Methanocatella gottschalkii HO: Difference between revisions

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== Introduction ==
== Introduction ==
[[File:Mgot HO.gif|thumb|Phase-contrast (top) and epifluorescence (bottom) microscopy of ''Methanocatella gottschalkii'' HO. '''Source:''' Christian Heryakusuma.]]
''Methanocatella gottschalkii'' HO<sup>T</sup> (formerly ''Methanobrevibacter gottschalkii'') is a type strain isolated from the enrichment of horse faeces<ref>Miller, T. L. (2002). "Description of Methanobrevibacter gottschalkii sp. nov., Methanobrevibacter thaueri sp. nov., Methanobrevibacter woesei sp. nov. and Methanobrevibacter wolinii sp. nov". International Journal of Systematic and Evolutionary Microbiology. 52 (3): 819–822. https://doi.org/10.1099/00207713-52-3-819.</ref>. ''M. gottschalkii'', along with ''Methanobrevibacter ruminantium'', comprises about 74% of total archaeal OTUs from rumen samples collected across a wide range of geographical regions, hosts, and diets<ref>Henderson, G., Cox, F., Ganesh, S. ''et al.'' Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range. ''Sci Rep'' '''5''', 14567 (2015). https://doi.org/10.1038/srep14567. </ref>. This universality makes them methanogens of particular interest to the Global Methane Hub, as it enables strategies to mitigate methane emissions by targeting these dominant methanogens.
== Global Methane Hub Metadata ==
{| class="wikitable"
{| class="wikitable"
!style="text-align: left;"|'''Origin:'''
|Leibniz Institute DSMZ, Germany
|-
!style="text-align: left;"|'''Sequenced by:'''
|Plasmidsaurus, ONT sequencing
|-
!style="text-align: left;"|'''Assembled by:'''
|Christian Heryakusuma
|-
!style="text-align: left;"|'''Methylome called by:'''
|William G. Alexander
|-
!style="text-align: left;"|'''Methyltransferases cloned by:'''
|Guss Lab, ORNL
|-
!style="text-align: left;"|'''MIC and Culturing Conditions by:'''
|Christian Heryakusuma
|-
!style="text-align: left;"|'''Transformation by:'''
|
|}
'''''Abbreviation:''''' ONT, Oxford Nanopore Technologies; ORNL, Oak Ridge National Laboratory.
== Genome ==
[[File:Circos Methanocatella gottschalkii HO-edt.png|thumb|left|Circos plot of ''Methanocatella gottschalkii'' HO. '''Source:''' Christian Heryakusuma|550x550px]]
{| class="wikitable" style="float: left; margin-right: 2em;"
|+
|+
! colspan="2" |Scientific Classification
! colspan="2" |Assembly Summary
|-
|-
|Domain:
|Completeness
|Archaea
|99.97%
|-
|-
|Kingdom:
|Contamination
|Methanobacteriati
|0.16%
|-
|-
|Phylum:
|Total contigs
|Methanobacteriota
|1
|-
|-
|Class:
|Max contig length
|Methanobacteria
|1,887,794 bp
|-
|-
|Order:
|GC content
|Methanobacteriales
|30.07%
|}
{| class="wikitable" style="float: left;"
|+
! colspan="4" |Annotation Summary
|-
|-
|Family:
|Coding density
|Methanobrevibacteraceae
|style="border-right: 3px solid gray;"| 88.1%
|CDSs
|1847
|-
|-
|Genus:
|tRNAs
|''Methanocatella''
|style="border-right: 3px solid gray;"| 33
|Pseudogenes
|2
|-
|-
|Species:
|tmRNAs
|''Methanocatella gottschalkii''
|style="border-right: 3px solid gray;"| 0
|Hypotheticals
|1585
|-
|-
|Strain
|rRNAs
|HO
|style="border-right: 3px solid gray;"| 7
|sORFs
|0
|-
|ncRNAs
|style="border-right: 3px solid gray;"| 1
|Gaps
|0
|-
|ncRNAs regions
|style="border-right: 3px solid gray;"| 1
|oriCs
|0
|-
|CRISPR arrays
|style="border-right: 3px solid gray;"| 0
|oriVs
|0
|-
|
|style="border-right: 3px solid gray;"|
|oriTs
|0
|}
|}
<nowiki>*</nowiki>NOTE: to keep these strain pages as uncluttered as possible, these type of info should be in the species page. Simply clone the species page and any associated templates as instructed and these data will automatically appear. See [[Sharpea azabuensis|Sharpea azubuensis]] as a species page example.
<div style="clear: both;"></div>


