Ligilactobacillus ruminis WC1T17
Introduction
Strain characteristics and function in the rumen
Ligilactobacillus ruminis WC1T17 is a Gram-positive, rod-shaped, aerotolerant anaerobic bacterium that typically grows in a single bacillus or diplo-bacillus cellular arrangement. Lactobacillus species are of increasing interest for methane mitigation research in ruminant animals as their metabolic network in the rumen can function as a hydrogen sink. Ligilactobacillus are lactic acid bacteria which ferment glucose in the rumen and produce lactate, this lactate can be utilized by syntrophic lactate utilizing bacteria to produce propionate, an important metabolite for the host animal, and this process also requires absorbs hydrogen thus reducing available substrate for methanogenesis. L. ruminis is of particular interest to the Global Methane Hub as it is the only known member of the Lactobacillus genus to be motile making it an attractive prospect for ruminant methane mitigation research as it is theoretically better suited to translocate to multiple niches within the rumen biome.
Strain history and information
L. ruminis WC1T17 was isolated from the rumen of a healthy cow in Palmerston North, New Zealand by the Institute for Bioeconomy Science, formerly known as AgResearch. The strain contains a circular 1.9 Mb genome with no extrachromosomal elements such as plasmids. This strain can be readily cultured in MRS broth and on MRS agar plates under anaerobic conditions with an average doubling time of 40 minutes. Minimum inhibitory concentration (MIC) screening of antibiotics and counter-selective agents in liquid media shows that the strain is sensitive to the following tested antibiotics: chloramphenicol, ampicillin, tetracycline, and streptomycin and the following counter-selective agents: 8-azehypoxanthine, 2-deoxy-d-galactose, 5-fluordeoxyuridine, and sucrose.
L. ruminis WC1T17 is genetically tractable and has been successfully transformed via electroporation with the replicative shuttle vector pMTL89151. This vector confers resistance to chloramphenicol and contains the pAMB1 Gram-positive origin of replication machinery in addition to a multiple cloning sight for integration of reporter genes or biological payloads of interest.
Global Methane Hub Metadata
| Strain origin: | New Zealand1 |
| Isolated from: | Cow rumen |
| Sequenced by: | Rhiannon Cecil2 |
| Assembled by: | William G. Alexander3 |
| Genome and methylome analysis by: | William G. Alexander3 & Ashley Hall2 |
| MIC and culturing conditions by: | Rhiannon Cecil & Ashley Howes2 |
| Transformed by: | Ashley Howes |
1Institute for Bioeconomy Science, Palmerston North, New Zealand. 2BiomEdit, Indianapolis, Indiana, United States. 3Oak Ridge National Lab, Oak Ridge, Tennessee, United States.
Genome
| L. ruminis WC1T17 Genome Summary | |
| Sequencing method | Oxford Nanopore |
| Contigs | 1 |
| Base pairs | 1.9 Mb |
| CDSs | 1,830 |
| CRISPR | 1 |
| Genes | 1,952 |
| rRNA | 19 |
| tRNA | 73 |
| Miscellaneous RNA | 29 |
| Antimicrobial resistance genes | None |
Methylome
Methylome analysis summary results
| L. ruminis WC1T17 Methylome Summary | |
| Isolation Motif | % Motif Methylation |
| GTTGN(6mA)C | 99.13 |
| CAC(6mA)G | 92.58 |
| CAGR(6mA)G | 99.54 |
DefenseFinder Results
| L. ruminis WC1T17 DefenseFinder Results | |
| DefenseFinder System | # Found |
| Cas (Class I, Type I-E) | 1 |
| RM Type III | 1 |
| PD-T7-2 | 1 |
| RM Type IIG | 3 |
Methylating Escherichia coli strains
E. coli strain AGXXXXX engineered to carry methytransferases X, X, X for methylating plasmid DNA for L. ruminis WC1T17.
Culturing Conditions
L. ruminis WC1T17 can be readily cultured in MRS broth or agar plates under anaerobic conditions (75% N2, 5% H2, and 20% CO2) at 39°C for 12 – 48 hours of incubation. L. ruminis WC1T17 colonies typically form on MRS agar plates overnight and colonies inoculated from an agar plate or transferred from an active liquid culture will also grow overnight; however, when reviving from cryostock the strain takes ~48 hours to exhibit growth in broth. The strain will remain viable in liquid or solid medium for ~1 week at room temperature under anaerobic conditions. For long-tern storage 1mL of active culture with an OD600 >0.8 should be combined with anaerobic 50% glycerol to a final glycerol concentration of 25% in anaerobic screw-cap cryovials then stored at -80°C. Revival of cryostocked cells is achieved by transferring a small amount of the cryostock to either an MRS agar plate or to MRS broth followed by incubation under anaerobic conditions.
