UNIT 3D.1 Laboratory Maintenance of Agrobacterium

Abstract

Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for laboratory cultivation of A. tumefaciens.

Agrobacterium tumefaciens is a member of the α-Proteobacteria within the family Rhizobiaceae. It is a soil microbe and a facultative phytopathogen capable of causing crown gall disease on plants. A. tumefaciens is closely related to symbiotic species of nitrogen-fixing rhizobia. Its virulence depends on the presence of a specific plasmid known as the Ti (tumor-inducing) plasmid. For a comprehensive review of Agrobacterium biology, see Tzfira and Citovsky, 2008.

Due to the amount of genetic exchange and variation between species of Agrobacterium, the nomenclature of the group continues to be a point of controversy. Large plasmids such as the Ti plasmid, although not essential for survival in the laboratory, contribute enormously to the ecology and relevant phenoytpes of the bacteria. In addition, these plasmids can be transferred horizontally between species, further complicating the issue. Historically, members of the genus Agrobacterium were classified at the species level according to their plasmid-determined phytopathogenic properties (i.e. tumefaciens for crown gall, rhizogenes for hairy root, vitis for grape infections, and radiobacter for non-pathogenic derivatives), and further subdivided into biovars I, II, and III (Table 1), based on other phenotypic and physiological characteristics. Genetic and genomic analyses have helped to more accurately define the phylogenetic relationship between different species so that in a more polyphasic method of nomenclature, A. tumefaciens, A. vitis, and A. rubi fall into one group, whereas A. rhizogenes and most Rhizobium species are associated with a second distinct lineage (Table 1). There is still some debate as to whether all Agrobacterium species should be classified as the same genus Rhizobium, but for now the most widely accepted methodology for classification distinguishes between the two genera.

Table 1.

Common strains of Agrobacterium by opine utilization and biovar type.

Species	Strain	Opines	Biovar
A. tumefaciens	C58	nopaline, agrocinopines A + B	1
	A6, 15955, B6, R10	octopine, mannityl opines	1
	A348a	octopine, mannityl opines	1
	Bo542	agropine-succimamopine, agrocinopines C + D	1
	Chry5	chrysopine	1
A. radiobacter	K27	agrocinopines A + B	2
	K84	nopaline, agrocinopines A + B	2
A. rhizogenes	A4	agropine	2
A. vitis	S4	vitopine

^aNopaline genetic background with pTiA6^NC Ti plasmid

Within the A. tumefaciens species, strains and plasmids have also historically been classified by compounds called opines, which are produced by infected neoplastic tissue in response to the disease (Table 1). The incorporated T-DNA encodes genes that cause the plant to synthesize the opines, which can serve as sources of carbon, nitrogen and phosphorus for the infecting A. tumefaciens and certain other soil microbes. Opines are metabolized by A. tumefaciens via other genes encoded on the Ti plasmid that are not transferred to the plant. Historically plasmids have been named by their dominant opine, such as the broadly studied nopaline-type Ti plasmids and the octopine-type Ti plasmids (Table 1). Several other opine-based classification groups have also been commonly used. Upon isolation from infected plants, the isolates of A. tumefaciens were grouped by this scheme, and thus this classification is often also used to refer to thegenomic background in which the Ti plasmid of a specific opine type was first isolated. For example, a commonly used laboratory strain is A. tumefaciens A348, which has a nopaline-type genetic background from which the resident nopaline-specifiying Ti plasmid had been cured, and into which an octopine-type Ti plasmid was introduced. This informal opine-based classification remains in common usage today, despite the fact that most Ti plasmids specify the synthesis of more than one type of opine, and also that this catabolic capacity is encoded on the inherently unstable and horizontally transferrable Ti plasmid. One of the most commonly studied strains is A. tumefaciens C58 (isolated from a cherry tree in 1958 in upstate New York), which contains a nopaline-type Ti plasmid. Other common laboratory strains, 15955, R10, and A348 contain an octopine-type Ti plasmid. Different Ti plasmids possess the same incompatibility region and thus multiple types generally do not occur together in the same strain (Soberon, 2004).

The detection of a subset of opines by A. tumefaciens cells proximal to the infected tissue leads to the activation of the genes responsible for conjugal transfer of the Ti plasmid (tra and trb) and copy number (rep). This control is indirect, as opines induce the response to an externalized diffusable signal, N-β-oxo-octanoyl-L-homoserine lactone (3-oxo-C8 HSL), that functions in quorum sensing control of plasmid functions. This signal molecule interacts with specific transcription factors to increase copy number and conjugal transfer.

