Working with Snodgrassella alvi

Snodgrassella alvi is a member of the honey bee (Apis mellifera) gut core microbiota that can be cultivated in vitro. Its isolation and characterization were recently carried out by Waldan Kwong in the laboratory of Dr. Nancy Moran and are described in depth in here.

In vitro Culture Conditions

Growth in Solid Media/Agar Plates

Snodgrassella alvi strain wkB2 (type strain) is generally cultured at 35°C and 5% CO₂. It grows robustly on agar plates after 48 hours on the following media:

• Brain Heart Infusion Agar (BHI)
• Brain Heart Infusion Agar (BHI) supplement with 5% Sheep's Blood
• Columbia Agar supplement with 5% Sheep's Blood (CBA) - PREFERRED
• R2 Agar

Liquid Media

Snodgrassella alvi can be cultivated in the following liquid media (same general culture conditions as above, with or without shaking):

• Insectagro (proprietary media)
• SFM4Insect with L-Glutamine (proprietary media)
• Salt-free LB
• Columbia broth
• R2 Liquid Media
• Brain Heart Infusion (BHI) media

Insectagro and SFM4Insect media induces maximum biofilm formation in S. alvi cultures.

Recently, Philip Engel's group published on using a modified version of M9 minimal media called the Bee9 medium to be able to robustly culture S. alvi in liquid (Quin et al 2024).

Transformation

Conjugation

Snodgrassella alvi can be conjugated using E. coli donor strains and mobilizable rsf1010-based plasmids. Refer to the Bee Microbiome Toolkit paper for full description of working with this species.
Perform conjugation as described here: Conjugation, with the following specifics: use 10:1 ratio of S. alvi to donor E. coli pooled on CBA plates. Incubate overnight, then resuspend and plate on selective CBA plates.

Electroporation

• Electrocompetent Cell Preparation:

S. alvi can be transformed by electroporation (Chhun et al 2024; Lariviere et al 2023). Briefly, streak wkB2 cells using overlapping streaks on CBA and grow at 35°C in 5% CO₂ for 2-3 days (recommended 72 hours). Once colonies have appeared, pick colonies, re-streak multiple CBA plates, and incubate them for 24-48 hours (not exceeding 48 hours) in the same condition. Scrape all the growth from the plate into 1.5 ml tube(s) with 1 ml PBS and resuspend/mix cells, and pellet by centrifuging at 5000 RCF (xg) for 10 mins. Mix the cells in the tube by pipetting up and down. Wash twice in 1 ml chilled autoclaved DI water or 10% glycerol and resuspend the pellet after the final wash in 10% glycerol. Keep the cells on ice from here on out. Make 70-100 µl aliquots and either freeze at -80°C or use right away.

• Electroporation:

To electroporate S. alvi (Chhun et al 2024; Lariviere et al 2023), obtain and thaw an aliquot of electrocompetent wkB2 cells and add the appropriate amount of DNA (0.5-1 µg recommended if using RSF1010-based plasmids). After adding plasmid DNA, incubate the cells on ice for 20 minutes. Afterwards, transfer to a 1 mm electroporation cuvette and electroporate (Ec1 on BioRad Micropulser). Immediately following electroporation, add 900 µl of SFM4 media or Columbia broth to the cuvette and transfer the cells to a 50 ml falcon tube, with the cap loosely taped on (to allow for gas exchange). Allow cells to recover overnight at 35°C in 5% CO₂ for 2-3 days. The next day, plate 100 µl of undiluted cells on selective media. Pellet the remainder of the cells, resuspend in 100 µl of media, and plate the concentrate on selective media as well. Grow selective plates at 35°C in 5% CO₂ for 2-3 days or until colonies form. Once colonies have formed, make an isolation streak on a new plate and grow at 35°C in 5% CO₂ for 2-3 days. Once isolation streaks have grown up, make glycerol stocks and store at -80°C.

• Genomic Integration by Electroporation:

To insert genes directly into S. alvi genome by electroporation, (Lariviere et al 2023), below is the recommended DNA concentrations based on the type of DNA:

• Gene fragment/Plasmid - 1 µg
• Purified Genomic DNA - 2 µg

Antibiotic Concentrations and Stock Solutions

This table lists “standard” concentrations of antibiotics commonly used in microbiology laboratories. These concentrations apply to S. alvi (wkB2). Antibiotic selection in other organisms may use different concentrations, so check primary literature for more info.

