopore, then assembled with Flye[5] and polished with medaka (https://github.com/nanoporetech/medaka). The assembled genome was evaluated for completeness and contamination using QUAST[6] and CheckM2[7]. Bakta v1.12.0[8] with database v6.0 full (https://github.com/oschwengers/bakta) was used for genome annotation. All these bioinformatics analyses were performed at the Virginia Tech Advanced Research Computing (VT-ARC) facility.
Methylome
Methylation pattern
| #motif | methylCounts | totalCounts | %motifsMethyl |
|---|---|---|---|
| G(6mA)TC | 16042 | 17258 | 92.95 |
| GC(6mA)NNNNNNCTC | 465 | 489 | 95.09 |
| G(6mA)GNNNNNNTGC | 477 | 489 | 97.55 |
Methylation motifs were analyzed using MIJAMP[9]. 4mC and 5mC motifs were not present in this isolate.
DefenseFinder Results
| Type | Subtype | Components |
|---|---|---|
| CBASS | CBASS Type II with a Cap4 effector | Cap2 - E1 (ThiF/adenylyltransferase) module, CD-NTase / cyclase (Type II allele), Cap3 - JAB/JAMM isopeptidase, Cap4 - SAVED and HNH effector |
| CRISPR-Cas | CAS Class1 Subtype IB | cas1_I_II_III_IV_V_VI_7, cas2_I_II_III_IV_V_VI_5, cas3_I_5, cas4_I_II_III_IV_V_VI_6, cas5_I-B_17, cas6_I_II_III_IV_V_VI_10, cas7b_I-B_I-C_2, cas8b1_I-B_5 |
| Prometheus | Prometheus | ProA |
| RM Type I | N6-adenine methylation (m6A) | Type I RM methyltransferase (HsdM), Type I RM restriction endonuclease (HsdR), Type I RM specificity subunit (HsdS) |
| Avs | Avs family 2 | Avs2 |
| CRISPR-Cas | CAS Class1 Subtype IB | DnaQ/DEDDh exonuclease, cas1_I_II_III_IV_V_VI_7, cas2_I_II_III_IV_V_VI_5, cas3_I_2, cas4_I_II_III_IV_V_VI_6, cas5_I-B_6, cas6_I_II_III_IV_V_VI_10, cas7_I-B_6, cas8b1_I-B_13 |
Abbreviation: CBASS, cyclic-oligonucleotide-based antiphage signalling system[10]; CRISPR-Cas, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzymes; Prometheus, DNA replication helicase/nuclease 2 (DNA2)-containing anti-phage defense protein[11]; RM, restriction-modification; Avs, antiviral STAND (Signal Transduction ATPases with Numerous Domains)[12].
Methylating E. coli
E. coli strain AG16322 developed by ORNL was used as a host to generate plasmids with M. formicicum BRM9 methylation motifs shown above.
Culturing Conditions
M. formicicum BRM9 is cultivated in GMH-VT2 medium at 37°C and 50 rpm with a 30 psig gas mixture of H2:CO2 (80:20, v/v) as an energy substrate. Growth is typically observed within 72 to 96 h. This methanogen can also be cultivated on GMH-VT2 plates using 1.5% gelrite or 2% agar as a gelling agent with a 12 to 30 psig H2:CO2 (80:20, v/v) gas mixture as an energy substrate. Visible growth on plate is usually observable within 5 to 7 days.
Antimicrobial MIC Tests
| Antibiotics | Concentration | Observation Period | |
|---|---|---|---|
| Liquid | Solid | ||
| Sodium sulfite | 0.1 mM | 0.1 mM | ~240 h |
| Mevinolin | Insensitive | Insensitive | ~240 h |
| 5-fluoroorotic acid (5-FOA) | Insensitive | Insensitive | ~240 h |
| 8-azahypoxanthine (8-AH) | Insensitive | Insensitive | ~240 h |
| 6-mercaptopurine (6-MP) | Insensitive | Insensitive | ~240 h |
| 5-fluorouracil (5-FU) | Insensitive | Insensitive | ~240 h |
| Sodium 2-bromoethanesulfonate (BES) | 2 mM | 1 mM | ~240 h |
Susceptibility testing was evaluated in GMH-VT2 medium inoculated with a 4% M. formicicum BRM9 culture that has a starting OD600 of ~0.4. M. formicicum BRM9 was considered sensitive to the selection or counterselection agents if there was no increase in the OD600 during the ~240 h (10 days) observation period.
