Methanobrevibacter millerae ZA-10T

Global Methane Hub Metadata

Origin:	AgResearch, New Zealand
Sequenced by:	Sharik Khan, University of Illinois at Urbana-Champaign
Assembled by:	Glen Cronan, University of Illinois at Urbana-Champaign
Methylome called by:	William G. Alexander, Oak Ridge National Laboratory
Methyltransferases cloned by:	Adam Guss, Oak Ridge National Laboratory
MIC and Culturing Conditions by:	Sharik Khan, University of Illinois at Urbana-Champaign
Transformation by:	Sharik Khan, University of Illinois at Urbana-Champaign Komal Aggrawal, University of Illinois at Urbana-Champaign

Genome

Single contig of 2,823,065 bp.

Methylome

#6mA modified motifs
#motif	methylCounts	totalCounts	%motifsMethyl
CA(6mA)NNNNNNNTTG	1905	1944	97.99
CRT(6mA)NNNNNNCCT	428	460	93.04
(6mA)GGNNNNNNTAYG	432	460	93.91
G(6mA)TC	4913	6856	71.66
GCT(6mA)NNNNNNRTTA	103	106	97.17
TA(6mA)YNNNNNNTAGC	104	106	98.11
#4mC modified motifs
#motif	methylCounts	totalCounts	%motifsMethyl
TC(4mC)AT	3383	8313	40.70

Methylating E. coli Strains

Two E. coli methylation strains, AG22054 (dam^-) and AG22055 (dam⁺), expressing M. millerae ZA-10T methylases have been constructed by Adam Guss's group at the Oak Ridge National laboratory.

Culturing Conditions

M. millerae ZA-10T grows well in both RMO2-medium and MSM (Methanobrevibacter smithii medium) topped with H₂/CO₂ (80:20, v/v). Growth of M. millerae ZA-10T requires rumen fluid but does not need volatile fatty acids (VFAs), or sodium sulfide. The plating efficiency of the strain on MSM-agar plates is > 90% and colonies are visible in 5-7 days when incubated at 37°C in Wolfe incubators filled with a gas mixture of H₂/CO₂ (80:20) supplemented with 1000ppm H₂S .

Antibiotic MIC Tests

antibiotic	concentration (ug/mL)
Puromycin
Nourseothricin
Neomycin
8-ADP
5-Fluorouracil
2-BES
5-FOA

Protocols

• Removal of extracellular material via chelation washing