Reagent	Abb	Stock Conc.	Working Conc.	Dilution	Solvent	MSDS	Notes
Ampicillin	Amp	100 mg/mL	20 µg/mL	5,000×	ddH2O	link	Ampicillin degrades quickly in both plates and stock solutions. Culture plates with Amp can be stored at 4°C for about 2 weeks. Stock solutions can be stored at 4°C for 2 weeks but can last as long as 4-6 months when stored at -20°C. In general, use carbenicillin for ampicillin selection, because it is more chemically stable.
Carbenicillin	Crb	100 mg/mL	50 µg/mL	2,000×	50% EtOH	link	Carbenicillin can be used in place of Ampicillin at the same working concentration. Carbenicillin is more stable than Ampicillin, and is generally preferable.
Chloramphenicol	Cam	20 mg/mL	20 µg/mL	1,000×	EtOH	link
Gentamicin	Gen	20 mg/mL	25 µg/mL	800×	ddH2O	link	For non-canonical experiments: Use 20 µg/mL in culture from 20mg/mL 1000× stock
Kanamycin	Kan	50 mg/mL	25 µg/mL	2,500×	ddH2O	link	Plates start to lose sensitivity after 2 weeks; recommended Kanamycin plates only last a few weeks at 4°C; best to make fresh Kan plates every 2 - 4 weeks.
Nalidixic Acid	Nal	30 mg/mL	30 µg/mL	1,000×	1M NaOH	link
Rifampicin	Rif	50 mg/mL	30 µg/mL	1667×	MeOH + drops of 10M NaOH	link	Toxic to humans, use PPE! Rifampicin is TOXIC to humans, light-sensitive, and degrades in solution rather quickly. It needs to be stored wrapped in foil and replaced every 2 months.
Spectinomycin	Spec	60 mg/mL	30 µg/mL	2,000×	ddH2O	link
Streptomycin	Str	100 mg/mL	20 µg/mL	5,000×	ddH2O	link
Tetracycline	Tet	10 mg/mL	10 µg/mL	1,000×	ddH2O	link	S. alvi is naturally resistant to Tet. Use 10 µg/mL. Can be used to isolate S. alvi from other bacteria or contaminants during bee colonization assays without other antibiotics.
Trimethoprim	TMP	10 mg/mL	20 µg/mL	500×	DMSO	link	S. alvi is naturally resistant to TMP. Use 20 µg/mL.
Tylosine	Tyl	5 mg/mL	5 µg/mL	1,000×	ddH2O	link	S. alvi wkB2 and S. communis wkB12 are naturally resistant to Tyl. Use 5 µg/mL.

References:

• Kwong WK, Moran NA. Cultivation and characterization of the gut symbionts of honey bees and bumble bees: description of Snodgrassella alvi gen. nov., sp. nov., a member of the family Neisseriaceae of the Betaproteobacteria, and Gilliamella apicola gen. nov., sp. nov., a member of Orbaceae fam. nov., Orbales ord. nov., a sister taxon to the order 'Enterobacteriales' of the Gammaproteobacteria. Int J Syst Evol Microbiol. 2013 Jun;63(Pt 6):2008-2018. doi: 10.1099/ijs.0.044875-0. Epub 2012 Oct 5. PMID: 23041637.

• Leonard SP, Perutka J, Powell JE, Geng P, Richhart DD, Byrom M, Kar S, Davies BW, Ellington AD, Moran NA, Barrick JE. Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids. ACS Synth Biol. 2018 May 18;7(5):1279-1290. doi: 10.1021/acssynbio.7b00399. Epub 2018 Apr 13. PMID: 29608282; PMCID: PMC5963681.

• Quinn, A., El Chazli, Y., Escrig, S. et al. Host-derived organic acids enable gut colonization of the honey bee symbiont Snodgrassella alvi. Nat Microbiol 9, 477–489 (2024). https://doi.org/10.1038/s41564-023-01572-y.

• Lariviere PJ, Leonard SP, Horak RD, Powell JE, Barrick JE. Honey bee functional genomics using symbiont-mediated RNAi. Nat Protoc. 2023 Mar;18(3):902-928. doi: 10.1038/s41596-022-00778-4. Epub 2022 Dec 2. PMID: 36460809.

• Chhun A, Moriano-Gutierrez S, Zoppi F, Cabirol A, Engel P, Schaerli Y. An engineered bacterial symbiont allows noninvasive biosensing of the honey bee gut environment. PLoS Biol. 2024 Mar 5;22(3):e3002523. doi: 10.1371/journal.pbio.3002523. PMID: 38442124; PMCID: PMC10914260.

• Single-step genome engineering in the bee gut symbiont Snodgrassella alvi. Patrick J. Lariviere, A. H. M. Zuberi Ashraf, Sean P. Leonard, Laurel G. Miller, Nancy A. Moran, Jeffrey E. Barrick. bioRxiv 2023.09.19.558440; doi: https://doi.org/10.1101/2023.09.19.558440