== Global Methane Hub Metadata ==
Genomic DNA (gDNA) of ''M. gottschalkii'' HO (collection number: DSM 11977, OCM 817) was extracted using NEB Monarch<sup>®</sup> 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)<ref>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. </ref>.
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== Genome ==
The extracted gDNA was sequenced with Oxford Nanopore, then assembled with Flye<ref>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.</ref> and polished with medaka (https://github.com/nanoporetech/medaka). The assembled genome was evaluated for completeness and contamination using QUAST<ref>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.</ref> and CheckM2<ref>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.</ref>. Bakta v1.12.0<ref>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.</ref> 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.
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== DefenseFinder Results ==
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== Methylome ==
== Methylome ==
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== Methylating ''E. coli'' ==
=== Methylation pattern ===
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{| class="wikitable"
|+
!'''#motif'''
!'''methylCounts'''
!'''totalCounts'''
!'''%motifsMethyl'''
|-
|C(6mA)YNNNNNRTTG
| align="right" |1495
| align="right" |1558
| align="right" |95.96
|-
|CA(6mA)YNNNNNRTG
| align="right" |1522
| align="right" |1558
| align="right" |97.69
|}
Methylation motifs were analyzed using MIJAMP<ref>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. </ref>. 4mC and 5mC motifs were not present in this isolate.
 
=== DefenseFinder Results ===
{| class="wikitable"
|+
!Type
!Subtype
!Components
|-
|CRISPR-Cas
|CAS Cluster
|cas1_I_II_III_IV_V_VI_10, <br>cas2_I_II_III_IV_V_VI_3
|-
|RM Type I
|N6-adenine methylating (m6A) Type I RM
|Type I RM methyltransferase (HsdM), <br>Type I RM restriction endonuclease (HsdR), <br>Type I RM specificity subunit (HsdS)
|-
|Prometheus
|Prometheus
|ProA
|-
|RAZR
|RAZR
|PD-T7-4
|-
|RM Type II
|DNA methylase and Type II REase of the PD-(D/E)xK superfamily
|Type II DNA methyltransferase, <br>Type II restriction endonuclease (profile 12)
|}
 
'''''Abbreviation:''''' CRISPR-Cas, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzymes; RM, restriction-modification; Prometheus, DNA replication helicase/nuclease 2 (DNA2)-containing anti-phage defense protein<ref>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.</ref>; RAZR, ring-activated zinc-finger RNase<ref>Zhang, T., Lyu, Y., Beck, C.R. ''et al.'' Bacterial immune activation via supramolecular assembly with phage triggers.                    ''Nature'' '''651''', 1051–1059 (2026). https://doi.org/10.1038/s41586-025-10060-8. </ref>; PD-T7-4, phage defense selecting against T7 bacteriophage system 4<ref>Vassallo CN, Doering CR, Littlehale ML, Teodoro GIC, Laub MT. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol. 2022 Oct;7(10):1568-1579. doi: 10.1038/s41564-022-01219-4. Epub 2022 Sep 19. Erratum in: Nat Microbiol. 2024 Oct;9(10):2760-2761. https://doi.org/10.1038/s41564-024-01724-8. </ref>; REase, restriction endonuclease.
=== Methylating ''E. coli'' ===
''E. coli'' strain AG21365 developed by ORNL was used as a host to generate plasmids with ''M. gottschalkii'' HO methylation motifs shown above.