For nopaline-type Ti plasmids, such as in C58, the transcriptional regulator of the tra operons, traR, is repressed in the absence of the conjugal opines, agrocinopine A and B. When sufficient agrocinopines are present transcription of traR is derepressed, and if the local population density is high, a proportional increase in 3-oxo-C8-HSL leads to activation of TraR. TraR is stabalized by forming a complex with 3-oxo-C8-HSL molecules, and subsequently activates the transcription of Ti plasmid interbacterial conjugal transfer (trb) genes and increases itscopy number. Because conjugation is completely dependent on the presence of opines, it is a process that only occurs in the tumor environment. For a second species of megaplasmid also harbored by many agrobacteria called the At plasmid, conjugation is not dependent on opines and contains regulatory elements more similar to Sinorhizobium meliloti’s mega plasmids, pSymA and pSymB (Blanca-Ordóñez et al. 2010)

All media described in this unit support the growth of A. tumefaciens. It is an obligately aerobic organism and thus, growing with proper aeration is critical. AT is a standard defined medium for growth, and thus is less likely to result in the development of auxotrophic mutants. The other media described in this unitare complex formulations or serve more specific purposes (e.g. biofilm-promoting and vir-inducing media), which are specified in the section.

Growth occurs optimally at 28°C. At temperatures above 30°C, A. tumefaciens begins to experience heat shock and is likely to result in errors in cell division (and is in fact an alternate strategy to Unit 3D.2: Protocol6 for plasmid curing).

If growth is slow in media containing sugar alcohols (e.g. mannitol), it may be due to iron limitation.

A. Growth Conditions

A. tumefaciens grows optimally at 28°C. The doubling time can range from 2.5–4h depending on the media, culture format and level of aeration.

AT Minimal Media (Tempe et al. 1977)

AT is a common laboratory medium used to support growth of A. tumefaciens. This minimal medium provides a non-stressful environment with all essential nutrients and limits the development of auxotrophic mutants. AT medium is the standard growth medium and will allow for a doubling time ranging from 2.5h 4h (dependent on specific carbon/nitrogen sources and antibiotics) and will approach stationary phase at an OD₆₀₀ of 0.9.

20X AT Buffer	Grams added/L	Final conc. (1X)
KH2PO4	214	0.079 M
pH to 7.0 w/ NaOH	35	0.044 M
20X AT Salts
(NH4)2SO4	40	0.015 M
MgSO4-7H2O	3.2	0.6 mM
CaCl2-2H2O	0.2	0.06 mM
MnSO4-H2O	0.024	0.0071 mM
50X Iron Stock
FeSO4-7H20	34.75	0.125 M
1X AT Medium	Volume/L	Final conc. (1X)
20X Buffer	50 ml/L	1X
20X Salts	50 ml/L	1X
50% glucose	10 ml/L	0.5% (28 mM)
*50X Iron	20 ml/L	1X
+/−Bacto-Agar	15 g	1.5%

Prepare sterile, autoclaved stocks of 20X Salts, 20X buffer, iron solution and the carbon source (most often glucose). Add the appropriate amounts to either sterile deionized water (AT broth) or molten (55°C) sterile 1.5% water-agar (AT agar), mix gently but thoroughly, and use or pour plates.

The carbon and nitrogen sources can be varied. A. tumefaciens will utilize a variety of sugars, α-keto acids, amino acids and sugar alcohols as carbon sources, as well as a wide range of diverse nitrogen sources. AT minimal medium with glucose and (NH₄)₂SO₄ is generally referred to as ATGN. AT medium with octopine as both carbon and nitrogen sourceis called ATO, as carbon source alone ATON and as nitrogen source alone ATGO.

Iron should be added prior to pouring for solid medium, and in most cases to liquid medium as well. Only use an iron stock that does not have a great amount of precipitate.

Induction Broth (IB medium) (Winans et al., 1988)

This growth medium is thought to mimic aspects of the vir-inducing conditions of a plant wound. In this medium, Ti plasmid bearing cells grow markedly slower with a lower yield (due to the high cost of vir gene expression and phosphorous limitation) and will reach stationary phase at an OD₆₀₀ of approximately 0.2–0.4.

In shaken liquid cultures, clumps of cells can be easily visualized in the medium. It has been observed that even in very light cultures that appear clear, there are high numbers of cells likely attributable to this clumping and perhaps smaller cell size.

1X IB Medium	volume/L	Final conc (1x)
1M MES 2-(N-morpholino) ethane sulfonic acid (pH 5.6)	20 ml	20 mM
20X AT Salts	50 ml	1X
0.2 M acetosyringone	1 ml	200 μM
50% glucose	10 ml	0.5% (28 mM)
0.1 M phosphate buffer (pH 7.0)	500 μl	50 μM
mqH2O	up to 1L

Higg Medium (Poindexter, 1978)

This medium stimulates rapid growth and robust biofilm formation. HIGG will lead to shorter doubling times and can support higher cell densities.