Protocols
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Transformation Results
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External Links
- ↑ Jarvis, G., Strömpl, C., Burgess, D. et al. Isolation and Identification of Ruminal Methanogens from Grazing Cattle. Curr Microbiol 40, 327–332 (2000). https://doi.org/10.1007/s002849910065.
- ↑ Janssen PH, Kirs M. Structure of the archaeal community of the rumen. Appl Environ Microbiol. 2008 Jun;74(12):3619-25. https://doi.org/10.1128/AEM.02812-07. Epub 2008 Apr 18. PMID: 18424540; PMCID: PMC2446570.
- ↑ Kelly WJ, Leahy SC, Li D, Perry R, Lambie SC, Attwood GT, Altermann E. The complete genome sequence of the rumen methanogen Methanobacterium formicicum BRM9. Stand Genomic Sci. 2014 Dec 8;9:15. https://doi.org/10.1186/1944-3277-9-15. PMID: 25780506; PMCID: PMC4335013.
- ↑ Luo Y, Pfister P, Leisinger T, Wasserfallen A. Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains. FEMS Microbiol Lett. 2002 Feb 19;208(1):47-51. https://doi.org/10.1111/j.1574-6968.2002.tb11059.x.
- ↑ Kolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019 May;37(5):540-546. https://doi.org/10.1038/s41587-019-0072-8. Epub 2019 Apr 1. PMID: 30936562.
- ↑ Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics. 2013 Apr 15;29(8):1072-5. https://doi.org/10.1093/bioinformatics/btt086. Epub 2013 Feb 19. PMID: 23422339; PMCID: PMC3624806.
- ↑ Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015 Jul;25(7):1043-55. https://doi.org/10.1101/gr.186072.114. Epub 2015 May 14. PMID: 25977477; PMCID: PMC4484387.
- ↑ Schwengers O, Jelonek L, Dieckmann MA, Beyvers S, Blom J, Goesmann A. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb Genom. 2021 Nov;7(11):000685. https://doi.org/10.1099/mgen.0.000685. PMID: 34739369; PMCID: PMC8743544.
- ↑ Alyssa K Tidwell, Evelyn Faust, Carrie A Eckert, Adam M Guss, William G Alexander, Discovering methylated DNA motifs in bacterial nanopore sequencing data with MIJAMP, Journal of Industrial Microbiology and Biotechnology, Volume 52, 2025, kuaf022, https://doi.org/10.1093/jimb/kuaf022.
- ↑ Cohen, D., Melamed, S., Millman, A. et al. Cyclic GMP–AMP signalling protects bacteria against viral infection. Nature 574, 691–695 (2019). https://doi.org/10.1038/s41586-019-1605-5.
- ↑ van den Berg DF, Costa AR, Esser JQ, Stanciu I, Geissler JQ, Zoumaro-Djayoon AD, Haas PJ, Brouns SJJ. Bacterial homologs of innate eukaryotic antiviral defenses with anti-phage activity highlight shared evolutionary roots of viral defenses. Cell Host Microbe. 2024 Aug 14;32(8):1427-1443.e8. https://doi.org/10.1016/j.chom.2024.07.007.
- ↑ Gao LA, Wilkinson ME, Strecker J, Makarova KS, Macrae RK, Koonin EV, Zhang F. Prokaryotic innate immunity through pattern recognition of conserved viral proteins. Science. 2022 Aug 12;377(6607):eabm4096. https://doi.org/10.1126/science.abm4096.