== Culturing Conditions ==
== Culturing Conditions ==
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''M. gottschalkii'' HO is cultivated in GMH-VT2 medium at 37°C and 50 rpm with a 30 psig gas mixture of H<sub>2</sub>:CO<sub>2</sub> (80:20, v/v) as an energy substrate. Growth is typically observed within 24 to 48 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 H<sub>2</sub>:CO<sub>2</sub> (80:20, v/v) gas mixture as an energy substrate. Visible growth on plate is usually observable within 2 to 5 days. The viability of ''M. gottschalkii'' HO on agar plates is 10-30 times higher than that of those grown on gelrite plates.


== Antimicrobial MIC Tests ==
== Antimicrobial MIC Tests ==
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[[File:Antimicrobial MIC Tests - M. gottschalkii HO.png|thumb|395x395px|Antimicrobial susceptibility tests on ''M. gottschalkii'' HO'''. Source:''' Christian Heryakusuma.]]
 
{| class="wikitable"
|+
! rowspan="2" |Antibiotics
! colspan="2" |Concentration
! rowspan="2" |Observation Period
|-
!style="text-align: center;" |'''Liquid'''
!style="text-align: center;" |'''Solid'''
|-
|Sodium sulfite
|0.5 mM
|0.3 mM
|style="text-align: center;" | ~240 h
|-
|Mevinolin
|40 μM
|20 μM
|style="text-align: center;" | ~240 h
|-
|5-fluoroorotic acid (5-FOA)
|style="color: red;"| ''Insensitive''
|style="color: red;"| ''Insensitive''
|style="text-align: center;" | ~240 h
|-
|8-azahypoxanthine (8-AH)
|style="color: red;"| ''Insensitive''
|style="color: red;"| ''Insensitive''
|style="text-align: center;" | ~240 h
|-
|6-mercaptopurine (6-MP)
|style="color: red;"| ''Insensitive''
|style="color: red;"| ''Insensitive''
|style="text-align: center;" | ~240 h
|-
|5-fluorouracil (5-FU)
|0.25 mg/mL
|0.15 mg/mL
|style="text-align: center;" | ~240 h
|-
|Sodium 2-bromoethanesulfonate (BES)
|2 mM
|1 mM
|style="text-align: center;" | ~240 h
|}
 
Susceptibility testing was evaluated in GMH-VT2 medium inoculated with a 4% ''M. gottschalkii'' HO culture that has a starting OD<sub>600</sub> of ~0.4. ''M. gottschalkii'' HO was considered sensitive to the selection or counterselection agents if there was no increase in the OD<sub>600</sub> during the ~240 h (10 days) observation period.
 


== Protocols ==
== Protocols ==
Line 61: Line 217:


== External Links ==
== External Links ==
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Latest revision as of 20:35, 3 November 2026

Introduction

Phase-contrast (top) and epifluorescence (bottom) microscopy of Methanocatella gottschalkii HO. Source: Christian Heryakusuma.

Methanocatella gottschalkii HOT (formerly Methanobrevibacter gottschalkii) is a type strain isolated from the enrichment of horse faeces[1]. M. gottschalkii, along with Methanobrevibacter ruminantium, comprises about 74% of total archaeal OTUs from rumen samples collected across a wide range of geographical regions, hosts, and diets[2]. This universality makes them methanogens of particular interest to the Global Methane Hub, as it enables strategies to mitigate methane emissions by targeting these dominant methanogens.

Global Methane Hub Metadata

Origin: Leibniz Institute DSMZ, Germany
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: ONT, Oxford Nanopore Technologies; ORNL, Oak Ridge National Laboratory.


Genome

Circos plot of Methanocatella gottschalkii HO. Source: Christian Heryakusuma
Assembly Summary
Completeness 99.97%
Contamination 0.16%
Total contigs 1
Max contig length 1,887,794 bp
GC content 30.07%
Annotation Summary
Coding density 88.1% CDSs 1847
tRNAs 33 Pseudogenes 2
tmRNAs 0 Hypotheticals 1585
rRNAs 7 sORFs 0
ncRNAs 1 Gaps 0
ncRNAs regions 1 oriCs 0
CRISPR arrays 0 oriVs 0
oriTs 0

Genomic DNA (gDNA) of M. gottschalkii HO (collection number: DSM 11977, OCM 817) 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)[3].