1X Higg Medium	Volume/L	Final conc. (1x)
1M imidazole (pH 7.0)	5 ml	5 mM
Hunter Base Concentrate	20 ml	1X
1M CaCl2	1 ml	100 mM
50% glucose	3 ml	0.15% (83 mM)
10% Sodium Glutamate	15 ml	1.5% (89 mM)
1M NH4Cl	8.9 ml	8.9 mM
0.5M Phosphate Buffer (pH 7.0)	20 ml	10 mM
mqH2O	up to 1L

^*Filter sterilize and store in foil

Metals 44

Amount required	Component
250 mg	Disodium EDTA
1.095 g	ZnSO4•7H2O
500 mg	FeSO4•7H2O
154.0 mg	MnSO4•H2O
39.2 mg	CuSO4•5H2O
24.8 mg	Co(NO3)2•6H2O
17.7 mg	Na2B4O7•10H2O
to 100 ml	mqH2O

-mix components

-Add a few drops of H₂SO₄ toretard precipitation

-Filter sterilize

Hutner Base Concentrate (100 ml)

→ Prepare stock of ammonium molybdate (92.5mg/ml) in sterile mqH2O and filter sterilize. Add 10 μl per 100 ml Hutner Base

1.0 g	Nitrilotriacetic acid
1.445 g	MgSO4
0.3335 g	CaCl2•2H2O
0.925 mg	(NH4)6Mo7O24•4H2O
5 ml	Metals 44
to 100 mL	H2O

Dissolve nitrilotriacetic acid in water and neutralize with KOH (~10ml of 10N KOH)

Add remaining components, and adjust pH to 6.6–6.8

Adjust volume to 100 mL

Filter sterilize

Store in foil to prevent precipitation

500 mM Phosphate Buffer pH7.0
61 ml	0.5M Na2HPO4
39 ml	0.5M KH2PO4

Mix and autoclave; pH should be ~7.0

Yeast Extract Mannitol (YEM)

This is a rich medium that supports A. tumefaciens growth very well. Alternatives includeLB, although it has been demonstrated to lead to some issues with irregular cell division. A. tumefaciens grows particularly well using mannitol as a carbon source, but it should be noted that iron can become limiting during sugar alcohol catabolism. YEM will lead to shorter doubling times and can support higher cell densities.

1X YEM Medium	grams/L	Final conc (1x)
Mannitol	5	27 mM
Yeast Extract	0.5	--
MgSO4 × 7 H20	0.2	0.8 mM
NaCl	0.1	0.017 mM
K2HPO4	0.5	0.027 mM
Sodium gluconate	5	22.9 mM
mqH2O	up to 1L

Prepare 16.6% CaCl₂ solution and autoclave.

Add 1 ml of the 16.6% CaCl₂ solution to 1L YEM medium.

COMMON ANTIBIOTICS – WORKING CONCENTRATIONS FOR AGROBACTERIUM

Concentrations of antibiotics that prevent growth of wild-type bacteria have been optimized for work in A. tumefaciens C58 and may vary with other strains of Agrobacterium.

Antibiotic	Working concentration (μg/ml)
Kanamycin	150
Gentamycin	300
Streptomycin	2500
Spectinomycin	150
Ampicillin	100
Tetracycline*	3–4
Rifampicin	25

Working concentrations provided are for liquid media conditions. They should be doubled when working with solid media.

^*Tetracycline has been demonstrated for C58 to be problematic due to spontaneous resistance (see UNIT 3D.2, PROTOCOL 5 ‘Creation of Markerless Deletions by Allelic Replacement’).

B. Storage Conditions

Short-term storage

For short-term storage of Agrobacterium, cells can be kept on agar media and stored at 4°C for several weeks. It is important to keep in mind however, that cells are still in a metabolically active state at these temperatures, so storage under these conditions should be limited. Cells should not be maintained by long-term passaging on plates because cells are likely to incur mutations.

Long-term storage

The ideal way to store Agrobacterium strains, ensuring that genetic changes due to laboratory selection are limited, is to keep them frozen at −80°C in 30% glycerol or DMSO (cryopreservants that prevent membrane damage caused by freezing and thawing). Once frozen permanents are generated, it is best to limit the amount of freezing and thawing as much as possible. For starting cultures from frozen permanents, it is recommended to keep samples cold and use a sterile stick, loop or needle to remove a small clump of cells (1–5 μl) and inoculate directly into liquid or solid media.