The extracted gDNA was sequenced with Oxford Nanopore, then assembled with Flye[4] and polished with medaka (https://github.com/nanoporetech/medaka). The assembled genome was evaluated for completeness and contamination using QUAST[5] and CheckM2[6]. Bakta v1.12.0[7] 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
C(6mA)YNNNNNRTTG 1495 1558 95.96
CA(6mA)YNNNNNRTG 1522 1558 97.69

Methylation motifs were analyzed using MIJAMP[8]. 4mC and 5mC motifs were not present in this isolate.

DefenseFinder Results

Type Subtype Components
CRISPR-Cas CAS Cluster cas1_I_II_III_IV_V_VI_10,
cas2_I_II_III_IV_V_VI_3
RM Type I N6-adenine methylating (m6A) Type I RM Type I RM methyltransferase (HsdM),
Type I RM restriction endonuclease (HsdR),
Type I RM specificity subunit (HsdS)
Prometheus Prometheus ProA
RAZR RAZR PD-T7-4
RM Type II DNA methylase and Type II REase of the PD-(D/E)xK superfamily Type II DNA methyltransferase,
Type II restriction endonuclease (profile 12)

Abbreviation: CRISPR-Cas, Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzymes; RM, restriction-modification; Prometheus, DNA replication helicase/nuclease 2 (DNA2)-containing anti-phage defense protein[9]; RAZR, ring-activated zinc-finger RNase[10]; PD-T7-4, phage defense selecting against T7 bacteriophage system 4[11]; REase, restriction endonuclease.

Methylating E. coli

E. coli strain AG21365 developed by ORNL was used as a host to generate plasmids with M. gottschalkii HO methylation motifs shown above.

Culturing Conditions

M. gottschalkii HO 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 24 to 48 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 2 to 5 days. The viability of M. gottschalkii HO on agar plates is 10-30 times higher than that of those grown on gelrite plates.

Antimicrobial MIC Tests

Antimicrobial susceptibility tests on M. gottschalkii HO. Source: Christian Heryakusuma.
Antibiotics Concentration Observation Period
Liquid Solid
Sodium sulfite 0.5 mM 0.3 mM ~240 h
Mevinolin 40 μM 20 μM ~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) 0.25 mg/mL 0.15 mg/mL ~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. gottschalkii HO culture that has a starting OD600 of ~0.4. M. gottschalkii HO 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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  1. ↑ Miller, T. L. (2002). "Description of Methanobrevibacter gottschalkii sp. nov., Methanobrevibacter thaueri sp. nov., Methanobrevibacter woesei sp. nov. and Methanobrevibacter wolinii sp. nov". International Journal of Systematic and Evolutionary Microbiology. 52 (3): 819–822. https://doi.org/10.1099/00207713-52-3-819.
  2. ↑ Henderson, G., Cox, F., Ganesh, S. et al. Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range. Sci Rep 5, 14567 (2015). https://doi.org/10.1038/srep14567.
  3. ↑ 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.
  4. ↑ 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.
  5. ↑ 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.
  6. ↑ 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.
  7. ↑ 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.
  8. ↑ 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.
  9. ↑ 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.
  10. ↑ Zhang, T., Lyu, Y., Beck, C.R. et al. Bacterial immune activation via supramolecular assembly with phage triggers. Nature 651, 1051–1059 (2026). https://doi.org/10.1038/s41586-025-10060-8.
  11. ↑ Vassallo CN, Doering CR, Littlehale ML, Teodoro GIC, Laub MT. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat Microbiol. 2022 Oct;7(10):1568-1579. doi: 10.1038/s41564-022-01219-4. Epub 2022 Sep 19. Erratum in: Nat Microbiol. 2024 Oct;9(10):2760-2761. https://doi.org/10.1038/s41564-024-01724-8.
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